Circulating small noncoding RNAs as biomarkers of aging

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ARR-501; No. of Pages 13

Ageing Research Reviews xxx (2014) xxx–xxx

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Review

Circulating small noncoding RNAs as biomarkers of aging Joseph M. Dhahbi a,b,∗ a b

Department of Biochemistry, University of California at Riverside, Riverside, CA 92521, USA Center for Genetics, Children’s Hospital Oakland Research Institute, Oakland, CA 94609, USA

a r t i c l e

i n f o

Article history: Received 10 January 2014 Received in revised form 17 February 2014 Accepted 24 February 2014 Available online xxx Keywords: Aging Calorie restriction Circulating miRNAs Small RNAs Y RNA fragments tRNA halves

a b s t r a c t Small noncoding RNAs (sncRNAs) mediate a variety of cellular functions in animals and plants. Deep sequencing has made it possible to obtain highly detailed information on the types and abundance of sncRNAs in biological specimens, leading to the discovery that sncRNAs circulate in the blood of humans and mammals. The most abundant types of circulating sncRNAs are microRNAs (miRNAs), 5 transfer RNA (tRNA) halves, and YRNA fragments, with minute amounts of other types that may nevertheless be significant. Of the more abundant circulating sncRNAs only miRNAs have well described functions, but characteristics of the others suggest specific processing and secretion as complexes that protect the RNA from degradation. The properties of circulating sncRNAs are consistent with their serving as signaling molecules, and investigations of circulating miRNAs support the view that they can enter cells and regulate cellular functions. The serum levels of specific sncRNAs change markedly with age, and these changes can be mitigated by calorie restriction (CR), indicating that levels are under physiologic control. The ability of circulating sncRNAs to transmit functions between cells and to regulate a broad spectrum of cellular functions, and the changes in their levels with age, implicate them in the manifestations of aging. Our understanding of the functions of circulating sncRNA, particularly in relation to aging, is currently at a very early stage; results to date suggest that more extensive investigation will yield important insights into mechanisms of aging. © 2014 Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . miRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. miRNAs and the advent of deep sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Intracellular miRNAs in senescence, aging, and calorie restriction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Circulating miRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Circulating miRNAs in aging and CR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . tRNA-derived small RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. tRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Intracellular tRNA-derived small RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. tRFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. tRNA halves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Circulating tRNA-derived small RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. 5 tRNA halves may be immune signaling molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Circulating levels of specific 5 tRNA halves are altered by aging and calorie restriction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . YRNA-derived small RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. YRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Intracellular YRNA-derived small RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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∗ Correspondence to: Department of Biochemistry, University of California at Riverside, Riverside, CA 92521, USA. Tel.: +1 951 827 3553. E-mail addresses: [email protected], [email protected] http://dx.doi.org/10.1016/j.arr.2014.02.005 1568-1637/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Dhahbi, J.M., Circulating small noncoding RNAs as biomarkers of aging. Ageing Res. Rev. (2014), http://dx.doi.org/10.1016/j.arr.2014.02.005

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4.3. Circulating YRNA-derived small RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Circulating YRNA-derived small RNAs in aging and CR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Noncoding RNAs are complex classes of structural and functional RNAs that are never translated into protein; they account for the majority of the transcripts in the cell. Small noncoding RNAs (sncRNAs) are a diverse group of noncoding RNAs that are shorter than 400 nucleotides (nt) in length. Some sncRNAs [i.e., microRNAs (miRNAs), small interfering RNAs (siRNAs), and Piwiinteracting RNAs (piRNAs)] are bound by Argonaute proteins, and have the common property of directing protein complexes to nucleic acids with sequence complementarity, where they may cleave or otherwise alter the target (Joshua-Tor and Hannon, 2011). Through interaction with target genes, sncRNAs control a wide variety of key cellular functions and biological processes including gene expression regulation, chromatin remodeling, genome stability, and development (Amaral and Mattick, 2008; Carthew and Sontheimer, 2009; Krude, 2010; Li, 2013; Okamura, 2012; Wery et al., 2011; Zhang, 2009). Disruption of sncRNAs expression and functions, especially miRNAs, has been associated with cancer, and other diseases including cardiovascular, neurological, autoimmune, and developmental diseases (reviewed in Esteller, 2011; Taft et al., 2010). Recent improvements in the throughput of deep sequencing (also called next generation sequencing) have allowed the discovery of new types of small RNAs that are different from the well-established classes of sncRNAs. Particularly, well-known functional sncRNAs such as tRNA, ribosomal RNA (rRNA), small nucleolar RNA (snoRNA) and YRNA, can undergo processing into smaller RNA molecules (Rother and Meister, 2011; Rutjes et al., 1999; Sobala and Hutvagner, 2011). Although initially regarded as degradation products, accumulating evidence suggests that these RNA fragments, while derived from pre-existing sncRNAs, are themselves functional in normal biology and in pathology (Fu et al., 2009; Haussecker et al., 2010; Lee et al., 2009; Martens-Uzunova et al., 2013; Rutjes et al., 1999; Wang et al., 2013; Zhao et al., 1999). These new sncRNAs have only recently emerged from small RNA data because the experiments that produced the sequence datasets were designed to study miRNAs. Sequences whose length exceeded the size of mature miRNAs (18–24 nt) were systematically discarded; also, sequencing reads that align with tRNAs, rRNAs, and snoRNAs were considered degradation products and excluded from further analysis. sncRNAs and their derivatives can be released into the extracellular environment and thereby travel between tissues within an organism, thus transferring their functions to other cells (Dhahbi et al., 2013a,b,c and reviewed in Hoy and Buck, 2012). There is evidence that miRNAs circulating in the bloodstream can be taken up by cells and alter gene expression (Hergenreider et al., 2012; Vickers et al., 2011; Zernecke et al., 2009; Zhang et al., 2010). This suggests that circulating miRNAs are factors of a newly discovered system of cell-to-cell communication and signaling that can operate in normal biological processes and in disease (Cortez et al., 2011; Kosaka et al., 2013; Shah and Calin, 2013; Turchinovich et al., 2012). Thus, there is great interest in the possibility that circulating miRNAs and other sncRNAs can serve as markers of specific disease states, especially cancer (Allegra et al., 2012; Etheridge et al., 2011; Zen and Zhang, 2012).

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Deep sequencing has facilitated the discovery and characterization of circulating sncRNAs. Our deep sequencing studies have consistently detected 3 major classes of circulating small RNAs (Fig. 1). These are miRNAs of size 20–24 nt, tRNA-derived RNAs of size 30–33 nt, and YRNA-derived RNAs of sizes 27 nt and 30–33 nt. Other types of sncRNAs including rRNAs, snoRNAs, and small nuclear RNAs (snRNAs) are present in the bloodstream at very low levels, in aggregate less than 1% of all small RNAs. This review will focus on the three major types, but it should be kept in mind that the low levels of the other types do not necessarily imply that they lack biological significance. Aging is characterized by extensive changes in gene expression linked to age-related pathologies specific to tissue types (Cao et al., 2001; Dhahbi et al., 2004; Spindler and Dhahbi, 2007; Weindruch et al., 2001). Calorie restriction (CR), a decreased caloric intake without malnutrition, delays the age-induced alterations in gene expression and ameliorates the age-related dysfunctions (McCay et al., 1989). Given the pervasive role of sncRNAs in the regulation of gene expression and the pathogenesis of disease, it is very likely that sncRNAs participate in changes in gene expression that accompany the progression of aging and its deleterious effects; furthermore they have the potential to mediate the beneficial antiaging effects of CR. Consistent with this view, cellular senescence, and organismal aging and its related diseases, are associated with alterations in miRNA expression that could have multifarious physiological effects (Boon et al., 2013; Dimmeler and Nicotera, 2013; ElSharawy et al., 2012; Gombar et al., 2012; Grillari and GrillariVoglauer, 2010; Hackl et al., 2010; Lafferty-Whyte et al., 2009; Liu et al., 2012; Mercken et al., 2013; Serna et al., 2012). However studies addressing the link between circulating sncRNAs, aging, and CR have just begun. Here we review intracellular and circulating miRNAs and the tRNA- and YRNA-derived sncRNAs, the most abundant classes of circulating sncRNAs identified by deep sequencing, and highlight their relation to aging and CR. 2. miRNAs 2.1. miRNAs and the advent of deep sequencing miRNAs are ∼22-nt sncRNAs that repress gene expression at the post-transcriptional level under normal and pathological conditions (Grosshans and Filipowicz, 2008; Nilsen, 2007; Silahtaroglu and Stenvang, 2010). They direct Argonaute protein complexes to messenger RNAs (mRNAs) to incite translational repression or mRNA degradation, leading to decreased protein synthesis (Fabian et al., 2010; Thomas et al., 2010). There are hundreds of miRNAs; through targeting of a vast number of distinct mRNAs, they regulate critical physiological and pathological processes including cell differentiation and signaling, development, tumorigenesis, and the pathogenesis of non-neoplastic diseases (Avraham and Yarden, 2012; da Costa Martins et al., 2010; Farazi et al., 2011; Heinrich and Dimmeler, 2012; Ichimura et al., 2011; Iorio and Croce, 2012; Ivey and Srivastava, 2010; Koturbash et al., 2011; Saugstad, 2010; Townley-Tilson et al., 2010). As potent regulators of gene expression and modulators of fundamental physiological and pathological processes, miRNAs are likely to play a role in the biology of aging and the deleterious changes that occur in cells and tissues, as we get older. Deep sequencing has become the technology of choice

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Fig. 1. Major types of circulating small noncoding RNAs. The length distribution and annotation of reads obtained by deep sequencing of small RNAs extracted from human (A) and mouse (B) serum. Reads were obtained by pooling several sequenced serum samples and mapped to the human hg19 or mouse mm10 genomes. Length is plotted against abundance of the reads annotated as miRNAs, YRNAs, tRNAs, rRNAs, or other sncRNAs (snRNAs and snoRNAs). Reads in the 20–24 nt peak were derived from miRNAs in both human and mouse. The 30–33 nt peak consists of reads mapping to both YRNA and tRNA genes in the human and only to tRNA genes in the mouse. The 27 nt peak exists only in the human and its reads map to YRNA genes.

for analysis of miRNA expression and discovery of novel miRNAs (McCormick et al., 2011; Wittmann and Jack, 2010). This technology is able to sequence a full-length miRNA with a single read, and produces precise counts of each type of miRNA. The steady improvement of bioinformatics analysis tools has facilitated accurate annotation of miRNA sequence datasets, consistent expression measurement with rigorous statistics, prediction of the targeted mRNAs, and functional annotation of the pathways regulated by specific miRNAs. In addition, unlike microarrays deep sequencing both discovers novel miRNAs and detects expression of rare but functionally significant types; only the depth of sequencing limits its sensitivity. The new miRNAs currently being discovered by deep sequencing have been missed by traditional techniques because they tend to be located within poorly annotated regions of the genome, and are expressed at low levels; such limitations are easily overcome by deep sequencing technology. The more abundant miRNAs have largely already been discovered; now, because of its higher sensitivity, deep sequencing is discovering the less abundant ones. 2.2. Intracellular miRNAs in senescence, aging, and calorie restriction The relationship between miRNAs and age-related diseases is a function of their ability to change gene expression without impairing the function of the gene product, extending the range of gene regulation beyond the limits imposed by transcriptional regulation. miRNAs are known for their regulation of critical steps in the pathogenesis of cancer, the most common age-associated disease. They have been implicated in all stages of neoplastic progression, including proliferation, apoptosis, initiation, progression, invasion, chemo/radiotherapy resistance, metastasis, and relapse (reviewed in Cheng et al., 2013; Di Leva and Croce, 2013; Farazi et al., 2013; Iuliano et al., 2013; Pencheva and Tavazoie, 2013; Shen et al., 2013; Uchino et al., 2013). This intimate involvement in tumorigenesis

reflects the pervasive roles of miRNAs in cellular physiology, and also makes them attractive candidates for diagnostic markers and therapeutic targets in cancer (Uchino et al., 2013; Yang et al., 2013). Expression of miRNAs is also altered during cellular senescence, in the course of normal and healthy organismal aging, and in agerelated diseases (Boon et al., 2013; Dimmeler and Nicotera, 2013; ElSharawy et al., 2012; Gombar et al., 2012; Grillari and GrillariVoglauer, 2010; Gupta et al., 2014; Hackl et al., 2010; He et al., 2007; Lafferty-Whyte et al., 2009; Liu et al., 2012; Maes et al., 2009; Mercken et al., 2013; Serna et al., 2012). Moreover, miRNAs that display age-dependent expression patterns tend to change expression in age-related diseases, and their potential targets are usually enriched in disease-associated pathways (ElSharawy et al., 2012; Hackl et al., 2010; Noren Hooten et al., 2010). Application of deep sequencing to the study of miRNAs in cellular senescence and organismal aging has only recently begun. Using deep sequencing, we have found that senescence in fibroblasts is associated with changes in the expression of a group of miRNAs (miR-1246, miR-584 and miR-323) (Dhahbi et al., 2011) known to play a role in cancer (Dixon-McIver et al., 2008; Wang et al., 2010b; Witten et al., 2010); these have not been reported to change in previous senescence studies. Other miRNAs whose levels change during senescence include miR-432, which has previously been reported only as an abundant miRNA in the earliest stage of fetal development (McDaneld et al., 2009), and which may regulate endothelial cell-specific genes (Bhasin et al., 2010), and miR-145, whose involvement in breast and colon cancers suggests that it can function as a tumor suppressor (Gotte et al., 2010; Zhang et al., 2011b). These newly identified senescence-regulated miRNAs further emphasize the involvement of miRNAs in cellular senescence. Functional annotation of the genes predicted to be regulated by these miRNAs revealed regulation of cell adhesion and cytoskeleton remodeling, suggesting that miRNAs may influence the morphological changes characteristic of the senescence phenotype (Dhahbi et al., 2011). Other pathways targeted by the senescence-regulated

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miRNAs include cell proliferation and apoptosis; these processes are important in the cellular decision to commit to senescence instead of apoptosis (Campisi and d’Adda di Fagagna, 2007). Evidence implicating a miRNA in aging was first observed in Caenorhabditis elegans: the miRNA lin-4 influences lifespan, and regulates insulin, insulin-like growth factor-1 signaling, and cell cycle checkpoints for DNA damage pathways, all of which are known to participate in the aging process (Boehm and Slack, 2005). Numerous subsequent studies have established that miRNA levels are altered during organismal aging (ElSharawy et al., 2012; Grillari and Grillari-Voglauer, 2010; Hackl et al., 2010; Liang et al., 2009; Liu et al., 2012; Noren Hooten et al., 2010; Smith-Vikos and Slack, 2012; Xu and Tahara, 2013). The mRNAs targets of miRNAs whose expression changes with age are involved in tissue-specific age-related functions, including oxidative stress defense and mitochondrial maintenance in the liver, apoptosis in the brain, and cell cycle regulation and proliferation in skeletal muscle (reviewed in Smith-Vikos and Slack, 2012). Despite the increasing evidence that miRNAs play a role in aging, there is at present little evidence that CR exerts any positive effects on the age-induced alterations in miRNAs or the pathophysiological consequences of those alterations. One study of aged mouse brain showed that CR prevented increases of miR-181a-1, miR-30e and miR-34a, along with the reciprocal upregulation of their target Bcl-2 gene (Khanna et al., 2011). The study further suggested that CR might decrease apoptosis and induce a gain in neuronal survival by modulating miRNA levels. 2.3. Circulating miRNAs miRNAs can circulate in the bloodstream, either as cargo of plasma exosomes or complexed with protein or lipoprotein factors (Arroyo et al., 2011; Turchinovich and Burwinkel, 2012; Vickers et al., 2011; Vickers and Remaley, 2012). Exosomes carry miRNAs from peripheral to recipient tissues, and protect them against RNase activity (Lasser et al., 2011; Pant et al., 2012; Zampetaki et al., 2012). miRNAs can be stable outside exosomes, indicating the existence of two transport systems of miRNAs, exosomal and extraexosomal (Arroyo et al., 2011; Turchinovich and Burwinkel, 2012). High-density lipoprotein (HDL) has been reported to carry and deliver miRNAs to recipient cells (Arroyo et al., 2011; Turchinovich and Burwinkel, 2012; Vickers et al., 2011). Stable Argonaute2miRNA complexes, independent of exosomes or microvesicles, are present in plasma and serum (Arroyo et al., 2011). In addition to HDL and Argonaute proteins, circulating miRNAs can be complexed to nucleophosmin-1 or ribosomal proteins L10a and L5 (Arroyo et al., 2011; Turchinovich and Burwinkel, 2012; Turchinovich et al., 2011; Vickers et al., 2011; Wang et al., 2010a; Zernecke et al., 2009). These packaging components may confer target specificity on the exported miRNAs, through interaction with receptors on the recipient cells (Turchinovich et al., 2012). More miRNAs are complexed with lipid/ribonucleoprotein than encapsulated in vesicles, indicating that if there is targeting specificity, it does not generally require factors on an exosomal membrane. There is currently little information about the tissues/cells that produce circulating small sncRNAs, the mechanisms by which they are delivered, and their functions once inside recipient cells. However, information about the properties of circulating miRNAs is emerging. Because of their contact with plasma, blood cells were at first thought to be the predominant source of extracellular miRNAs (Chen et al., 2008). However, the discovery in plasma of miRNAs specific to the liver, muscle, heart, and brain indicates a multi-tissue origin of extracellular miRNAs (Turchinovich et al., 2012). Furthermore, tumors can release miRNAs into the bloodstream (Healy et al., 2012; Ma et al., 2012; Zen and Zhang, 2012). Vickers and colleagues were able to deliver exogenous HDL–miRNA complexes into hepatocytes, with subsequent

alterations in the expression of genes involved in lipid metabolism, inflammation, and atherosclerosis (Vickers et al., 2011). In another study, extracellular miR-126 secreted by endothelial cells triggered the production of the chemokine CXCL12 in recipient vascular cells (Zernecke et al., 2009), and miR-143/145 altered gene expression in co-cultured smooth muscle cells to reduce atherosclerotic lesion formation in the aortas of ApoE(−/−) mice (Hergenreider et al., 2012). Similarly, miR-150 secreted by human blood cells and cultured monocytic THP-1 cells reduced c-Myb expression and enhanced cell migration after delivery into HMEC-1 cells (Zhang et al., 2010). Thus extracellular miRNAs can enter target cells and alter gene expression, with functional consequences. 2.4. Circulating miRNAs in aging and CR The systemic circulation of miRNAs raises the question of whether they function in a manner similar to hormones. There is increasing evidence that circulating miRNAs take part in cell-to-cell communication and signaling in normal biology and in pathophysiology (Cortez et al., 2011; Kosaka et al., 2013; Shah and Calin, 2013; Turchinovich et al., 2012). Changes in the circulating levels of miRNAs are closely associated with various types of cancer (Healy et al., 2012; Ma et al., 2012; Reid et al., 2011; Zen and Zhang, 2012). Non-neoplastic disorders can also exhibit alterations of circulating miRNA levels, which change during inflammatory, cardiovascular, and neurological disorders, including sepsis, rheumatoid arthritis, myocardial infarction, and Alzheimer’s disease (Filkova et al., 2012; Lehmann et al., 2012; Reid et al., 2011; Salic and De Windt, 2012; Xu et al., 2012; Zampetaki et al., 2012). Given the well-established regulatory role of miRNAs in carcinogenesis (Lages et al., 2012) and common diseases (Li and Kowdley, 2012; Mo, 2012), together with their stability and accessibility in the bloodstream, miRNAs hold great promise as systemic markers for diagnosis and prognosis of cancer and other diseases (Redova et al., 2013; Reid et al., 2011). The first observations of altered circulating miRNA levels during aging was an increase in miR-34a in the plasma of old mice (Li et al., 2011). miR-34a was also increased in PBMCs and brains of the old mice, with a reciprocal decrease of its target SIRT1 mRNA, suggesting that circulating miR-34a can be used as biomarker of brain aging. In flies, loss of miR-34 accelerates brain aging and decreases survival, while miR-34 upregulation extends median lifespan and mitigates neurodegeneration (Liu et al., 2012). Another study assessed plasma levels of 365 miRNAs in healthy young and old humans including centenarians, and in older patients with cardiovascular disease, and reported that transforming growth factor-beta signaling is the main pathway potentially regulated by the differentially abundant circulating miRNAs; the study proposed circulating miR-21 as an inflammatory biomarker linking the aging process to cardiovascular diseases (Olivieri et al., 2012). The scarcity of information on circulating miRNAs during aging prompted us to use deep sequencing to carry out a comprehensive assessment of the effects of age and CR on the levels of circulating miRNAs in the mouse (Dhahbi et al., 2013c). We found that the levels of many miRNAs circulating in the mouse are increased with age, and that CR antagonizes the age-induced increases (Dhahbi et al., 2013c). Whether these changes are of etiological origin, or merely consequences of deleterious age-induced dysfunctions, remains to be established. Moreover, the changes can be causal of pathophysiological alterations of aging only if the circulating miRNAs enter peripheral tissues, retain their functional mRNA targeting capabilities, and modulate gene expression. Zhang and coworkers showed that miR-150 was transferred from THP-1 to HMEC-1 cells, where its target c-Myb was silenced, thereby enhancing migration of the recipient HMEC-1 cells (Zhang et al., 2010). Also, Vickers et al. (2011) demonstrated that HDL–miRNA complexes are delivered into hepatocytes where they alter the expression of genes involved

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in lipid metabolism, inflammation, and atherosclerosis. This evidence that circulating miRNAs are taken up by targeted cells, and repress the translation of target genes, strongly supports the potential for functional significance of age- and CR-associated changes in the circulating levels of miRNAs. In our mouse study, functional analysis showed that the targets of the miRNAs whose circulating levels are increased with age and antagonized by CR include genes with key functions in energy metabolism, apoptosis, and Wnt signaling (Dhahbi et al., 2013c). We and others have shown that CR alleviates age-induced changes in the activity of key metabolic enzymes and the expression of genes involved in energy metabolism (Cao et al., 2001; Dhahbi et al., 2004; Lee et al., 1999; Weindruch et al., 2001). It has been suggested that CR ameliorates age-associated metabolic dysfunctions by reprogramming metabolism, causing a shift to an altered state of metabolism that delays the onset of age-associated pathologies and sustains the beneficial effects of CR on health and longevity (Anderson and Weindruch, 2007, 2010). It may be speculated that the changes in systemic miRNAs contribute to establishing and maintaining this beneficial metabolic shift. Apoptosis is suppressed during tumorigenesis; this may be related to the age-associated increase in prevalence of cancers (Hursting et al., 2003). CR increases apoptosis, decreases cellular proliferation, and eliminates preneoplastic and neoplastic cells from tissues (reviewed in Spindler, 2010). Thus, if the circulating miRNAs discussed here are indeed delivered to and directly regulate gene expression in recipient cells, they could take part in the regulation of apoptosis in peripheral tissues during aging and in response to CR. Wnt signaling regulates cell proliferation and differentiation, apoptosis, and stem cell renewal, and plays a complex role in aging that involves ␤-catenin, FOXO, and TCF/LEF (reviewed in Arthur and Cooley, 2012). In addition, FOXO transcription factors regulate the rate of aging and may mediate the anti-neoplastic effects of CR (Lin et al., 1997; Yamaza et al., 2010). Thus, CR-associated changes in the circulating levels of miRNAs could exert fine-tuning functions on Wnt signaling in peripheral tissues, to preclude the age-induced senescence of stem and proliferating cells and delay the onset of age-related disorders. The genes targeted by the age- and CR-modulated miRNAs seem to regulate biological processes directly relevant to aging; however, a causal relationship between changes in the levels of circulating miRNAs and manifestations of aging remains to be established. Since the publication of our mouse study mentioned above, Noren Hooten and colleagues reported that 3 serum miRNAs were significantly decreased with age in humans (miR-151a-3p, miR181a-5p and miR-1248) (Noren Hooten et al., 2013). These miRNAs are involved in development and organismal survival, and are predicted to mediate inflammation, suggesting that they may play a role in the age-associated inflammatory processes. In contrast to this human study (Noren Hooten et al., 2013), we found that aging in the mouse is associated with changes in serum levels of many miRNAs (Dhahbi et al., 2013c). Specifically, we found that aging increased the circulating levels of 73 known miRNAs, 45 of which were mitigated by CR, and decreased the levels of 47 known miRNAs, 3 of which were mitigated by CR. In addition to the small number of age-affected miRNAs, which might be explained by the use of different statistical significance criteria, Noren Hooten and colleagues reported that the miRDeep2 algorithm detected only 23 miRNAs, and of these only 5 were present in the serum of both young and old individuals in their study (Noren Hooten et al., 2013). This can be compared with our study in which miRDeep2 detected 553 known miRNAs and in addition predicted 79 potential novel miRNAs (Dhahbi et al., 2013c), using the relatively stringent score cut-off of 4 and signal-to-noise ratio of 19.6 (Friedlander et al., 2012; Mackowiak, 2011). The Noren Hooten findings are also low compared to other reports of human serum miRNAs, e.g.

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1594 miRNAs were detected using deep sequencing (Ninomiya et al., 2013). Poor detection of circulating miRNAs may be an effect of sample quality: we have found that the quality of small RNAs in frozen serum samples decreases with time. There is a growing list of circulating miRNAs whose levels are altered in association with aging. However, more work is needed to identify the sources of these miRNAs and their target tissues, the mechanisms of secretion, transport, and transfer into target cells, and the contribution of the transferred miRNAs to the functional declines observed during aging. Such extensive studies are justified by the great potential of these miRNAs as diagnostic tools and therapeutic targets in age-related diseases. 3. tRNA-derived small RNAs 3.1. tRNAs tRNAs are 73–93 nucleotide-long small noncoding RNAs that are transcribed by RNA polymerase III. Post-transcriptional processing and modifications of tRNA primary transcripts lead to the folding into a cloverleaf structure characterized by base-paired stems, unpaired loops including the anticodon loop, and a 3 CCA tail to which an amino acid is covalently linked. tRNAs function as adapter molecules that translate the genetic information in the messenger RNA to the amino acid sequence of protein. In addition to their role in translation, tRNAs can regulate gene expression (reviewed in Li and Zhou, 2009). In bacteria, yeast, and human cells, tRNAs have been shown to act as sensors of nutritional stress; they play a role in the amino acid starvation response during which global gene expression is adjusted to promote cell survival (Green et al., 2010; Murguia and Serrano, 2012; Shaheen et al., 2007). 3.2. Intracellular tRNA-derived small RNAs A size-based classification distinguishes two types of tRNAderived fragments (Martens-Uzunova et al., 2013; Sobala and Hutvagner, 2011): tRNA halves, 30–40 nt long, produced by cleavage of full length mature tRNAs at the anticodon loop, and shorter tRNA-derived fragments (tRFs), 18–22 nt in length, produced from both mature and pre-tRNAs by Dicer, RNase Z, or other as yet undetermined nucleases (Cole et al., 2009; Fu et al., 2009; Lee et al., 2009; Pederson, 2010; Sobala and Hutvagner, 2011; Thompson et al., 2008; Thompson and Parker, 2009). 3.2.1. tRFs This class is further subdivided into 3 types of tRNA-derived fragments: 3 U tRFs, 3 CCA tRFs, and 5 tRFs (Sobala and Hutvagner, 2011). RNase Z produces 3 U tRFs during processing of the pre-tRNA transcript. The other two result from Dicer cleavage at the 3 or 5 ends of mature tRNAs to produce 3 CCA tRFs or 5 tRFs, respectively. There is increasing evidence that tRFs are functional. 5 tRFs were detected by deep sequencing of RNA extracted after immunoprecipitation with anti-Argonaute antibodies (Burroughs et al., 2011). Dicer products, 5 tRFs and 3 CCA tRFs, also can associate with Argonaute proteins and may silence reporter transgenes (Burroughs et al., 2011; Haussecker et al., 2010; Yeung et al., 2009). This suggests that tRFs may regulate gene expression in a miRNAlike fashion. However if tRFs are involved in cellular RNAi pathways, their gene targets remain to be determined. Other functions have been reported for tRFs. For example, a tRF derived from the 3 end of a Ser-TGA tRNA precursor was found to be highly expressed in cancer cell lines, and its depletion impaired cell proliferation (Lee et al., 2009). tRFs of size ∼18 nt were identified in prostate cancer small RNA sequencing libraries, with significantly higher levels in cases having lymph node metastases than in organ-confined cases (Martens-Uzunova et al., 2012). More

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Fig. 2. Cleavage sites of tRNAs and YRNAs. (A) Cloverleaf structure of a tRNA gene (downloaded from http://gtrnadb.ucsc.edu) showing cleavage sites upstream of the AGC anticodon (arrowhead). (B) Secondary structure of YRNAs as predicted by Vienna (Hofacker, 2003) from the RF00019 Y RNA Family (http://rfam.sanger.ac.uk/family/RF00019) and drawn by Varna (Darty et al., 2009). The putative cleavage sites at the predicted internal loop are denoted by an arrow.

recently, 5 tRFs were shown to inhibit translation in a reporter assay system, through a mechanism that requires a universally conserved “GG” dinucleotide in the tRF (Sobala and Hutvagner, 2013). This translation inhibition does not require mRNA targets; instead the underlying mechanism seems to be a direct inhibition at the translation elongation step (Sobala and Hutvagner, 2013). Taken together, these findings strongly suggest that tRFs may be effectors in new mechanisms of gene expression regulation by sncRNAs. 3.2.2. tRNA halves The second class of tRNA-derived fragments includes 5 - and 3 tRNA halves of 30–40 nt in length. They are produced in cultured cells by cleavage of mature tRNA at the anticodon loop (Fig. 2A) in response to stresses such as arsenite, heat shock, hypoxia, amino acids depletion, or ultraviolet irradiation (Fu et al., 2009; Lee and Collins, 2005; Thompson et al., 2008; Yamasaki et al., 2009); they have recently been detected after infection of airway epithelial cells with respiratory syncytial virus (Wang et al., 2013). The cleavage of tRNA is mediated by angiogenin, a ribonuclease that was originally known for its promotion of new blood vessel growth (Fu et al., 2009). The cleavage of tRNA by angiogenin is not a general degradation mechanism of cellular tRNA caused by stress, but it is rather part of a well-controlled and conserved cellular response (Thompson et al., 2008). The stress-induced 5 tRNA halves promote assembly of stress granules and inhibit translation (Emara et al., 2010; Ivanov et al., 2011; Yamasaki et al., 2009). Normally, cells suppress protein synthesis during stress as a strategy to preserve energy for damage repair. Many stressors activate kinases that phosphorylate eIF2␣, which in its phosphorylated state becomes unable to bind the 43S pre-initiation complex, leading to inhibition of translation. Stress-induced 5 tRNA halves act independently of this eIF2␣ phosphorylation pathway; instead, they

inhibit translation by associating with the translational repressor YB-1 and displacing eIF4G/eIF4A from the translation initiation complex (Ivanov et al., 2011). 5 tRNA halves and 5 tRFs are produced by different pathways under different cellular conditions, and have different functions. In contrast to the constitutive nature of 5 tRF production, 5 tRNA halves are produced under conditions of cellular stress. 5 tRNA halves inhibit translation at the initiation step while 5 tRFs seem to inhibit translation elongation. The functions of 5 tRFs extend beyond translation inhibition: they play a role in cell proliferation. Finally, the angiogenin-mediated cleavage that produces 5 tRNA halves can be regulated by RNA methylation; Drosophila Dnmt2 methylates C38 in the anticodon loop of a number of tRNAs, and inhibits their cleavage by angiogenin (Schaefer et al., 2010). 3.3. Circulating tRNA-derived small RNAs Until a recent study by our group (Dhahbi et al., 2013b), tRNAderived fragments had been described only in cultured cells, with the exception of one report (Fu et al., 2009) in which tRNA halves were unexpectedly detected on cloning of miRNAs from human fetal liver. By deep sequencing mouse serum small RNAs, we discovered a class of 30–33 nt tRNA-derived fragments that resemble the 5 tRNA halves previously described in stressed cell cultures (Dhahbi et al., 2013b). These circulating 5 tRNA halves are derived from a distinct subset of tRNAs, and are as abundant as miRNAs in mouse serum. They circulate outside exosomes/microvesicles as 100–300 kDa complexes that can be destabilized with the chelating agent EDTA. The 3 fragment of the tRNA molecules is present in serum only in trace quantities, and tRFs are not detectable at the sequencing depths we used. Deep sequencing of human serum small RNAs produced very similar findings (Dhahbi et al., 2013b).

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Tissue distribution analysis revealed that 5 tRNA halves are highly expressed in hematopoietic and lymphoid tissues of the mouse; they are present in other tissues at significantly lower levels that may reflect the presence of residual blood cells (Dhahbi et al., 2013b). Although this suggests that immune cells release the circulating 5 tRNA halves, the specific cell type that produces serum 5 tRNA halves, their destinations, and the factors with which they complex in serum are yet to be determined. Angiogenin is the ribonuclease that mediates the stress-induced cleavage of tRNA to produce tRNA halves that inhibit translation in cultured cells. However, recombinant angiogenin can induce tRNA half production and inhibit protein synthesis in the absence of stress (Yamasaki et al., 2009). Thus, angiogenin or similar endonucleases may mediate the biogenesis of the 5 tRNA halves in immune tissues under nonstress physiologic conditions in the context of a whole organism. This issue could be addressed with a mouse angiogenin knockout experiment, but the angiogenin gene is duplicated in the mouse, making it difficult not only to generate knockout mice, but also to obtain accurate functional information.

3.4. 5 tRNA halves may be immune signaling molecules The evidence discussed above prompts this speculation regarding the function of circulating 5 tRNA halves. Since they are present only in mouse hematopoietic and lymphoid tissues, and circulate in the bloodstream (Dhahbi et al., 2013b), it is likely that they are released into the bloodstream by immune cells. We speculate that circulating 5 tRNA halves act as secreted signals that take part in cell-to-cell communications analogous to the mechanisms involving the release, transport, and delivery of miRNAs from donor to recipient tissues. Unlike circulating miRNAs, which seem to be secreted by all types of peripheral tissues, circulating 5 tRNA halves may originate only from immune tissues. Thus, they may function as systemic signaling molecules for communication between hematopoietic and lymphoid tissues, and/or as carriers of signals from immune to non-immune tissues. We base this idea on our findings: (i) 5 tRNA halves are expressed at the whole organism level; all previous reports of 5 tRNA halves originated from cell culture, except for a single study in which tRNA halves were detected incidentally during the cloning of miRNAs from human fetal liver (a hematopoietic tissue) (Fu et al., 2009). (ii) 5 tRNA halves circulate in the serum and concurrently are present in hematopoietic and lymphoid tissues; there is no clear evidence that they are expressed in other tissues. (iii) Their occurrence at the organismal level, in immune tissues and in circulation, takes place under a normal, non-stressed physiologic state. This is in contrast to previous observations in cultured cells, where tRNA halves are expressed only in response to stresses such as arsenite, heat shock, or ultraviolet irradiation (Thompson et al., 2008; Yamasaki et al., 2009). Furthermore, both 5 and 3 tRNA halves are induced by stress in cultured cells, while under normal physiologic conditions in the mouse we observed almost exclusively 5 tRNA halves in immune tissues and in the blood (Dhahbi et al., 2013b). In support of this idea, the largest human tRNA gene cluster is located in the major histocompatibility complex (MHC), the genomic region that is crucial in adaptive and innate immunity. It has been suggested that clustering of tRNA genes in the MHC reflects a role for tRNAs in the immune system (Horton et al., 2004; Mungall et al., 2003). Other genes with immune-related functions, including inflammation and stress response genes, also co-localize with MHC (Horton et al., 2004; Mungall et al., 2003). If tRNAs play a role in immunity (Horton et al., 2004; Mungall et al., 2003), then the 5 tRNA halves released by hematopoietic and lymphoid tissues in the systemic environment are plausible effectors of the presumed immune function of tRNAs.

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We stress that this is speculation, and furthermore it is clear that much work will be required to test the idea. In particular, it remains to be demonstrated that 5 tRNA halves exert immune-related functions upon uptake by recipient cells in peripheral tissues. Further investigation of the production, secretion, uptake, and cellular functions of circulating 5 tRNA halves will be necessary before any firm conclusions can be reached. 3.5. Circulating levels of specific 5 tRNA halves are altered by aging and calorie restriction We have found that aging increases or decreases the circulating levels of 5 tRNA halves derived from specific tRNA isoacceptors (Dhahbi et al., 2013b). Aging increased the circulating levels of 5 tRNA halves derived from tRNA-His(GTG), while it decreased the levels of 5 tRNA halves derived from the tRNAs of Arg(CCG), Cys(GCA), Gly(GCC), Lys(CTT), and Val(AAC) (Fig. 3). As discussed above, the only known function of 5 tRNA halves is the stressinduced inhibition of translation initiation in cultured cells. Ivanov and colleagues compared the translational inhibitory activity of individual 5 tRNA halves, and found that the ones derived from Alaand Cys-tRNAs suppressed translation more potently than others (Ivanov et al., 2011). They also reported that translational repression requires a terminal oligoguanine motif of 4–5 guanine residues at the 5 end of the molecule, which is present only in Ala- and Cys-tRNAs. This suggests that 5 tRNA halves may have other biological effects, since only a subset represses translation. In support of this, 5 tRNA halves are present in cells and in the circulation under normal physiological conditions, and not only in stressed cells where they repress translation (Dhahbi et al., 2013b). Whether the age-induced changes in the circulating levels of 5 tRNA halves are linked to age-associated changes in translation or other cellular processes remains to be elucidated. Notably, CR mitigated the age-related changes in circulating levels of 5 tRNA halves, with the exception of tRNA-Gly(GCC) (Dhahbi et al., 2013b) (Fig. 3). This reversal of the age-associated changes by CR is of particular significance because it validates the ageassociated alterations in the levels of circulating 5 tRNA halves, and provides further evidence that circulating 5 tRNA halves are physiologically regulated. CR opposes molecular and biological changes that occur during aging, especially age-associated alterations in gene expression (Dhahbi et al., 1998, 1999, 2004, 2006, 2012, 2013c; Lee et al., 1999; Spindler and Dhahbi, 2007; Weindruch et al., 2001). The underlying mechanisms by which age-associated changes in the levels of circulating 5 tRNA halves may affect biological processes in target tissues has yet to be determined, and a causal relationship between the changes of circulating 5 tRNA halves and the manifestations of aging needs to be established. In the meantime, our findings strongly suggest that the circulating levels of 5 tRNA halves have a role, whether as markers or effectors, in the manifestations of aging. 4. YRNA-derived small RNAs 4.1. YRNAs YRNAs are 84–112 nt sncRNAs transcribed by RNA polymerase III from four genes in man (hY1, hY3, hY4 and hY5), and two genes in mice (mY1 and mY3) (Wolin and Steitz, 1983). Approximately 1000 “pseudogenes” derived from YRNAs are present in the human genome, while the mouse has few or none (Perreault et al., 2005, 2007). YRNAs fold into stem-loop structures and complex with Ro60 protein and other proteins to form ribonucleoproteins (RoRNPs) of uncertain function. The Ro60 component of cellular RoRNPs is recognized by autoantibodies found in the serum

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Fig. 3. Abundance of 5 tRNA-derived sequencing reads from sera of young, old control, and old CR mice. The Y-axis shows levels of circulating 5 tRNA halves derived from the tRNA genes indicated in the X-axis. The serum levels of 5 tRNA halves are reported as the average counts per million (cpm) reads in the sequenced libraries from the 3 experimental groups: young control (black bars), old control (blue bars), and old CR (red bars). The tRNA genes from which the circulating 5 tRNA halves are derived are identified by their genomic positions in the mouse mm10 genome, and by the corresponding tRNA isoacceptor identity. CR mitigates age-associated changes in levels of specific 5 tRNA halves.

of patients with inflammatory connective tissue diseases such as Sjogren’s syndrome and systemic lupus erythematosus (Bouffard et al., 1996; Lerner et al., 1981). The YRNA moiety in RoRNPs may contribute to the tissue injury mediated by the anti-Ro60 autoantibodies. YRNAs were found to be essential for TNF-␣ secretion and initiation of inflammatory responses in macrophages treated with immune complexes consisting of YRNAs, Ro60, and anti-Ro60 antibodies (Clancy et al., 2010). Moreover, depletion of mouse YRNA in apoptotic cells prevented surface translocation of Ro60, a critical step in the formation of the autoimmune complexes and the subsequent activation of inflammatory pathways (Reed et al., 2013). More recently, a YRNA gene, 99% identical to human hY4, was found in a quantitative trait locus (QTL) that is strongly associated with mastitis, an inflammation-driven disease of the bovine mammary gland (Meredith et al., 2013). YRNAs were the first example of a sncRNA shown to regulate DNA replication (reviewed in Krude, 2010). Fractionation and reconstitution of cytosolic extract revealed YRNAs as essential soluble factors required for chromosomal DNA replication; depletion of YRNAs in proliferating cells by RNAi prevented DNA replication (Christov et al., 2006; Krude et al., 2009; Zhang et al., 2011a). Furthermore, antisense oligonucleotide inhibition of YRNA function in Xenopus laevis and zebrafish embryos showed that DNA replication becomes YRNA-dependent after midblastula transition, suggesting that YRNAs may control DNA replication during development (Collart et al., 2011). The function of YRNAs in DNA replication does not require Ro protein (Langley et al., 2010); other factors might complex with YRNAs in DNA replication, since naked YRNAs are unstable. This function of YRNAs in DNA replication seems to be redundant: YRNAs can replace each other in DNA replication (Chen et al., 2003; Christov et al., 2006; Garcia et al., 2009; Gardiner et al., 2009). Association of Ro60 with misfolded sncRNAs in the nuclei of animal cells suggested that Ro60 might function in “quality control” of sncRNAs (Chen et al., 2003; Hogg and Collins, 2007; O’Brien and Wolin, 1994; Shi et al., 1996). Bacterial YRNAs tether Ro proteins to a nuclease to form a degradation apparatus that targets misfolded sncRNAs (Chen et al., 2013; Wolin et al., 2013). Similar tethering mechanisms may take place in mammalian cells (Wolin et al., 2013). Accumulating evidence indicates that this function of

YRNAs and Ro60 in RNA metabolism may be part of a broader stress response that involves RoRNPs. YRNA and Ro60 increase in bacteria exposed to radiation, heat, oxidative stresses, desiccation, and starvation (reviewed in Wolin et al., 2013). Also, RoRNPs enhance viability after UV irradiation of bacteria and mammalian cells (Chen et al., 2000, 2003). The C. elegans Ro ortholog rop-1 was shown to be involved in the formation of dauer larvae, suggesting a role for RoRNPs in response to starvation (Labbe et al., 2000). However, more work is needed to resolve the putative roles of YRNAs and Ro protein in the cellular response to environmental stress. There is some evidence that YRNAs are involved in cancer. Human YRNAs are significantly upregulated in carcinomas and adenocarcinomas of the lung, kidney, bladder, prostate, colon, and cervix (Christov et al., 2008), and were found to be required for proliferation of cancer cells (Christov et al., 2008). Of note, cancer risk is higher in patients with autoimmune diseases characterized by the presence of autoantibodies that target RoRNPs containing YRNAs (Abu-Shakra et al., 1996; Bernatsky et al., 2013; Routsias et al., 2013; Voulgarelis and Moutsopoulos, 2001). If YRNAs were indeed demonstrated to have a direct function in tumorigenesis, they could serve as new markers for diagnosis and/or as targets for cancer treatment. The material discussed above relates to the expression and function of full-length YRNAs. We have found that YRNA fragments circulate in the blood; the function of these fragments is completely unknown. 4.2. Intracellular YRNA-derived small RNAs As discussed above, relatively little is known about YRNAs and their functions. Even less is known about YRNA-derived fragments. They were first observed in cells exposed to apoptotic stimuli (Rutjes et al., 1999), and later in cells treated with the stressor poly(I:C), a double-stranded RNA mimic immunostimulant chemical (Nicolas et al., 2012). The cleavage of YRNAs (Fig. 2B) and production of YRNA-derived fragments during apoptosis are caspase-dependent (Rutjes et al., 1999). It has been suggested that the nucleases that cleave YRNAs are stressactivated, and might be caspase-activated nucleases responsible for inter-nucleosomal cleavage of chromatin that produces the DNA

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ladder during apoptosis (Rutjes et al., 1999). YRNA fragments are also detected under unstressed conditions in cultured cells, the brain and other tissues, and in tumors (Chen and Heard, 2013; Meiri et al., 2010; Nicolas et al., 2012; Schotte et al., 2009; Verhagen and Pruijn, 2011). Some YRNA-derived fragments were initially classified as a new type of miRNAs generated by processing of full length YRNAs (Meiri et al., 2010; Nicolas et al., 2012; Verhagen and Pruijn, 2011). However, no gene silencing activity and no targets were identified for these proposed miRNAs. In addition, their biogenesis is independent of the Dicer pathway and they are unable to associate with Argonaute proteins (Chen and Heard, 2013; Langenberger et al., 2013; Meiri et al., 2010; Nicolas et al., 2012). Specifically, two 25-nt fragments derived from RNY5 and RNY3 were misannotated as miR-1975 and miR-1979 (Meiri et al., 2010; Verhagen and Pruijn, 2011). These YRNA-derived fragments, mistakenly reported as miRNAs, were identified in human and rhesus macaque brain tissues (Shao et al., 2010), induced during viral infections (Cui et al., 2010; Qi et al., 2010), and differentially regulated in renal cell carcinoma subtypes (Youssef et al., 2011), and liver cancer tissues (Lin et al., 2013). However, their role during stress and cancer, and in normal tissues, is at present unknown. 4.3. Circulating YRNA-derived small RNAs We recently provided the first evidence that YRNA-derived small RNAs are present in human serum and plasma (Dhahbi et al., 2013a). As part of our deep sequencing and analysis of circulating small RNAs, we found abundant cell-free YRNA fragments that are not encapsulated in exosomes or microvesicles. Similarly to tRNA halves, YRNA fragments circulate as large complexes of 100–300 kDa. In contrast to the particles carrying 5 tRNA halves, the chelating agent EDTA does not destabilize complexes carrying YRNA fragments (Dhahbi et al., 2013a,b). This differential sensitivity to ion chelation suggests that YRNA fragments and tRNA halves are complexed to different factors, the nature and identity of which remain to be determined. Circulating YRNA fragments are derived mostly from the 5 ends of YRNAs; levels of fragments derived from 3 ends are drastically lower than those derived from the 5 ends. It seems that YRNAs are cleaved at an internal loop to produce stable 5 YRNA fragments and short-lived 3 end fragments. The preponderance of 5 - over 3 -end fragments may reflect functional differences in addition to differences in stability. At least in the case of tRNA-derived fragments, it was shown that 5 but not 3 tRNA halves inhibit protein synthesis in cultured cell lines (Yamasaki et al., 2009). It was proposed that the presence of 5 terminal monophosphates in the 5 but not in the 3 tRNA halves might account for differences in stability and/or function (Emara et al., 2010). The 5 monophosphate was shown to be required for optimal stress granules assembly, which is a component of a stress response program mediated by angiogenin and tRNA halves (Emara et al., 2010). A similar mechanism could underlie the differential abundance of 5 - and 3 -end YRNA fragments, but clarification will require new experimental approaches. The circulating YRNA-derived fragments reported in our study are 27 nt and 30–33 nt in size (Fig. 1); we did not detect shorter miRNA-size YRNA species in the circulation (Dhahbi et al., 2013a). This is in contrast with the findings of Meiri and colleagues (Meiri et al., 2010); they reported that deep sequencing detected two miRNA-size fragments derived from RNY1 and RNY3 RNAs in solid tumors, and using qRT-PCR detected them in serum of healthy people. Since Meiri and colleagues filtered out sequences whose length exceeded 17–25 nt, one cannot rule out the presence of YRNA-derived fragments larger than the miRNA-size in the solid tumors and serum (Meiri et al., 2010). In another study, 28 nt YRNA fragments were found in vesicles released into culture medium by

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immune cells, along with full length YRNAs, as well as full length SRP-RNA and vault-RNA and derivatives of both (Nolte-’t Hoen et al., 2012). The vesicular small RNAs were enriched relative to cellular RNA, suggesting their selective release into the extracellular space. We found that the 5 YRNA fragments circulating in human serum and plasma are mostly derived from RNY4 and its “pseudogenes” (Dhahbi et al., 2013a). The overrepresentation of RNY4 in the circulation may imply a type-specific biogenesis and/or release of the circulating 5 YRNA fragments. Our data demonstrate that human RNY4 sequences that have been annotated as “pseudogenes” are transcribed, and that the transcripts are processed and secreted, calling into question their annotation as “pseudogenes”. Also, a significant proportion of circulating 5 YRNA fragments were derived from YRNAs computationally predicted in the Rfam database (http://rfam.sanger.ac.uk), demonstrating that these are expressed and supporting the validity of the Rfam predictions (Dhahbi et al., 2013a). Since circulating 5 YRNA fragments have no known functions as yet, the significance of the “pseudogenes” expression is unclear. A puzzling feature noted when we compared human and mouse circulating small RNAs is that 5 YRNA fragments are drastically more abundant in human than in mouse serum (Fig. 1) (Dhahbi et al., 2013a). This may reflect the much greater copy number of YRNA genes and pseudogenes in humans (Perreault et al., 2005, 2007). Furthermore the bulk of circulating 5 YRNA fragments are derived from RNY4, which is absent from the mouse genome. Although YRNAs have been conserved, some YRNA genes were lost during evolution, leading to disproportionate numbers of active YRNA genes in vertebrates (Mosig et al., 2007). The biological significance of the disparity in levels of circulating 5 YRNA fragments between mice and humans is another mystery that will need to be explored. 4.4. Circulating YRNA-derived small RNAs in aging and CR Nothing is currently known about the effects of aging and CR on the circulating levels of YRNA-derived small RNAs. Moreover, no work has been done yet to elucidate the secretion pathways of circulating YRNA fragments, the cells that produce and release them in the systemic environment, their packaging inside cells, their transport and delivery to their destination, and the functions they may exert once inside recipient cells. Since full length YRNAs or their fragments are linked to processes relevant to aging such as inflammatory responses (Clancy et al., 2010; Meredith et al., 2013; Reed et al., 2013), DNA replication and cell proliferation (Christov et al., 2006; Krude et al., 2009; Zhang et al., 2011a), stress responses (Nicolas et al., 2012), degradation of misfolded ncRNAs (Wolin et al., 2013), apoptosis (Rutjes et al., 1999), viral infections (Cui et al., 2010; Qi et al., 2010), and several types of cancer (Meiri et al., 2010; Schotte et al., 2009), and since YRNA fragments are secreted in the systemic environment, and they are associated with age-relevant processes, it is conceivable that aging may affect the circulating levels of YRNA fragments. Indeed, our group has preliminary evidence for changes in the circulating levels of YRNA fragments in older people (J.M Dhahbi, unpublished observations). 5. Concluding remarks It is by now clear that small noncoding RNAs circulate in the blood of humans and other vertebrates, and there is sufficient evidence to justify the view that at least some of these RNAs act as signaling molecules or direct effectors of cellular changes. The major classes of small RNAs circulating in human and mouse blood are miRNAs, 5 tRNA halves, and YRNA fragments; other small RNAs,

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while much less abundant, may nevertheless carry out functions in target cells. In regard to miRNAs, the abundant evidence about their effects on cellular physiology provides a basis for supposing that their transport in the blood can transfer these functions from one cell to another; in regard to 5 tRNA halves and YRNA fragments, much more detailed knowledge of their cellular functions is needed, but the evidence that specific fragments are processed and secreted as nucleoprotein particles argues strongly for some physiological function. The evidence we have discussed indicates that circulating populations of miRNAs and 5 tRNA halves change dramatically with age, and that CR can ameliorate these changes; similar effects may also occur with YRNA fragments. The known functions of these molecules suggest that they may be active participants in the manifestations of aging, and consequently that they are candidate biomarkers and therapeutic targets. Acknowledgements I thank Stephen R. Spindler and David I.K. Martin for their help in the preparation of this review. References Abu-Shakra, M., Gladman, D.D., Urowitz, M.B., 1996. Malignancy in systemic lupus erythematosus. Arthritis and Rheumatism 39, 1050–1054. Allegra, A., Alonci, A., Campo, S., Penna, G., Petrungaro, A., Gerace, D., Musolino, C., 2012. Circulating microRNAs: new biomarkers in diagnosis, prognosis and treatment of cancer (Review). International Journal of Oncology 41, 1897–1912. Amaral, P.P., Mattick, J.S., 2008. Noncoding RNA in development. Mammalian Genome: Official Journal of the International Mammalian Genome Society 19, 454–492. Anderson, R.M., Weindruch, R., 2007. Metabolic reprogramming in dietary restriction. Interdisciplinary Topics in Gerontology 35, 18–38. Anderson, R.M., Weindruch, R., 2010. Metabolic reprogramming, caloric restriction and aging. Trends in Endocrinology and Metabolism: TEM 21, 134–141. Arroyo, J.D., Chevillet, J.R., Kroh, E.M., Ruf, I.K., Pritchard, C.C., Gibson, D.F., Mitchell, P.S., Bennett, C.F., Pogosova-Agadjanyan, E.L., Stirewalt, D.L., Tait, J.F., Tewari, M., 2011. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proceedings of the National Academy of Sciences of the United States of America 108, 5003–5008. Arthur, S.T., Cooley, I.D., 2012. The effect of physiological stimuli on sarcopenia: impact of Notch and Wnt signaling on impaired aged skeletal muscle repair. International Journal of Biological Sciences 8, 731–760. Avraham, R., Yarden, Y., 2012. Regulation of signalling by microRNAs. Biochemical Society Transactions 40, 26–30. Bernatsky, S., Ramsey-Goldman, R., Labrecque, J., Joseph, L., Boivin, J.F., Petri, M., Zoma, A., Manzi, S., Urowitz, M.B., Gladman, D., Fortin, P.R., Ginzler, E., Yelin, E., Bae, S.C., Wallace, D.J., Edworthy, S., Jacobsen, S., Gordon, C., Dooley, M.A., Peschken, C.A., Hanly, J.G., Alarcon, G.S., Nived, O., Ruiz-Irastorza, G., Isenberg, D., Rahman, A., Witte, T., Aranow, C., Kamen, D.L., Steinsson, K., Askanase, A., Barr, S., Criswell, L.A., Sturfelt, G., Patel, N.M., Senecal, J.L., Zummer, M., Pope, J.E., Ensworth, S., El-Gabalawy, H., McCarthy, T., Dreyer, L., Sibley, J., St Pierre, Y., Clarke, A.E., 2013. Cancer risk in systemic lupus: an updated international multi-centre cohort study. Journal of Autoimmunity 42, 130–135. Bhasin, M., Yuan, L., Keskin, D.B., Otu, H.H., Libermann, T.A., Oettgen, P., 2010. Bioinformatic identification and characterization of human endothelial cell-restricted genes. BMC Genomics 11, 342. Boehm, M., Slack, F., 2005. A developmental timing microRNA and its target regulate life span in C. elegans. Science 310, 1954–1957. Boon, R.A., Iekushi, K., Lechner, S., Seeger, T., Fischer, A., Heydt, S., Kaluza, D., Treguer, K., Carmona, G., Bonauer, A., Horrevoets, A.J., Didier, N., Girmatsion, Z., Biliczki, P., Ehrlich, J.R., Katus, H.A., Muller, O.J., Potente, M., Zeiher, A.M., Hermeking, H., Dimmeler, S., 2013. MicroRNA-34a regulates cardiac ageing and function. Nature 495, 107–110. Bouffard, P., Laniel, M.A., Boire, G., 1996. Anti-Ro (SSA) antibodies: clinical significance and biological relevance. The Journal of Rheumatology 23, 1838–1841. Burroughs, A.M., Ando, Y., de Hoon, M.J., Tomaru, Y., Suzuki, H., Hayashizaki, Y., Daub, C.O., 2011. Deep-sequencing of human Argonaute-associated small RNAs provides insight into miRNA sorting and reveals Argonaute association with RNA fragments of diverse origin. RNA Biology 8, 158–177. Campisi, J., d’Adda di Fagagna, F., 2007. Cellular senescence: when bad things happen to good cells. Nature Reviews. Molecular Cell Biology 8, 729–740. Cao, S.X., Dhahbi, J.M., Mote, P.L., Spindler, S.R., 2001. Genomic profiling of short- and long-term caloric restriction effects in the liver of aging mice. Proceedings of the National Academy of Sciences of the United States of America 98, 10630–10635. Carthew, R.W., Sontheimer, E.J., 2009. Origins and mechanisms of miRNAs and siRNAs. Cell 136, 642–655. Chen, C.J., Heard, E., 2013. Small RNAs derived from structural non-coding RNAs. Methods 63, 76–84.

Chen, X., Ba, Y., Ma, L., Cai, X., Yin, Y., Wang, K., Guo, J., Zhang, Y., Chen, J., Guo, X., Li, Q., Li, X., Wang, W., Zhang, Y., Wang, J., Jiang, X., Xiang, Y., Xu, C., Zheng, P., Zhang, J., Li, R., Zhang, H., Shang, X., Gong, T., Ning, G., Wang, J., Zen, K., Zhang, J., Zhang, C.Y., 2008. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Research 18, 997–1006. Chen, X., Quinn, A.M., Wolin, S.L., 2000. Ro ribonucleoproteins contribute to the resistance of Deinococcus radiodurans to ultraviolet irradiation. Genes & Development 14, 777–782. Chen, X., Smith, J.D., Shi, H., Yang, D.D., Flavell, R.A., Wolin, S.L., 2003. The Ro autoantigen binds misfolded U2 small nuclear RNAs and assists mammalian cell survival after UV irradiation. Current Biology: CB 13, 2206–2211. Chen, X., Taylor, D.W., Fowler, C.C., Galan, J.E., Wang, H.W., Wolin, S.L., 2013. An RNA degradation machine sculpted by Ro autoantigen and noncoding RNA. Cell 153, 166–177. Cheng, Q., Yi, B., Wang, A., Jiang, X., 2013. Exploring and exploiting the fundamental role of microRNAs in tumor pathogenesis. OncoTargets and Therapy 6, 1675–1684. Christov, C.P., Gardiner, T.J., Szuts, D., Krude, T., 2006. Functional requirement of noncoding Y RNAs for human chromosomal DNA replication. Molecular and Cellular Biology 26, 6993–7004. Christov, C.P., Trivier, E., Krude, T., 2008. Noncoding human Y RNAs are overexpressed in tumours and required for cell proliferation. British Journal of Cancer 98, 981–988. Clancy, R.M., Alvarez, D., Komissarova, E., Barrat, F.J., Swartz, J., Buyon, J.P., 2010. Ro60-associated single-stranded RNA links inflammation with fetal cardiac fibrosis via ligation of TLRs: a novel pathway to autoimmune-associated heart block. Journal of Immunology 184, 2148–2155. Cole, C., Sobala, A., Lu, C., Thatcher, S.R., Bowman, A., Brown, J.W., Green, P.J., Barton, G.J., Hutvagner, G., 2009. Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs. RNA 15, 2147–2160. Collart, C., Christov, C.P., Smith, J.C., Krude, T., 2011. The midblastula transition defines the onset of Y RNA-dependent DNA replication in Xenopus laevis. Molecular and Cellular Biology 31, 3857–3870. Cortez, M.A., Bueso-Ramos, C., Ferdin, J., Lopez-Berestein, G., Sood, A.K., Calin, G.A., 2011. MicroRNAs in body fluids – the mix of hormones and biomarkers. Nature Reviews. Clinical Oncology 8, 467–477. Cui, L., Guo, X., Qi, Y., Qi, X., Ge, Y., Shi, Z., Wu, T., Shan, J., Shan, Y., Zhu, Z., Wang, H., 2010. Identification of microRNAs involved in the host response to enterovirus 71 infection by a deep sequencing approach. Journal of Biomedicine & Biotechnology 2010, 425939. da Costa Martins, P.A., Leptidis, S., Salic, K., De Windt, L.J., 2010. MicroRNA regulation in cardiovascular disease. Current Drug Targets 11, 900–906. Darty, K., Denise, A., Ponty, Y., 2009. VARNA: Interactive drawing and editing of the RNA secondary structure. Bioinformatics 25, 1974–1975. Dhahbi, J.M., Atamna, H., Boffelli, D., Magis, W., Spindler, S.R., Martin, D.I., 2011. Deep sequencing reveals novel microRNAs and regulation of microRNA expression during cell senescence. PLoS One 6, e20509. Dhahbi, J.M., Atamna, H., Boffelli, D., Martin, D.I., Spindler, S.R., 2012. mRNA-Seq reveals complex patterns of gene regulation and expression in the mouse skeletal muscle transcriptome associated with calorie restriction. Physiological Genomics 44, 331–344. Dhahbi, J.M., Kim, H.J., Mote, P.L., Beaver, R.J., Spindler, S.R., 2004. Temporal linkage between the phenotypic and genomic responses to caloric restriction. Proceedings of the National Academy of Sciences of the United States of America 101, 5524–5529. Dhahbi, J.M., Mote, P.L., Wingo, J., Tillman, J.B., Walford, R.L., Spindler, S.R., 1999. Calories and aging alter gene expression for gluconeogenic, glycolytic, and nitrogen-metabolizing enzymes. The American Journal of Physiology 277, E352–E360. Dhahbi, J.M., Spindler, S.R., Atamna, H., Boffelli, D., Mote, P., Martin, D.I., 2013a. 5 YRNA fragments derived by processing of transcripts from specific YRNA genes and pseudogenes are abundant in human serum and plasma. Physiological Genomics 45 (21), 990–998. Dhahbi, J.M., Spindler, S.R., Atamna, H., Yamakawa, A., Boffelli, D., Mote, P., Martin, D.I., 2013b. 5 tRNA halves are present as abundant complexes in serum, concentrated in blood cells, and modulated by aging and calorie restriction. BMC Genomics 14, 298. Dhahbi, J.M., Spindler, S.R., Atamna, H., Yamakawa, A., Guerrero, N., Boffelli, D., Mote, P., Martin, D.I., 2013c. Deep sequencing identifies circulating mouse miRNAs that are functionally implicated in manifestations of aging and responsive to calorie restriction. Aging 5, 130–141. Dhahbi, J.M., Tillman, J.B., Cao, S., Mote, P.L., Walford, R.L., Spindler, S.R., 1998. Caloric intake alters the efficiency of catalase mRNA translation in the liver of old female mice. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences 53, B180–B185. Dhahbi, J.M., Tsuchiya, T., Kim, H.J., Mote, P.L., Spindler, S.R., 2006. Gene expression and physiologic responses of the heart to the initiation and withdrawal of caloric restriction. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences 61, 218–231. Di Leva, G., Croce, C.M., 2013. miRNA profiling of cancer. Current Opinion in Genetics & Development 23, 3–11. Dimmeler, S., Nicotera, P., 2013. MicroRNAs in age-related diseases. EMBO Molecular Medicine 5, 180–190. Dixon-McIver, A., East, P., Mein, C.A., Cazier, J.B., Molloy, G., Chaplin, T., Andrew Lister, T., Young, B.D., Debernardi, S., 2008. Distinctive patterns of microRNA

Please cite this article in press as: Dhahbi, J.M., Circulating small noncoding RNAs as biomarkers of aging. Ageing Res. Rev. (2014), http://dx.doi.org/10.1016/j.arr.2014.02.005

G Model ARR-501; No. of Pages 13

ARTICLE IN PRESS J.M. Dhahbi / Ageing Research Reviews xxx (2014) xxx–xxx

expression associated with karyotype in acute myeloid leukaemia. PLoS One 3, e2141. ElSharawy, A., Keller, A., Flachsbart, F., Wendschlag, A., Jacobs, G., Kefer, N., Brefort, T., Leidinger, P., Backes, C., Meese, E., Schreiber, S., Rosenstiel, P., Franke, A., Nebel, A., 2012. Genome-wide miRNA signatures of human longevity. Aging Cell 11, 607–616. Emara, M.M., Ivanov, P., Hickman, T., Dawra, N., Tisdale, S., Kedersha, N., Hu, G.F., Anderson, P., 2010. Angiogenin-induced tRNA-derived stress-induced RNAs promote stress-induced stress granule assembly. The Journal of Biological Chemistry 285, 10959–10968. Esteller, M., 2011. Non-coding RNAs in human disease. Nature Reviews. Genetics 12, 861–874. Etheridge, A., Lee, I., Hood, L., Galas, D., Wang, K., 2011. Extracellular microRNA: a new source of biomarkers. Mutation Research 717, 85–90. Fabian, M.R., Sonenberg, N., Filipowicz, W., 2010. Regulation of mRNA translation and stability by microRNAs. Annual Review of Biochemistry 79, 351–379. Farazi, T.A., Hoell, J.I., Morozov, P., Tuschl, T., 2013. MicroRNAs in human cancer. Advances in Experimental Medicine and Biology 774, 1–20. Farazi, T.A., Spitzer, J.I., Morozov, P., Tuschl, T., 2011. miRNAs in human cancer. Journal of Pathology 223, 102–115. Filkova, M., Jungel, A., Gay, R.E., Gay, S., 2012. MicroRNAs in rheumatoid arthritis: potential role in diagnosis and therapy. BioDrugs: Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy 26, 131–141. Friedlander, M.R., Mackowiak, S.D., Li, N., Chen, W., Rajewsky, N., 2012. miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades. Nucleic Acids Research 40, 37–52. Fu, H., Feng, J., Liu, Q., Sun, F., Tie, Y., Zhu, J., Xing, R., Sun, Z., Zheng, X., 2009. Stress induces tRNA cleavage by angiogenin in mammalian cells. FEBS Letters 583, 437–442. Garcia, E.L., Onafuwa-Nuga, A., Sim, S., King, S.R., Wolin, S.L., Telesnitsky, A., 2009. Packaging of host mY RNAs by murine leukemia virus may occur early in Y RNA biogenesis. Journal of Virology 83, 12526–12534. Gardiner, T.J., Christov, C.P., Langley, A.R., Krude, T., 2009. A conserved motif of vertebrate Y RNAs essential for chromosomal DNA replication. RNA 15, 1375–1385. Gombar, S., Jung, H.J., Dong, F., Calder, B., Atzmon, G., Barzilai, N., Tian, X.L., Pothof, J., Hoeijmakers, J.H., Campisi, J., Vijg, J., Suh, Y., 2012. Comprehensive microRNA profiling in B-cells of human centenarians by massively parallel sequencing. BMC Genomics 13, 353. Gotte, M., Mohr, C., Koo, C.Y., Stock, C., Vaske, A.K., Viola, M., Ibrahim, S.A., Peddibhotla, S., Teng, Y.H., Low, J.Y., Ebnet, K., Kiesel, L., Yip, G.W., 2010. miR145-dependent targeting of junctional adhesion molecule A and modulation of fascin expression are associated with reduced breast cancer cell motility and invasiveness. Oncogene 29, 6569–6580. Green, N.J., Grundy, F.J., Henkin, T.M., 2010. The T box mechanism: tRNA as a regulatory molecule. FEBS Letters 584, 318–324. Grillari, J., Grillari-Voglauer, R., 2010. Novel modulators of senescence, aging, and longevity: small non-coding RNAs enter the stage. Experimental Gerontology 45, 302–311. Grosshans, H., Filipowicz, W., 2008. Molecular biology: the expanding world of small RNAs. Nature 451, 414–416. Gupta, S.K., Piccoli, M.T., Thum, T., 2014. Non-coding RNAs in cardiovascular ageing. Ageing Research Reviews (pii: S1568-1637(14)00003-8). Hackl, M., Brunner, S., Fortschegger, K., Schreiner, C., Micutkova, L., Muck, C., Laschober, G.T., Lepperdinger, G., Sampson, N., Berger, P., Herndler-Brandstetter, D., Wieser, M., Kuhnel, H., Strasser, A., Rinnerthaler, M., Breitenbach, M., Mildner, M., Eckhart, L., Tschachler, E., Trost, A., Bauer, J.W., Papak, C., Trajanoski, Z., Scheideler, M., Grillari-Voglauer, R., Grubeck-Loebenstein, B., Jansen-Durr, P., Grillari, J., 2010. miR-17, miR-19b, miR-20a, and miR-106a are down-regulated in human aging. Aging Cell 9, 291–296. Haussecker, D., Huang, Y., Lau, A., Parameswaran, P., Fire, A.Z., Kay, M.A., 2010. Human tRNA-derived small RNAs in the global regulation of RNA silencing. RNA 16, 673–695. He, L., He, X., Lim, L.P., de Stanchina, E., Xuan, Z., Liang, Y., Xue, W., Zender, L., Magnus, J., Ridzon, D., Jackson, A.L., Linsley, P.S., Chen, C., Lowe, S.W., Cleary, M.A., Hannon, G.J., 2007. A microRNA component of the p53 tumour suppressor network. Nature 447, 1130–1134. Healy, N.A., Heneghan, H.M., Miller, N., Osborne, C.K., Schiff, R., Kerin, M.J., 2012. Systemic mirnas as potential biomarkers for malignancy. International Journal of Cancer. Journal International du Cancer 131, 2215–2222. Heinrich, E.M., Dimmeler, S., 2012. MicroRNAs and stem cells: control of pluripotency, reprogramming, and lineage commitment. Circulation Research 110, 1014–1022. Hergenreider, E., Heydt, S., Treguer, K., Boettger, T., Horrevoets, A.J., Zeiher, A.M., Scheffer, M.P., Frangakis, A.S., Yin, X., Mayr, M., Braun, T., Urbich, C., Boon, R.A., Dimmeler, S., 2012. Atheroprotective communication between endothelial cells and smooth muscle cells through miRNAs. Nature Cell Biology 14, 249–256. Hofacker, I.L., 2003. Vienna RNA secondary structure server. Nucleic Acids Research 31, 3429–3431. Hogg, J.R., Collins, K., 2007. Human Y5 RNA specializes a Ro ribonucleoprotein for 5S ribosomal RNA quality control. Genes & Development 21, 3067–3072. Horton, R., Wilming, L., Rand, V., Lovering, R.C., Bruford, E.A., Khodiyar, V.K., Lush, M.J., Povey, S., Talbot Jr., C.C., Wright, M.W., Wain, H.M., Trowsdale, J., Ziegler, A., Beck, S., 2004. Gene map of the extended human MHC. Nature Reviews. Genetics 5, 889–899. Hoy, A.M., Buck, A.H., 2012. Extracellular small RNAs: what, where, why? Biochemical Society Transactions 40, 886–890.

11

Hursting, S.D., Lavigne, J.A., Berrigan, D., Perkins, S.N., Barrett, J.C., 2003. Calorie restriction, aging, and cancer prevention: mechanisms of action and applicability to humans. Annual Review of Medicine 54, 131–152. Ichimura, A., Ruike, Y., Terasawa, K., Tsujimoto, G., 2011. miRNAs and regulation of cell signaling. The FEBS Journal 278, 1610–1618. Iorio, M.V., Croce, C.M., 2012. microRNA involvement in human cancer. Carcinogenesis 33, 1126–1133. Iuliano, R., Vismara, M.F., Dattilo, V., Trapasso, F., Baudi, F., Perrotti, N., 2013. The role of microRNAs in cancer susceptibility. BioMed Research International 2013, 591931. Ivanov, P., Emara, M.M., Villen, J., Gygi, S.P., Anderson, P., 2011. Angiogenininduced tRNA fragments inhibit translation initiation. Molecular Cell 43, 613–623. Ivey, K.N., Srivastava, D., 2010. MicroRNAs as regulators of differentiation and cell fate decisions. Cell Stem Cell 7, 36–41. Joshua-Tor, L., Hannon, G.J., 2011. Ancestral roles of small RNAs: an Ago-centric perspective. Cold Spring Harbor Perspectives in Biology 3, a003772. Khanna, A., Muthusamy, S., Liang, R., Sarojini, H., Wang, E., 2011. Gain of survival signaling by down-regulation of three key miRNAs in brain of calorie-restricted mice. Aging 3, 223–236. Kosaka, N., Yoshioka, Y., Hagiwara, K., Tominaga, N., Katsuda, T., Ochiya, T., 2013. Trash or treasure: extracellular microRNAs and cell-to-cell communication. Frontiers in Genetics 4, 173. Koturbash, I., Zemp, F.J., Pogribny, I., Kovalchuk, O., 2011. Small molecules with big effects: the role of the microRNAome in cancer and carcinogenesis. Mutation Research 722, 94–105. Krude, T., 2010. Non-coding RNAs: new players in the field of eukaryotic DNA replication. Sub-cellular Biochemistry 50, 105–118. Krude, T., Christov, C.P., Hyrien, O., Marheineke, K., 2009. Y RNA functions at the initiation step of mammalian chromosomal DNA replication. Journal of Cell Science 122, 2836–2845. Labbe, J.C., Burgess, J., Rokeach, L.A., Hekimi, S., 2000. ROP-1, an RNA qualitycontrol pathway component, affects Caenorhabditis elegans dauer formation. Proceedings of the National Academy of Sciences of the United States of America 97, 13233–13238. Lafferty-Whyte, K., Cairney, C.J., Jamieson, N.B., Oien, K.A., Keith, W.N., 2009. Pathway analysis of senescence-associated miRNA targets reveals common processes to different senescence induction mechanisms. Biochimica et Biophysica Acta 1792, 341–352. Lages, E., Ipas, H., Guttin, A., Nesr, H., Berger, F., Issartel, J.P., 2012. MicroRNAs: molecular features and role in cancer. Frontiers in Bioscience: A Journal and Virtual Library 17, 2508–2540. Langenberger, D., Cakir, M.V., Hoffmann, S., Stadler, P.F., 2013. Dicer-processed small RNAs: rules and exceptions. Journal of Experimental Zoology. Part B, Molecular and Developmental Evolution 320, 35–46. Langley, A.R., Chambers, H., Christov, C.P., Krude, T., 2010. Ribonucleoprotein particles containing non-coding Y RNAs, Ro60, La and nucleolin are not required for Y RNA function in DNA replication. PLoS One 5, e13673. Lasser, C., Alikhani, V.S., Ekstrom, K., Eldh, M., Paredes, P.T., Bossios, A., Sjostrand, M., Gabrielsson, S., Lotvall, J., Valadi, H., 2011. Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. Journal of Translational Medicine 9, 9. Lee, C.K., Klopp, R.G., Weindruch, R., Prolla, T.A., 1999. Gene expression profile of aging and its retardation by caloric restriction. Science 285, 1390–1393. Lee, S.R., Collins, K., 2005. Starvation-induced cleavage of the tRNA anticodon loop in Tetrahymena thermophila. The Journal of Biological Chemistry 280, 42744–42749. Lee, Y.S., Shibata, Y., Malhotra, A., Dutta, A., 2009. A novel class of small RNAs: tRNAderived RNA fragments (tRFs). Genes & Development 23, 2639–2649. Lehmann, S.M., Kruger, C., Park, B., Derkow, K., Rosenberger, K., Baumgart, J., Trimbuch, T., Eom, G., Hinz, M., Kaul, D., Habbel, P., Kalin, R., Franzoni, E., Rybak, A., Nguyen, D., Veh, R., Ninnemann, O., Peters, O., Nitsch, R., Heppner, F.L., Golenbock, D., Schott, E., Ploegh, H.L., Wulczyn, F.G., Lehnardt, S., 2012. An unconventional role for miRNA: let-7 activates Toll-like receptor 7 and causes neurodegeneration. Nature Neuroscience 15, 827–835. Lerner, M.R., Boyle, J.A., Hardin, J.A., Steitz, J.A., 1981. Two novel classes of small ribonucleoproteins detected by antibodies associated with lupus erythematosus. Science 211, 400–402. Li, L.C., 2013. Chromatin remodeling by the small RNA machinery in mammalian cells. Epigenetics: Official Journal of the DNA Methylation Society 9. Li, X., Khanna, A., Li, N., Wang, E., 2011. Circulatory miR34a as an RNAbased, noninvasive biomarker for brain aging. Aging 3, 985–1002. Li, Y., Kowdley, K.V., 2012. MicroRNAs in common human diseases. Genomics, Proteomics & Bioinformatics 10, 246–253. Li, Y., Zhou, H., 2009. tRNAs as regulators in gene expression. Science in China. Series C, Life sciences/Chinese Academy of Sciences 52, 245–252. Liang, R., Bates, D.J., Wang, E., 2009. Epigenetic control of MicroRNA expression and aging. Current Genomics 10, 184–193. Lin, K., Dorman, J.B., Rodan, A., Kenyon, C., 1997. daf-16: an HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278, 1319–1322. Lin, L., Lin, Y., Jin, Y., Zheng, C., 2013. Microarray analysis of microRNA expression in liver cancer tissues and normal control. Gene 523, 158–160. Liu, N., Landreh, M., Cao, K., Abe, M., Hendriks, G.J., Kennerdell, J.R., Zhu, Y., Wang, L.S., Bonini, N.M., 2012. The microRNA miR-34 modulates ageing and neurodegeneration in Drosophila. Nature 482, 519–523.

Please cite this article in press as: Dhahbi, J.M., Circulating small noncoding RNAs as biomarkers of aging. Ageing Res. Rev. (2014), http://dx.doi.org/10.1016/j.arr.2014.02.005

G Model ARR-501; No. of Pages 13 12

ARTICLE IN PRESS J.M. Dhahbi / Ageing Research Reviews xxx (2014) xxx–xxx

Ma, R., Jiang, T., Kang, X., 2012. Circulating microRNAs in cancer: origin, function and application. Journal of Experimental & Clinical Cancer Research: CR 31, 38. Mackowiak, S.D., 2011. Identification of novel and known miRNAs in deepsequencing data with miRDeep2. Current Protocols in Bioinformatics/Editoral Board (Andreas D. Baxevanis, et al. Chapter 12, Unit 12.10). Maes, O.C., Sarojini, H., Wang, E., 2009. Stepwise up-regulation of microRNA expression levels from replicating to reversible and irreversible growth arrest states in WI-38 human fibroblasts. Journal of Cellular Physiology 221, 109–119. Martens-Uzunova, E.S., Jalava, S.E., Dits, N.F., van Leenders, G.J., Moller, S., Trapman, J., Bangma, C.H., Litman, T., Visakorpi, T., Jenster, G., 2012. Diagnostic and prognostic signatures from the small non-coding RNA transcriptome in prostate cancer. Oncogene 31, 978–991. Martens-Uzunova, E.S., Olvedy, M., Jenster, G., 2013. Beyond microRNA – novel RNAs derived from small non-coding RNA and their implication in cancer. Cancer Letters 340 (2), 201–211. McCay, C.M., Crowell, M.F., Maynard, L.A., 1989. The effect of retarded growth upon the length of life span and upon the ultimate body size. 1935. Nutrition 5, 155–171 (discussion 172). McCormick, K.P., Willmann, M.R., Meyers, B.C., 2011. Experimental design, preprocessing, normalization and differential expression analysis of small RNA sequencing experiments. Silence 2, 2. McDaneld, T.G., Smith, T.P., Doumit, M.E., Miles, J.R., Coutinho, L.L., Sonstegard, T.S., Matukumalli, L.K., Nonneman, D.J., Wiedmann, R.T., 2009. MicroRNA transcriptome profiles during swine skeletal muscle development. BMC Genomics 10, 77. Meiri, E., Levy, A., Benjamin, H., Ben-David, M., Cohen, L., Dov, A., Dromi, N., Elyakim, E., Yerushalmi, N., Zion, O., Lithwick-Yanai, G., Sitbon, E., 2010. Discovery of microRNAs and other small RNAs in solid tumors. Nucleic Acids Research 38, 6234–6246. Mercken, E.M., Majounie, E., Ding, J., Guo, R., Kim, J., Bernier, M., Mattison, J., Cookson, M.R., Gorospe, M., de Cabo, R., Abdelmohsen, K., 2013. Age-associated miRNA alterations in skeletal muscle from rhesus monkeys reversed by caloric restriction. Aging 5, 692–703. Meredith, B.K., Berry, D.P., Kearney, F., Finlay, E.K., Fahey, A.G., Bradley, D.G., Lynn, D.J., 2013. A genome-wide association study for somatic cell score using the Illumina high-density bovine beadchip identifies several novel QTL potentially related to mastitis susceptibility. Frontiers in Genetics 4, 229. Mo, Y.Y., 2012. MicroRNA regulatory networks and human disease. Cellular and Molecular Life Sciences: CMLS 69, 3529–3531. Mosig, A., Guofeng, M., Stadler, B.M., Stadler, P.F., 2007. Evolution of the vertebrate Y RNA cluster. Theory in Biosciences = Theorie in den Biowissenschaften 126, 9–14. Mungall, A.J., Palmer, S.A., Sims, S.K., Edwards, C.A., Ashurst, J.L., Wilming, L., Jones, M.C., Horton, R., Hunt, S.E., Scott, C.E., Gilbert, J.G., Clamp, M.E., Bethel, G., Milne, S., Ainscough, R., Almeida, J.P., Ambrose, K.D., Andrews, T.D., Ashwell, R.I., Babbage, A.K., Bagguley, C.L., Bailey, J., Banerjee, R., Barker, D.J., Barlow, K.F., Bates, K., Beare, D.M., Beasley, H., Beasley, O., Bird, C.P., Blakey, S., Bray-Allen, S., Brook, J., Brown, A.J., Brown, J.Y., Burford, D.C., Burrill, W., Burton, J., Carder, C., Carter, N.P., Chapman, J.C., Clark, S.Y., Clark, G., Clee, C.M., Clegg, S., Cobley, V., Collier, R.E., Collins, J.E., Colman, L.K., Corby, N.R., Coville, G.J., Culley, K.M., Dhami, P., Davies, J., Dunn, M., Earthrowl, M.E., Ellington, A.E., Evans, K.A., Faulkner, L., Francis, M.D., Frankish, A., Frankland, J., French, L., Garner, P., Garnett, J., Ghori, M.J., Gilby, L.M., Gillson, C.J., Glithero, R.J., Grafham, D.V., Grant, M., Gribble, S., Griffiths, C., Griffiths, M., Hall, R., Halls, K.S., Hammond, S., Harley, J.L., Hart, E.A., Heath, P.D., Heathcott, R., Holmes, S.J., Howden, P.J., Howe, K.L., Howell, G.R., Huckle, E., Humphray, S.J., Humphries, M.D., Hunt, A.R., Johnson, C.M., Joy, A.A., Kay, M., Keenan, S.J., Kimberley, A.M., King, A., Laird, G.K., Langford, C., Lawlor, S., Leongamornlert, D.A., Leversha, M., Lloyd, C.R., Lloyd, D.M., Loveland, J.E., Lovell, J., Martin, S., Mashreghi-Mohammadi, M., Maslen, G.L., Matthews, L., McCann, O.T., McLaren, S.J., McLay, K., McMurray, A., Moore, M.J., Mullikin, J.C., Niblett, D., Nickerson, T., Novik, K.L., Oliver, K., Overton-Larty, E.K., Parker, A., Patel, R., Pearce, A.V., Peck, A.I., Phillimore, B., Phillips, S., Plumb, R.W., Porter, K.M., Ramsey, Y., Ranby, S.A., Rice, C.M., Ross, M.T., Searle, S.M., Sehra, H.K., Sheridan, E., Skuce, C.D., Smith, S., Smith, M., Spraggon, L., Squares, S.L., Steward, C.A., Sycamore, N., Tamlyn-Hall, G., Tester, J., Theaker, A.J., Thomas, D.W., Thorpe, A., Tracey, A., Tromans, A., Tubby, B., Wall, M., Wallis, J.M., West, A.P., White, S.S., Whitehead, S.L., Whittaker, H., Wild, A., Willey, D.J., Wilmer, T.E., Wood, J.M., Wray, P.W., Wyatt, J.C., Young, L., Younger, R.M., Bentley, D.R., Coulson, A., Durbin, R., Hubbard, T., Sulston, J.E., Dunham, I., Rogers, J., Beck, S., 2003. The DNA sequence and analysis of human chromosome 6. Nature 425, 805–811. Murguia, J.R., Serrano, R., 2012. New functions of protein kinase Gcn2 in yeast and mammals. IUBMB Life 64, 971–974. Nicolas, F.E., Hall, A.E., Csorba, T., Turnbull, C., Dalmay, T., 2012. Biogenesis of Y RNAderived small RNAs is independent of the microRNA pathway. FEBS Letters 586, 1226–1230. Nilsen, T.W., 2007. Mechanisms of microRNA-mediated gene regulation in animal cells. Trends in Genetics: TIG 23, 243–249. Ninomiya, M., Kondo, Y., Funayama, R., Nagashima, T., Kogure, T., Kakazu, E., Kimura, O., Ueno, Y., Nakayama, K., Shimosegawa, T., 2013. Distinct microRNAs expression profile in primary biliary cirrhosis and evaluation of miR 505-3p and miR197-3p as novel biomarkers. PLoS One 8, e66086. Nolte-’t Hoen, E.N., Buermans, H.P., Waasdorp, M., Stoorvogel, W., Wauben, M.H., t Hoen, P.A., 2012. Deep sequencing of RNA from immune cell-derived vesicles uncovers the selective incorporation of small non-coding RNA biotypes with potential regulatory functions. Nucleic Acids Research 40, 9272–9285.

Noren Hooten, N., Abdelmohsen, K., Gorospe, M., Ejiogu, N., Zonderman, A.B., Evans, M.K., 2010. microRNA expression patterns reveal differential expression of target genes with age. PLoS One 5, e10724. Noren Hooten, N., Fitzpatrick, M., Wood 3rd, W.H., De, S., Ejiogu, N., Zhang, Y., Mattison, J.A., Becker, K.G., Zonderman, A.B., Evans, M.K., 2013. Age-related changes in microRNA levels in serum. Aging 5, 725–740. O’Brien, C.A., Wolin, S.L., 1994. A possible role for the 60-kD Ro autoantigen in a discard pathway for defective 5S rRNA precursors. Genes & Development 8, 2891–2903. Okamura, K., 2012. Diversity of animal small RNA pathways and their biological utility. Wiley Interdisciplinary Reviews. RNA 3, 351–368. Olivieri, F., Spazzafumo, L., Santini, G., Lazzarini, R., Albertini, M.C., Rippo, M.R., Galeazzi, R., Abbatecola, A.M., Marcheselli, F., Monti, D., Ostan, R., Cevenini, E., Antonicelli, R., Franceschi, C., Procopio, A.D., 2012. Age-related differences in the expression of circulating microRNAs: miR-21 as a new circulating marker of inflammaging. Mechanisms of Ageing and Development 133, 675–685. Pant, S., Hilton, H., Burczynski, M.E., 2012. The multifaceted exosome: biogenesis, role in normal and aberrant cellular function, and frontiers for pharmacological and biomarker opportunities. Biochemical Pharmacology 83, 1484–1494. Pederson, T., 2010. Regulatory RNAs derived from transfer RNA? RNA 16, 1865–1869. Pencheva, N., Tavazoie, S.F., 2013. Control of metastatic progression by microRNA regulatory networks. Nature Cell Biology 15, 546–554. Perreault, J., Noel, J.F., Briere, F., Cousineau, B., Lucier, J.F., Perreault, J.P., Boire, G., 2005. Retropseudogenes derived from the human Ro/SS-A autoantigenassociated hY RNAs. Nucleic Acids Research 33, 2032–2041. Perreault, J., Perreault, J.P., Boire, G., 2007. Ro-associated Y RNAs in metazoans: evolution and diversification. Molecular Biology and Evolution 24, 1678–1689. Qi, Y., Tu, J., Cui, L., Guo, X., Shi, Z., Li, S., Shi, W., Shan, Y., Ge, Y., Shan, J., Wang, H., Lu, Z., 2010. High-throughput sequencing of microRNAs in adenovirus type 3 infected human laryngeal epithelial cells. Journal of Biomedicine & Biotechnology 2010, 915980. Redova, M., Sana, J., Slaby, O., 2013. Circulating miRNAs as new blood-based biomarkers for solid cancers. Future Oncology 9, 387–402. Reed, J.H., Sim, S., Wolin, S.L., Clancy, R.M., Buyon, J.P., 2013. Ro60 requires y3 RNA for cell surface exposure and inflammation associated with cardiac manifestations of neonatal lupus. Journal of Immunology 191, 110–116. Reid, G., Kirschner, M.B., van Zandwijk, N., 2011. Circulating microRNAs: association with disease and potential use as biomarkers. Critical Reviews in Oncology/Hematology 80, 193–208. Rother, S., Meister, G., 2011. Small RNAs derived from longer non-coding RNAs. Biochimie 93, 1905–1915. Routsias, J.G., Goules, J.D., Charalampakis, G., Tzima, S., Papageorgiou, A., Voulgarelis, M., 2013. Malignant lymphoma in primary Sjogren’s syndrome: an update on the pathogenesis and treatment. Seminars in Arthritis and Rheumatism 43, 178–186. Rutjes, S.A., van der Heijden, A., Utz, P.J., van Venrooij, W.J., Pruijn, G.J., 1999. Rapid nucleolytic degradation of the small cytoplasmic Y RNAs during apoptosis. The Journal of Biological Chemistry 274, 24799–24807. Salic, K., De Windt, L.J., 2012. MicroRNAs as biomarkers for myocardial infarction. Current Atherosclerosis Reports 14, 193–200. Saugstad, J.A., 2010. MicroRNAs as effectors of brain function with roles in ischemia and injury, neuroprotection, and neurodegeneration. Journal of Cerebral Blood Flow and Metabolism 30, 1564–1576. Schaefer, M., Pollex, T., Hanna, K., Tuorto, F., Meusburger, M., Helm, M., Lyko, F., 2010. RNA methylation by Dnmt2 protects transfer RNAs against stress-induced cleavage. Genes & Development 24, 1590–1595. Schotte, D., Chau, J.C., Sylvester, G., Liu, G., Chen, C., van der Velden, V.H., Broekhuis, M.J., Peters, T.C., Pieters, R., den Boer, M.L., 2009. Identification of new microRNA genes and aberrant microRNA profiles in childhood acute lymphoblastic leukemia. Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, UK 23, 313–322. Serna, E., Gambini, J., Borras, C., Mohammed, K., Belenguer, A., Sanchis, P., Avellana, J.A., Rodriguez-Manas, L., Vina, J., 2012. Centenarians, but not octogenarians, up-regulate the expression of microRNAs. Scientific Reports 2, 961. Shah, M.Y., Calin, G.A., 2013. The mix of two worlds: non-coding RNAs and hormones. Nucleic Acid Therapeutics 23 (1), 2–8. Shaheen, H.H., Horetsky, R.L., Kimball, S.R., Murthi, A., Jefferson, L.S., Hopper, A.K., 2007. Retrograde nuclear accumulation of cytoplasmic tRNA in rat hepatoma cells in response to amino acid deprivation. Proceedings of the National Academy of Sciences of the United States of America 104, 8845–8850. Shao, N.Y., Hu, H.Y., Yan, Z., Xu, Y., Hu, H., Menzel, C., Li, N., Chen, W., Khaitovich, P., 2010. Comprehensive survey of human brain microRNA by deep sequencing. BMC Genomics 11, 409. Shen, J., Stass, S.A., Jiang, F., 2013. MicroRNAs as potential biomarkers in human solid tumors. Cancer Letters 329, 125–136. Shi, H., O’Brien, C.A., Van Horn, D.J., Wolin, S.L., 1996. A misfolded form of 5S rRNA is complexed with the Ro and La autoantigens. RNA 2, 769–784. Silahtaroglu, A., Stenvang, J., 2010. MicroRNAs, epigenetics and disease. Essays in Biochemistry 48, 165–185. Smith-Vikos, T., Slack, F.J., 2012. MicroRNAs and their roles in aging. Journal of Cell Science 125, 7–17. Sobala, A., Hutvagner, G., 2011. Transfer RNA-derived fragments: origins, processing, and functions. Wiley Interdisciplinary Reviews. RNA 2, 853–862. Sobala, A., Hutvagner, G., 2013. Small RNAs derived from the 5 end of tRNA can inhibit protein translation in human cells. RNA Biology 10, 553–563.

Please cite this article in press as: Dhahbi, J.M., Circulating small noncoding RNAs as biomarkers of aging. Ageing Res. Rev. (2014), http://dx.doi.org/10.1016/j.arr.2014.02.005

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ARTICLE IN PRESS J.M. Dhahbi / Ageing Research Reviews xxx (2014) xxx–xxx

Spindler, S.R., 2010. Caloric restriction: from soup to nuts. Ageing Research Reviews 9, 324–353. Spindler, S.R., Dhahbi, J.M., 2007. Conserved and tissue-specific genic and physiologic responses to caloric restriction and altered IGFI signaling in mitotic and postmitotic tissues. Annual Review of Nutrition 27, 193–217. Taft, R.J., Pang, K.C., Mercer, T.R., Dinger, M., Mattick, J.S., 2010. Non-coding RNAs: regulators of disease. Journal of Pathology 220, 126–139. Thomas, M., Lieberman, J., Lal, A., 2010. Desperately seeking microRNA targets. Nature Structural & Molecular Biology 17, 1169–1174. Thompson, D.M., Lu, C., Green, P.J., Parker, R., 2008. tRNA cleavage is a conserved response to oxidative stress in eukaryotes. RNA 14, 2095–2103. Thompson, D.M., Parker, R., 2009. Stressing out over tRNA cleavage. Cell 138, 215–219. Townley-Tilson, W.H., Callis, T.E., Wang, D., 2010. MicroRNAs 1, 133, and 206: critical factors of skeletal and cardiac muscle development, function, and disease. International Journal of Biochemistry and Cell Biology 42, 1252–1255. Turchinovich, A., Burwinkel, B., 2012. Distinct AGO1 and AGO2 associated miRNA profiles in human cells and blood plasma. RNA Biology 9 (8), 1066–1075. Turchinovich, A., Weiz, L., Burwinkel, B., 2012. Extracellular miRNAs: the mystery of their origin and function. Trends in Biochemical Sciences 37 (11), 460–465. Turchinovich, A., Weiz, L., Langheinz, A., Burwinkel, B., 2011. Characterization of extracellular circulating microRNA. Nucleic Acids Research 39, 7223–7233. Uchino, K., Ochiya, T., Takeshita, F., 2013. RNAi therapeutics and applications of microRNAs in cancer treatment. Japanese Journal of Clinical Oncology 43, 596–607. Verhagen, A.P., Pruijn, G.J., 2011. Are the Ro RNP-associated Y RNAs concealing microRNAs? Y RNA-derived miRNAs may be involved in autoimmunity. BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology 33, 674–682. Vickers, K.C., Palmisano, B.T., Shoucri, B.M., Shamburek, R.D., Remaley, A.T., 2011. MicroRNAs are transported in plasma and delivered to recipient cells by highdensity lipoproteins. Nature Cell Biology 13, 423–433. Vickers, K.C., Remaley, A.T., 2012. Lipid-based carriers of microRNAs and intercellular communication. Current Opinion in Lipidology 23, 91–97. Voulgarelis, M., Moutsopoulos, H.M., 2001. Malignant lymphoma in primary Sjogren’s syndrome. The Israel Medical Association Journal: IMAJ 3, 761–766. Wang, K., Zhang, S., Weber, J., Baxter, D., Galas, D.J., 2010a. Export of microRNAs and microRNA-protective protein by mammalian cells. Nucleic Acids Research 38, 7248–7259. Wang, Q., Lee, I., Ren, J., Ajay, S.S., Lee, Y.S., Bao, X., 2013. Identification and functional characterization of tRNA-derived RNA fragments (tRFs) in respiratory syncytial virus infection. Molecular Therapy: The Journal of the American Society of Gene Therapy 21 (2), 368–379. Wang, X.Y., Wu, M.H., Liu, F., Li, Y., Li, N., Li, G.Y., Shen, S.R., 2010b. Differential miRNA expression and their target genes between NGX6-positive and negative colon cancer cells. Molecular and Cellular Biochemistry 345, 283–290. Weindruch, R., Kayo, T., Lee, C.K., Prolla, T.A., 2001. Microarray profiling of gene expression in aging and its alteration by caloric restriction in mice. The Journal of Nutrition 131, 918S–923S. Wery, M., Kwapisz, M., Morillon, A., 2011. Noncoding RNAs in gene regulation. Wiley Interdisciplinary Reviews. Systems Biology and Medicine 3, 728–738. Witten, D., Tibshirani, R., Gu, S.G., Fire, A., Lui, W.O., 2010. Ultra-high throughput sequencing-based small RNA discovery and discrete statistical biomarker

13

analysis in a collection of cervical tumours and matched controls. BMC Biology 8, 58. Wittmann, J., Jack, H.M., 2010. New surprises from the deep – the family of small regulatory RNAs increases. The Scientific World Journal 10, 1239–1243. Wolin, S.L., Belair, C., Boccitto, M., Chen, X., Sim, S., Taylor, D.W., Wang, H.W., 2013. Non-coding Y RNAs as tethers and gates: insights from bacteria. RNA Biology 10. Wolin, S.L., Steitz, J.A., 1983. Genes for two small cytoplasmic Ro RNAs are adjacent and appear to be single-copy in the human genome. Cell 32, 735–744. Xu, D., Tahara, H., 2013. The role of exosomes and microRNAs in senescence and aging. Advanced Drug Delivery Reviews 65 (3), 368–375. Xu, J., Zhao, J., Evan, G., Xiao, C., Cheng, Y., Xiao, J., 2012. Circulating microRNAs: novel biomarkers for cardiovascular diseases. Journal of Molecular Medicine (Berlin) 90, 865–875. Yamasaki, S., Ivanov, P., Hu, G.F., Anderson, P., 2009. Angiogenin cleaves tRNA and promotes stress-induced translational repression. The Journal of Cell Biology 185, 35–42. Yamaza, H., Komatsu, T., Wakita, S., Kijogi, C., Park, S., Hayashi, H., Chiba, T., Mori, R., Furuyama, T., Mori, N., Shimokawa, I., 2010. FoxO1 is involved in the antineoplastic effect of calorie restriction. Aging Cell 9, 372–382. Yang, J., Hao, Y., Xi, J.J., 2013. Therapeutic application of microRNAs against human cancers. Journal of Laboratory Automation 18, 30–33. Yeung, M.L., Bennasser, Y., Watashi, K., Le, S.Y., Houzet, L., Jeang, K.T., 2009. Pyrosequencing of small non-coding RNAs in HIV-1 infected cells: evidence for the processing of a viral-cellular double-stranded RNA hybrid. Nucleic Acids Research 37, 6575–6586. Youssef, Y.M., White, N.M., Grigull, J., Krizova, A., Samy, C., Mejia-Guerrero, S., Evans, A., Yousef, G.M., 2011. Accurate molecular classification of kidney cancer subtypes using microRNA signature. European Urology 59, 721–730. Zampetaki, A., Willeit, P., Drozdov, I., Kiechl, S., Mayr, M., 2012. Profiling of circulating microRNAs: from single biomarkers to re-wired networks. Cardiovascular Research 93, 555–562. Zen, K., Zhang, C.Y., 2012. Circulating microRNAs: a novel class of biomarkers to diagnose and monitor human cancers. Medicinal Research Reviews 32, 326–348. Zernecke, A., Bidzhekov, K., Noels, H., Shagdarsuren, E., Gan, L., Denecke, B., Hristov, M., Koppel, T., Jahantigh, M.N., Lutgens, E., Wang, S., Olson, E.N., Schober, A., Weber, C., 2009. Delivery of microRNA-126 by apoptotic bodies induces CXCL12dependent vascular protection. Science Signaling 2, ra81. Zhang, A.T., Langley, A.R., Christov, C.P., Kheir, E., Shafee, T., Gardiner, T.J., Krude, T., 2011a. Dynamic interaction of Y RNAs with chromatin and initiation proteins during human DNA replication. Journal of Cell Science 124, 2058–2069. Zhang, C., 2009. Novel functions for small RNA molecules. Current Opinion in Molecular Therapeutics 11, 641–651. Zhang, J., Guo, H., Zhang, H., Wang, H., Qian, G., Fan, X., Hoffman, A.R., Hu, J.F., Ge, S., 2011b. Putative tumor suppressor miR-145 inhibits colon cancer cell growth by targeting oncogene Friend leukemia virus integration 1 gene. Cancer 117, 86–95. Zhang, Y., Liu, D., Chen, X., Li, J., Li, L., Bian, Z., Sun, F., Lu, J., Yin, Y., Cai, X., Sun, Q., Wang, K., Ba, Y., Wang, Q., Wang, D., Yang, J., Liu, P., Xu, T., Yan, Q., Zhang, J., Zen, K., Zhang, C.Y., 2010. Secreted monocytic miR-150 enhances targeted endothelial cell migration. Molecular Cell 39, 133–144. Zhao, H., Bojanowski, K., Ingber, D.E., Panigrahy, D., Pepper, M.S., Montesano, R., Shing, Y., 1999. New role for tRNA and its fragment purified from human urinary bladder carcinoma conditioned medium: inhibition of endothelial cell growth. Journal of Cellular Biochemistry 76, 109–117.

Please cite this article in press as: Dhahbi, J.M., Circulating small noncoding RNAs as biomarkers of aging. Ageing Res. Rev. (2014), http://dx.doi.org/10.1016/j.arr.2014.02.005