Small RNAs derived from longer non-coding RNAs

Biochimie 93 (2011) 1905e1915

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Review

Small RNAs derived from longer non-coding RNAs Susanne Röther*, Gunter Meister University of Regensburg, Biochemistry I, Universitätsstrasse 31, 93053 Regensburg, Bavaria, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 April 2011 Accepted 29 July 2011 Available online 9 August 2011

Posttranscriptional gene regulation by small RNAs and its crucial impact on development, apoptosis, stem cell self-renewal and differentiation gained tremendous scientific attention since the discovery of RNA interference (RNAi) and microRNAs (miRNAs). However, in the last few years, many more examples for regulatory small RNAs were discovered, some of them even with miRNA-like functions. Even though these small RNA molecules were previously thought to be mere artifacts accumulating during the preparation of RNA libraries, advances in sequencing technology revealed that small RNAs derive from hairpin-fold RNA structures, for example. Mirtrons, short hairpin RNAs or small RNAs that are processed from longer non-coding RNAs such as tRNAs or snoRNAs have been found recently and some of them might be involved in the regulation of gene expression in different organisms. Furthermore, small RNAs originating from transposable elements, heterochromatic regions or convergent transcription units forming endogenous short interfering RNAs (endo-siRNAs) are the somatic equivalents of the germlinespecific Piwi-interacting RNAs (piRNAs) in mediating transposon silencing. This review will focus on several recent findings that have added new aspects to small RNA-guided gene silencing. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: miRNA Small RNAs miRNA-like small RNAs Non-coding RNAs Regulation of gene expression

1. Introduction When in 1998 RNA interference (RNAi) was discovered by A. Fire and C. Mello [1], this phenomenon was considered to be a revolutionary tool to study gene function. However, in the last decade endogenous small non-coding RNAs were identified and it became evident that small RNAs form a novel layer of gene regulation with fundamental roles for accurate cell function. MicroRNAs (miRNA) (endogenous) short interfering RNAs (siRNAs, endo-siRNAs) and piwi interacting RNAs (piRNAs) are now known as key regulators of processes as diverse as development, apoptosis, stem cell selfrenewal, differentiation and maintenance of cell integrity. Recently, more and more small RNA species deriving from other, longer noncoding RNAs were discovered. Considering the fact that although eukaryotic genomes transcribe up to 90e95% of the genomic DNA, less than 2e3% encode for proteins [2,3], it is evident that noncoding RNAs might have a more important role than previously thought. Indeed, deep sequencing technologies followed by extensive bioinformatics-based characterization of genomic locations and RNA secondary structure predictions revealed the existence of a vast amount of small RNAs some of them even having miRNA-like functions in posttranscriptional gene regulation. These exciting findings

* Corresponding author. Tel.: þ49 941 943 2847. E-mail address: [email protected] (S. Röther). 0300-9084/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2011.07.032

highlight the importance of small RNAs in gene regulation. This review will summarize the most recent findings in the field of small RNAs deriving from longer non-coding RNA species.

2. Non-coding RNAs (ncRNAs) Traditionally, ncRNAs are divided into long ncRNAs (>200e300 nucleotides (nt)) and shorter (<200e300 nt) species of ncRNAs (Table 1) [4,5]. Long ncRNAs, ranging from approximately 200 to several thousands of nt in size, are transcribed by RNA polymerase II, 50 capped, spliced and polyadenylated [6]. Most examples described to date (reviewed in [6e9]) are involved in genomic imprinting (such as for example Kcnq1ot1), an epigenetic phenomenon by which a subset of genes is monoallelically expressed in a parentalspecific manner [8,10e12] or epigenetic mechanisms, such as for example large intergenic non coding RNA p21 (lincRNA-p21). Expression of this ncRNA is induced by p53 upon DNA damage or oncogenic stress and results in global gene repression in the p53 transcriptional response inducing cellular apoptosis by physical interaction with hnRNP-K, thereby directing the repressor protein to promoters of p53 dependent genes to be repressed [13]. Short ncRNAs, ranging from 20 to approximately 300 nt in size, are involved in the most basic cellular mechanisms such as biosynthesis of proteins, removal of intronic sequences while

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Table 1 Examples for non-coding RNAs. ncRNA Long ncRNA lincRNA-p21 Kcnq1ot1

Function

Size

Ref

Tumor suppressor; global gene repression in 53 transcriptional response Genomic imprinting

3.1 kb

[13]

91.5 kb

[8,10e12]

Short ncRNA involved in basic cellular mechanisms tRNA mRNA translation, signal transduction, apoptosis snoRNA rRNA processing, 20 O-methylation and pseudouridylation of rRNA, snRNA, tRNA snRNA Splicing of pre-mRNA Telomerase RNA Telomere synthesis 7SK RNA Transcription elongation control

70e90 nt 70e90 nt

[15] [18e20]

90e220 450 nt (H. sapiens) 300

[18,24] [25] [26,27]

Chromatin associated ncRNA paRNA TSSaRNA PASR tiRNA spliRNA

>200 nt 20e90 nt <200 18 nt 17e18 nt

[28,29] [33] [34] [35,36] [38]

21e25 21e23 21 21 26e31 21e25 22 (H/ACA) 17e30 (C/D) 18e22 21e25

[41e46] [41,45,60e62] [63e66] [51,75e78, 91] [74,95e98] [91,115e117] [21,121e124]

Maintenance of transcription active state Maintenance of transcription active state Regulation Highlighting/Marking of highly transcribed genes Highlighting/Marking of highly transcribed genes

sRNAs involved in the regulation of gene expression miRNA Posttranscriptional gene silencing siRNA (exo) Gene-specific silencing endo-siRNA (plants, worms) Suppression of retrotransposition in somatic cells Endo-siRNA (fly, mammal) Suppression of retrotransposition in somatic cells piRNA Germ line-specific transposon silencing mirtrons Posttranscriptional gene silencing H/ACA and C/D sRNA Posttranscriptional gene silencing Type I/II tsRNA shRNA svRNA moR qiRNA

Posttranscriptional gene silencing Posttranscriptional gene silencing Drug metabolism Posttranscriptional gene silencing DNA damage response

splicing, site specific RNA modification, telomere synthesis, transcription, modulation of protein function and regulation of gene expression. In some cases, the molecular mechanisms are well understood, whereas in others they are completely unknown. Transfer RNAs (tRNAs) serve as key molecules to decode the genetic information stored in mRNAs by basepairing with cognate codons on the mRNA and delivering amino acids to the translation machinery [14e17]. However, recent research implied tRNAs also in signal transduction pathways responding to nutrient deprivation, regulation of apoptosis and in the retroviral life cycle (for review see [15] and references therein). Small nucleolar RNAs (snoRNAs) are involved in ribosomal RNA (rRNA) processing and are responsible for the 20 O-methylation or pseudouridylation of rRNAs, snRNAs and tRNAs (for review see [18e20]). Novel reports furthermore show that smaller processed forms of snoRNAs are able to act as miRNAs ([21], also see below) or regulate alternative splicing of the serotonin receptor 2C [22,23]. Small nuclear RNAs (snRNAs) are part of the spliceosome complex directing the accurate removal of intronic sequences of pre-mRNAs (for review see [18,24]). The synthesis of telomeres is accomplished by Telomerase, a ribonucleoprotein complex consisting of the ncRNA subunit telomerase RNA and the catalytic protein subunit TERT (telomerase reverse transcriptase), a process essential for genome stability (for review see [25]). The highly conserved 7SK RNA has been shown to be important for P-TEFb-mediated transcription elongation control by association of 7SK RNA with the P-TEFb-inhibitory HEXIM proteins, thereby interfering with transcription elongation (for review see [26,27]). More recently, deep sequencing approaches identified ncRNA species that specifically associate with chromatin. Promoterassociated RNAs (paRNAs) were found to be required for RNAdirected transcriptional gene silencing. PaRNAs complementary

19e20 20e21

[91,137e139] [91,143] [144] [91,145e147] [148]

to gene promoters recognize ncRNA transcripts deriving from the corresponding gene promoters, and thereby direct epigenetic silencing complexes to the target promoter [28e32]. Transcription start site-associated RNA (TSSaRNAs) were found to be transcribed in flanking regions of active promoters and may contribute to maintain a transcription-active open chromatin state in promoter regions of protein-coding genes [33]. 50 capped promoter associated small RNAs (PASRs) and 50 capped promoter associated large RNAs (PALRs) were identified to be short and large promoter associated RNAs; whereas PALRs seem to be the precursor molecules for the PASR, involved in regulation of cellular metabolism [34]. Transcription initiation RNAs (tiRNAs) are 18nt long ncRNAs generated from sequences immediately downstream of transcription start sites in animals. They were found to be generally associated with GC rich promoters, highly expressed transcripts and sites of RNAPII binding [35,36]. TiRNA biogenesis is dependent on RNAPII backtracking and transcription factor IIS (TFIIS) cleavage [33,37]. Splice-site RNAs (spliRNAs) show an average length between 17 and 18 nt, are associated with highly expressed genes and their 30 ends were found to map precisely to the splice donor site of internal exons [38]. Since spliRNAs expression differed in selected developmental stages and extremity, spliRNAs might be connected with distinct gene expression activities. Similar to tiRNAs, RNAPII pausing and backtracking due to the proximity of the downstream exon-associated nucleosome, followed by cleavage of TFIIS, generates this novel sRNA species. However, spliRNAs as byproducts of splicing or post transcriptional cleavage of longer capped RNAs are also discussed. Taft and colleagues propose that tiRNAs and spliRNAs might have evolved due to the intrinsic RNAPII backtracking capacity to mark positions of high transcriptional activity for future reference in parallel with transcription elongation [38]. Besides the aforementioned examples, deep

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sequencing approaches of various transcriptomes identified many more chromatin associated ncRNA species such as nuclear run-on RNAs (NRO-RNAs), promoter upstream transcripts (PROMPTs) [39], cryptic unstable transcripts (CUTs), stable unannotated transcripts (SUTs), 30 intergenic ncRNAs, transcripts of unknown function (TUFs), mitochondrial ncRNAs, satellite ncRNAs and repeat associated RNAs. However these novel types of ncRNAs have been subject to several reviews and interested readers are referred to [2,34,40]. 3. Small RNAs and the regulation of gene expression Small RNA-guided posttranscriptional regulation of gene expression is mainly covered by small RNAs (sRNAs) of 18e35 nt in size. In many organisms, the best studied representatives are miRNAs, siRNAs and piRNAs. More recently, ongoing research discovered new classes of sRNA such as endogenous siRNAs (endosiRNAs), mirtrons, miRNA-like sRNAs derived from longer ncRNAs (tRNAs or snoRNAs), short hairpin RNAs (shRNAs), small vault RNAs (svRNAs), microRNA-offset RNAs (moRs) and QDE2-interacting small RNAs (qiRNAs). Since research in terms of biogenesis and biological significance is mostly advanced in the field of miRNAs, other sRNA species are often classified based on their biogenesis in comparison to miRNAs (Fig. 1, see below). 3.1. miRNAs MiRNAs are the most well studied class of small non-coding RNAs with an average size of about 21e23 nt, regulating a huge amount of protein-coding and non-coding genes by posttranscriptional gene silencing in animals and plants. For example, it has been estimated that 30%e50% of all mRNAs are regulated by miRNAs (for review see [41e46]). MiRNAs are transcribed by RNAPII from independent genes or introns of protein coding genes, generating a 50 capped, spliced and polyadenylated stem-loop-structured primary miRNA (primiRNA). The RNase III enzyme Drosha and the double stranded

Fig. 1. Nucleases and enzymes involved in miRNA-like small RNA biogenesis. MiRNAs are generated through the subsequent processing by the RNase type III endonucleases Drosha and Dicer from precursors containing an imperfect hairpin RNA. Whereas Drosha is responsible for the generation of microRNA-offset RNAs (moRs), it is dispensable for the processing of (endo)siRNAs, mirtrons and miRNA-like sRNAs derived from snoRNAs (H/ACA sRNAs) or tRNAs (type I tsRNAs), short hairpin RNAs (shRNAs), and vault RNA (svRNAs). Endo-siRNAs in worms or plants and QDE-2 interacting small RNAs (qiRNAs) are produced through a combination of Dicer processing and the action of the RNA-dependent RNA polymerase (RdRP). PiRNAs are generated by the ping pong cycle, independent of Drosha and Dicer. C/D box snoRNAs derived small RNAs (C/D sRNAs) and type II tsRNAs are also produced independently by Ago2 and RNaseZ, respectively.

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RNA binding domain (dsRBD) protein DiGeorge syndrome critical region gene 8 (DGCR8, Pasha in Drosophila melanogaster) form the nuclear microprocessor complex that recognizes this stem loop structure and cleave at the stem of the hairpin, thus producing an approximately 70 nt stem loop miRNA precursor (pre-miRNA). Exportin-5 then transports the pre-miRNA to the cytoplasm, where a second RNase III enzyme termed Dicer generates a 21e25 nt long miRNA duplex intermediate with 50 phosphates and 2 nt 30 overhangs, referred to as miRNA/miRNA* (miRNA star). In all organisms analyzed so far, Dicer proteins function together with dsRBD protein partners. In Drosophila for example, Dcr1 is required for miRNA processing and cooperates with Loquacious (loqs). In human, Dicer interacts with TRBP (HIV transactivating response RNA-binding protein) and/or PACT (protein activator of dsRNA-activated protein kinase PKR). Following the thermodynamic asymmetry rule [47,48], one strand of the intermediate double stranded RNA, typically with a relatively lower stability of base pairing at the 50 end, is incorporated into a miRNA-protein complex (miRNP) by direct binding of the miRNA to a member of the Argonaute protein family, thus becoming the mature miRNA. The opposite strand, the miRNA*, is thought to be degraded; however there are also reports of functional miRNA* sequences, especially under distinct cellular conditions and in different tissues [49e53]. The incorporated mature miRNA then targets the miRNP to sequences within the target mRNA that are partially complementary to the miRNA. Sequence complementarity between the miRNA and the target site is often provided by the 50 proximal seed region of the miRNA (nt 2e8). MiRNA target interactions result in reduced mRNA stability or translation inhibition of the target mRNA. Mechanisms underlying translational repression are not yet clear, and research from different laboratories are quite contradictory. Studies from several labs suggested that miRNA-guided translational repression is based on preventing the circularization of mRNA, crucial for the onset of translation e however it has been shown that mRNAs lacking a polyA tail can still be targeted by miRNAs. Other models propose that miRNAs cause ribosomes to prematurely “drop off” the mRNA during ongoing translation or that the nascent polypeptide is cotranslationally degraded (for review see [42,43,46,54]). Destabilization of target mRNAs is much better understood, depending on a physical interaction between Argonaute and glycine-tryptophane protein of 182 kDa (GW182). In mammals, three different GW proteins exists which are referred to as TNRC6A-B. While the amino terminal part of GW proteins interact with Argonaute, the carboxyterminal part of GW proteins interact with the poly(A) binding protein (PABP) and recruits the deadenylases CCR4 and CAF1, components of the CCR-NOT deadenylase complex, required to the remove the poly(A) tail from mRNAs (for review see [42,43,55]). In many organisms, perfect complementation of the incorporated miRNA and target mRNA leads to cleavage of the target mRNA by endonucleolytically active Argonaute protein. In human, Ago2 is the catalytically active Ago protein and also referred to as Slicer (Fig. 2b) [56,57]. MiRNAs are regarded as key regulatory small molecules to ensure cell function and integrity. They can regulate crucial cell fate decisions and are thus implicated in biological processes ranging from apoptosis and cell signaling to organogenesis and development. Non-surprisingly, given the aforementioned importance in basic cellular mechanisms and the fact that one single miRNA can regulate tens to hundreds of genes, miRNAs are crucial for a correct function of the cell. Consequently, de-regulation of miRNA expression can lead to severe disorders including almost all types of cancer. Moreover, miRNA expression profiles very often correlate with disease status and can be used as novel biomarkers for diagnosis [58,59].

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Fig. 2. Biogenesis of endogenous and exogenous siRNAs, miRNAs and Mirtrons. A) Endogenous siRNAs (endo siRNAs) derive from transcriptional read through of inverted repeat sequences, or from intermolecular dsRNA precursors arising from transcription of either natural sense/antisense loci in the same (cis-nat siRNA) or different (trans-nat siRNA) position in the genome (gene/pseudogene) [91]. B) MiRNAs are transcribed as primary miRNAs. After Drosha cleavage, the precursor miRNAs (pre-miRNA) are transported to the cytoplasm by exportin 5 and undergo Dicer cleavage. The mature miRNA is loaded into the Argonaute ribonucleoprotein complex and depending on the degree of complementary between miRNA and target mRNA, the target mRNA is either cleaved and degraded (perfect complementary) or translationally repressed leading to either mRNA degradation or storage. C) Mirtrons derive from vertebrate short intron structures. After splicing, the pre-miRNA-like hairpin structures resemble the classical pre-miRNAs and are further processed as miRNAs. D) Exogenous dsRNA or artificial siRNAs are introduced into the cell and loaded into Argonaute RISC complexes, leading to subsequent mRNA cleavage and degradation.

3.2. Exogenous and endogenous short interfering RNAs (siRNAs, endo-siRNAs) The finding that RNAi is triggered by double stranded RNA [1] and the subsequent biochemical characterization of the short effector molecules, the siRNAs, led to the development of a powerful technique e and subsequently to the discovery of the

world of small RNAs. SiRNA processing from long double stranded RNA to 21 nt long siRNAs requires Dicer but is independent of Drosha. Similar to miRNAs, a short double stranded intermediate RNA is formed and one strand, referred to as the guide strand is incorporated into the Argonaute-containing RNA-induced silencing complex (RISC). Alternatively, siRNAs, the Dicer cleavage products, can be exogenously introduced into the cell. Since Dicer can use

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linear dsRNA or hairpin RNA (pre-miRNA-like RNAs) as substrates, DNA vectors expressing hairpin RNAs were developed for genespecific silencing. Perfect sequence complementary between the guide strand containing RISC and the target mRNA leads to endonucleolytic cleavage of the mRNA and subsequent degradation (Fig. 2d; for review see [41,45,60e62]). In addition to exogenously introduced siRNAs, endogenous siRNAs (endo-siRNAs) have been discovered in many species. EndosiRNAs are similar to miRNAs in their binding to the Ago subfamily proteins. However, they differ depending on their biogenesis pathway. In contrast to mammals and flies, where the production of endo-siRNAs appear to depend on Dicer (but not Drosha), worms (Caenorhabditis elegans) and plants (Arabidopsis thaliana) produce numerous endo-siRNAs with the help of Dicer in conjunction with RNA dependent RNA polymerases (RdRPs). In C. elegans, long dsRNAs are cleaved to primary siRNAs by the action of Dicer [63,64]. Association of the small RNA with an Argonaute family protein and the interaction with the target mRNA recruits an RdRP, using the target as template for de novo production of secondary siRNAs [65,66]. In A. thaliana, RdRPs together with specific Dicer family members convert single stranded RNA precursors to double stranded endo-siRNAs, resulting in the three major subclasses of endo-siRNAs known in plants: trans-acting siRNAs (ta-siRNAs) deriving from TAS1 or TAS3 non-coding RNAs, natural antisense transcript derived siRNAs (nat-siRNAs) originating from convergent transcription units and heterochromatic siRNAs (hc-siRNAs) deriving from repeats and transposable elements (for review see [67e70]). In flies and mammals, endo-siRNAs are mainly implicated in small RNA mediated silencing of transposable elements (TEs). Discovered in the 1940s by Barbara McClintock in maize, TEs are now known to be highly abundant in almost all genomes. In human, 45% of the genome consists of TEs [71]. Based on their structure and modes of integration, TEs are separated in two classes. Class 1 comprise retrotransposons and retrotransposonlike elements such as LINE and SINE, integrating via a copy and paste mechanism, whereas class 2 consists of DNA transposons moving directly via a cut and paste mechanism (for review see [72,73]). Although TEs can have beneficial roles for organisms, the mobilization of TEs is often considered to be crucial for cell fate because it might lead to chromatin deletions, duplications or inversions. However, organisms developed effective strategies to limit TE transposition. Among them, Argonaute-bound sRNAs play a pivotal role (for review see [74]). Whereas in the germline, PIWI/ piRNA complexes are responsible for the suppression of TE expression and mobilization (see below), in the fruit fly D. melanogaster non-gonadal somatic cells are protected from TEs by endosiRNAs. Similar to piRNAs (see below), endo-siRNAs can originate from TEs, heterochromatic regions, RNA transcripts with extensive hairpin structures or convergent transcription units forming a dsRNA [75e78]. Whereas miRNAs are produced by Dicer1 together with its dsRBD protein partner Loquacious (see above [79e81]), exogenous siRNA biogenesis is dependent on Dicer2 together with the dsRBD protein R2D2 [82,83], 21 nt long endosiRNAs are generated by a combination of Dicer2 in collaboration with an alternative form of Loquacious [84e86]. In mammals, endo-siRNAs were previously thought to be unlikely because the presence of long dsRNA precursors would trigger interferon response [87] within the cell. However interferon response does not occur in oocytes and embryonic stem cells [88,89], and indeed, the presence of endo-siRNAs was discovered in these cell types. Similar to flies, biogenesis of endo-siRNAs is dependent on Dicer [90e92], but not dependent on Drosha [91]. Whereas in oocytes, endo-siRNAs derived from hairpins and convergent transcription were identified [90,93,94], mouse

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embryonic stem cells are dominated by hairpin-derived endo-siRNAs [91,92]. Mouse embryonic stem cell-specific endo-siRNAs were mainly found to arise from hairpins produced from transcriptional read-through of inverted SINE retrotransposons. Interestingly, almost all of the identified endo-siRNAs mapped to the B1 Alu subclass [91,92], however, in oocytes, LINE and LTR retrotransposons are also sources for endo-siRNAs [90,93,94], even though endo-siRNAs produced from SINE elements were not as pronounced. In oocytes, endo-siRNAs are also produced from intermolecular dsRNA precursors as result of convergent transcription: cis-nat siRNAs deriving from transcription of natural antisense transcripts from the same loci, i.e. from an intermolecular dsRNA formed between oppositely oriented transcripts, and transnat siRNAs deriving from interchromosomal pairs of mRNAs and corresponding pseudogenes (Fig. 2a, [90,93,94]). The different kinds of endo-siRNA in mouse embryonic stem cells and oocytes suggest a tight regulation during transition from oocytes and blastocyst staged embryos. Since in Dicer knockout oocytes the expression level of certain retrotransposons was increased and the complementary endo-siRNA was decreased, the endo-siRNA pathway most likely, together with the piRNA pathway (see below) contributes to suppression of retrotransposition in these cell types [90,94]. 3.3. PIWI interacting RNAs (piRNAs) Another class of small RNAs comprises the germline-specific PIWI-interacting RNAs (piRNAs). PiRNAs are usually between 26 and 31 nt long, characterized by the presence of a 50 uridine and their association with PIWI proteins, the germline specific Argonaute subfamily (for review see [74,95e98]). In most organisms, mutations in PIWI lead to defects in controlling retrotransposition of transposable genomic elements in the germline [99e103]. How do PIWI proteins that are associated with piRNAs control retrotransposition? The biological mechanism behind this question is best understood in D. melanogaster (Fig. 3). In flies, piRNA clusters

repetitive elements, transposons piRNA cluster DNA

U

Maternal deposition, Cluster processing of primary piRNAs

3' 5' Aub/Piwi

3'

U A

5'

transposon / piRNA cluster transcript Ago3

U

5' 3' Aub/Piwi

secondary piRNA generation Ping-Pong model

3'

A 5' Ago3

Aub/Piwi 5'

3'

A 5'

3' transposon / piRNA cluster transcript

Ago3

Fig. 3. Ping-pong cycle for the generation of piRNAs and transposon repression. Maternally deposited antisense piRNAs or primary piRNAs derived from initial processing of piRNA clusters bound to Aubergine and Piwi proteins identify and cleave expressed transposon transcripts, resulting in new sense secondary piRNAs. Bound to Ago3, these newly generated piRNAs are targeting antisense transposon sequences and Ago3-mediated cleavage generates additional antisense piRNAs, leading to a feed forward amplification mechanism.

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lie within heterochromatin/euchromatin boundaries near the centromers, the most repeat-rich region of the genome containing ancient fragmented transposon copies [97]. PiRNAs arise from sequences that are antisense to transposons, consistent with recognition and silencing of transposon mRNA [102,104e106]. The PIWI protein family in Drosophila consists of three members: Piwi, Aubergine and Ago3. Interestingly, piRNAs bound to Piwi or Aubergine are antisense to transposon mRNAs, whereas the third Piwi protein Ago3 is loaded with piRNAs corresponding to sense transposon mRNAs sequences. A closer analysis of piRNAs targeting individual transposons revealed overlapping 50 ends separated by precisely 10 nt. Since Piwi proteins share the intrinsic property of Argonaute proteins to cleave a target 10 nt from the 50 end of the guide small RNA [104,107,108], and no Dicer dependency in generation of piRNAs could be observed [102] it was suggested that Piwi-mediated cleavage might be involved in the production of the 50 ends of sense and antisense piRNAs. All these observations led to the development of a model for a feed forward amplification mechanism, the ping-pong cycle, to explain the biogenesis of piRNAs [105,106]. Even though it is still unclear how the ping-pong cycle is initiated, it is widely accepted that transposon antisense piRNAs bound to Piwi and Aubergine associate with sense transposon transcripts and cleave 10 nt from the 50 end. A second cleavage event, accomplished by a so far unidentified nuclease creating the 30 end of the piRNA, releases a sense secondary piRNA, which associates with Ago3. Ago3 bound sense piRNAs subsequently then target antisense transposon transcripts and Ago3mediated cleavage, together with a second cleavage event generates antisense piRNAs, capable of both actively silencing the target and reinforcing the cycle by creating new/additional sense piRNAs. Like this, the ping-pong cycle not only produces piRNAs but also degrades transposable elements at the same time, representing a small RNA based immune system. Apparently, the piRNA generating and transposition regulating ping-pong cycle is conserved throughout evolution and can be also found in mouse [109,110] and most likely in human. Processing enzymes generating the initial antisense piRNAs are not identified yet, however since Aubergine and Piwi are maternally inherited, it is speculated that primary piRNAs initiating the cycle are supplied by maternally deposited piRNAs [111]. In mammals, about 17% of the piRNAs map to clusters of repetitive sequences, including SINEs, LINEs and DNA transposons throughout the genome [111e113], even though position but not sequence content is evolutionary conserved [97]. Besides silencing of transposable elements in the genome [111,112], they are also involved in epigenetics by regulating DNA methylation in mouse germ cells [110,114]. Transposons in mice are marked by DNA methylation and re-establishment of these marks is dependent on the mouse PIWI proteins Mili and Miwi2 [110,114], suggesting a role for piRNAs in modification and silencing of transposable elements, however the biochemical mechanism by which piRNAs, Piwi proteins and DNA or histone methylation are interconnected remains to be clarified. 3.4. Mirtrons Mirtrons, intron-encoded miRNA representing a subspecies of miRNAs, were firstly discovered in D. melanogaster [115,116] and soon thereafter also in mammals [91,117]. Even though mirtrons constitute 5e10% of miRNA genes in invertebrates [115,116] and vertebrates [117,118] they are expressed at much lower levels than typical canonical miRNAs. Mirtrons derive from vertebrate very short intron structures, ranging from 50e150 nt in size. Removal of introns located in pre-messenger RNA (pre-mRNA) transcripts and subsequent joining of exon structures is a crucial step in gene

expression and accomplished by the splicing machinery, a huge macromolecular complex consisting of over 200 individual proteins and 5 U-rich snRNAs (U1, U2, U4, U5 and U6 snRNA [24,119]). Introns are characterized by a 50 splice donor site (GU), a branch point located 20e50 nt upstream of the acceptor site and the 30 splice acceptor site (AG). The splicing process itself involves a twostep biochemical process: first, the 20 -OH of the branch point nt attacks the 50 splice site, resulting in a lariat intermediate. Then, the free 30 OH of the released 50 exon performs a nucleophilic attack on the splice acceptor site, thus joining the exons and releasing an intron lariat, characterized by an 20 e50 phosphodiester bond at the branch point. This structure is then cleaved by the lariat debranching enzyme and converted into a linear molecule, which is normally subject to degradation [119]. However, in the case of short introns, giving rise to mirtrons, these linearized structures adopt hairpin structures typical of pre-miRNAs. These mirtron-derived pre-miRNAs are exported into the cytoplasm by means of exportin 5, thus merging with the canonical miRNA biogenesis pathway (Fig. 2c). Further processing into miRNA/miRNA* duplexes is accomplished by Dicer, thus produces a Drosha independent but Dicer dependent mature miRNA. In 2010, a study from the Lai laboratory revealed a Drosophila mirtron-like locus where the 50 end is identical with the splice donor site, however, a tail separates the hairpin from the 30 splice acceptor site, representing a subclass of classical mirtrons, the tailed mirtrons. After splicing and debranching, an additional RNA exosome-mediated trimming event is required for the removal of these tails and the production of tailed-mirtron-derived miRNAs [120]. 3.5. H/ACA and C/D box snoRNA-derived small RNAs Small nucleolar RNAs (snoRNAs) are a highly evolutionary conserved class of non-coding RNAs concentrated in nucleoli where they function in either the modification of ribosomal RNA (rRNA) and U6 spliceosomal small nuclear RNA (snRNA) or participate in the processing of rRNA during ribosome subunit generation. Depending on sequence and function, two main classes of snoRNAs are known: C/D box snoRNAs and H/ACA box snoRNAs. Both kinds of RNA act in concert with a conserved subset of proteins in guiding enzymatic modification of target RNAs at sites determined by RNA:RNA antisense interactions. C/D box snoRNAs, together with the CD box snoRNP proteins NOP56, NOP58, 15.5 K and Fibrillarin, catalyze 20 O ribose methylation and H/ACA box snoRNAs, interact with GAR1, NHP2, NOP10, and Dyskerin to introduce pseudouridine modifications (for review see [18e20]). Recent bioinformatic analysis of small RNA libraries suggested that in addition to rRNA processing and site-specific modification of ncRNAs, snoRNAs can also give rise to miRNA-like sRNA species. The first snoRNA-derived sRNAs were described in the unicellular protozoan Giardia lamblia and in HEK293 T cells, the latter containing the ACA45 sRNA regulating CDC2L6 mRNA [21,121]. To date more and more groups revealed the existence of snoRNA-derived sRNAs that exhibit processing and mRNA silencing features similar to those of miRNAs [122e124]. Even more importantly, silencing activity of snoRNAderived sRNAs differs among cell types, a hallmark of regulation of gene expression by miRNAs [123,125]. But how are these snoRNAderived sRNAs generated? Analysis of deep sequencing data of sRNAs associated with human Ago1 und Ago2 together with secondary structure predictions of putative H/ACA snoRNA precursors revealed a characteristic secondary structure where two premiRNA-like hairpins are linked by a hinge and the identified snoRNA-derived sRNAs mostly derive from the 30 hairpin. Similar to endo-siRNAs and mirtrons the production of a functional H/ACAderived sRNA is independent of the Drosha microprocessor complex, but requires the action of Dicer (Fig. 4d, [21]). Box C/D

S. Röther, G. Meister / Biochimie 93 (2011) 1905e1915

A

B

C

D

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Fig. 4. Non-canonical pathways for the generation of miRNA-like small RNAs. A) Biogenesis of tRNA-derived small RNAs. Precursor tRNA transcripts are matured by consecutive action of RNaseP and RNaseZ, the latter releasing a type II tRNA-derived small RNA (type II tsRNA). After translocation of the mature tRNA to the cytoplasm, type I tsRNA are released by the action of Dicer [140]. B) microRNA-offset RNAs (moRs) are produced while canonical miRNA biogenesis of adjacent miRNAs by the action of the microprocessor complex [145,147]. C) Short hairpin RNAs (shRNAs) are directly transcribed as short hairpin stem loop structures and undergo Dicer maturation [91]. D) H/ACA snoRNA derived small RNAs (H/ACA sRNAs) are processed in dependence of Dicer [21,121], whereas C/D box derived small RNAs (C/D box sRNAs) are most likely processed by a different nuclease, maybe Argonaute [123].

snoRNAs on the contrary show a great variety of predicted secondary structures, including rather unstable hairpins, thus rendering a classical Dicer processing (which requires a stable RNA stem [126e128]) questionable. However, recent reports suggested a novel processing scheme for miRNAs in an Ago2-mediated, Dicer independent mechanism, which could also explain processing and miRNP loading of C/D box-derived sRNAs (Fig. 4d, [123]). Apparently, the endonuclease activity of mammalian Ago2 can cleave the 30 arm of pre-miR-451, thus forming an Ago2-cleaved precursor miRNA [129,130]. This view is furthermore supported by the finding that mass spectrometry analysis of Argonaute-associated proteins revealed a direct interaction with Nop56 and Fibrillarin e both of

which are subunits of the C/D Box snoRNP complex [131]. Further support for a Dicer-independent processing of C/D box-derived sRNAs comes from a systematical analysis of C/D box abundance in sRNA libraries from dicer1D/D and dgcr8D/D mouse embryonic stem cells [91]. Whereas classical miRNAs are approximately 100 fold and 20 fold less abundant in dicer1D/D and dgcr8D/D cells, respectively, C/D box-derived sRNAs are only mildly downregulated [124]. In stark contrast, H/ACA-derived sRNAs show pronounced responses to the loss of Dicer and to a lesser proportion to DGCR8 [124] e supporting the observation of the Meister lab for a Dicerdependent, but Drosha-independent maturation of the analyzed H/ACA-derived sRNA [21]. Apparently, H/ACA-derived sRNAs and

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C/D box-derived sRNAs use divergent biogenesis pathways, depending on the type of precursor snoRNA. Subcellular localization studies revealed nucleolar localization not only for the full length snoRNAs, but also for the small fragments deriving from them [38,122]. At a first glance this might be seen contradictory to the miRNA biogenesis pathway, however recent reports characterizing miRNAs revealed the existence of a nuclear [38,132] and even nucleolar localized subpopulation of mature miRNAs [133]. The fact that regulatory sRNAs can also emerge from other, functional snoRNAs is an exciting observation: apparently mammals developed even further extending regulatory mechanisms than previously thought. This finding is even more important, considering the presence of orphan snoRNAs in the genome that do not display antisense elements to any known rRNA or snRNA. Interestingly, the PradereWilli-Syndrome (PWS), a severe neurodevelopmental disorder, is characterized by the deletion of a paternally expressed C/D box-containing snoRNA cluster containing the orphan snoRNAs SNORD107/HBII-436, SNORD64/HBII13, SNORD108/HBII-437, SNORD116/HBII-85, SNORD115/HBII-52, SNORD109A/HBII-438A and SNORD109B/HBII-438B [134e136]. Interestingly, it has been shown that the MBII-52, the mouse homolog of HBII-52, is processed into smaller RNAs that regulate alternative splice-site selection, a process supposed to contribute to the etiology of this disease [22,23]. However, the finding that snoRNAs can produce sRNAs with miRNA-like functions also raise the possibility for a putative function of sRNAs derived from either type of these snoRNAs in the development of this disease. 3.6. tRNA-derived small RNAs (tsRNA) For a long time, some sRNA species found in deep sequencing libraries have been largely ignored as merely unimportant degradation products of abundant RNA species e especially since highly abundant RNAs undergo RNA modifications and were thus difficult to correctly align to the genomes. However, more detailed bioinformatic analysis started to reveal that sRNAs can also derive from tRNAs (tsRNAs), [91,137e140]. A study in 2010 by the Kay laboratory [140] divided this new class of sRNA in two subgroups: Dicer-dependent (type I tsRNA, also [91,137]) and Dicerindependent but tRNase Z-dependent (type II tsRNA, also [138]). tRNAs are transcribed by RNA polymerase III as precursors containing 50 leader and 30 trailer sequences that must be removed by processing and undergo a complex set of base modifications. Haussecker et al. [140] suggest that after RNA Polymerase III transcription, the 50 leader and 30 trailer are removed by RNase P and Z, respectively from the precursor tRNA. Cleavage by RNase Z thus releases the 30 trailer-contained type II tsRNA. After addition of the CCA on the 30 end of the tRNA, the mature tRNA is exported into the cytoplasm, where Dicer recognizes imperfectly base-paired tRNA stem structures, releasing a type I tsRNA (Fig. 4a, [140]). In accordance to this model, levels of type II tsRNA were not affected in Dicer knock down experiments [140] e as opposed to type I tsRNA [91,137,140]. Subcellular localization studies revealed that independently of the biogenesis pathway, type I and type II tsRNAs are present in the cytoplasm. Interestingly, enhanced RNA polymerase III transcription induced an upregulation of type II tsRNAs, with a concomitant downregulation of miRNAs [140]. This observation is in concordance with observations made in miRNA profiling experiments in various cancer tissues in comparison to normal tissues: miRNA abundance is decreased [141], but an increase in tRNA transcription rates [142] can be observed. Similarly, loss of DGCR8 or Dicer in mouse embryonic stem cells results in decreased miRNA levels, but is accompanied by increase in tsRNAs [91]. Apparently, competition between classes of small RNA depends on

the physiologic state of the cell and tsRNAs are able to compete with miRNAs for Ago, thereby regulating abundance of miRNPs and miRNAs. 3.7. Short hairpin RNAs (shRNAs), small vault RNAs (svRNAs), microRNA-offset RNAs (moR) and QDE-2-interacting small RNAs (qiRNAs) Short hairpin RNAs (shRNAs) derive from intergenic regions and were identified in mouse embryonic stem cells [91] and embryonic tissue [143]. Similar to exogenously introduced RNAs used in experimental knock down approaches, shRNAs are directly transcribed as short hairpin RNAs containing a mismatched stem loop structure and undergo Drosha-independent, but Dicer-dependent maturation [91,143]. Interestingly, one of the short hairpin loci corresponds to the tRNA Ile gene, which has the intrinsic potential to form an alternative secondary structure (cloverleaf and hairpin) and can therefore give rise to a either a mature tRNA or a sRNA. Similar to tailed mirtrons, tailed shRNA were identified [91]. These subspecies form two hairpins, however, only the second hairpin produced small RNA fragments; suggesting that an unknown nuclease removes the first hairpin up to the 50 end to produce a suitable Dicer substrate (Fig. 4c). Vault particles, conserved organelles implicated in multidrug resistance and intracellular transport in human, contain among different proteins non-coding vault RNAs (vRNAs). These ncRNAs can undergo a Drosha-independent but Dicer-mediated cleavage e before association with Ago proteins and formation with functional miRNPs, downregulating for example CYP3A4, a key enzyme in drug metabolism [144]. MoRs are highly conserved small RNAs derived from sequences immediately adjacent to mature miRNA/miRNA* loci. In most cases, moRs are located within the predicted pri-miRNA hairpin, they are 19e20 nt in length, developmentally regulated and appear to be produced by RNase III-like processing from the primary miRNA hairpin. Firstly discovered in the tunicate Ciano intestinalis [145], putative moRs were also identified in D. melanogaster, murine embryonic stem cells and human, even though in low numbers [91,146,147]. MoRs are most likely processed by Drosha while processing the pri-miRNA transcript (Fig. 4b, [145]). Consistently, mouse embryonic stem cells lacking Dicer show enriched levels of moRs, which are lost upon disruption of Drosha activity [91]. Interestingly, miRNA families with offset RNAs are significantly overrepresented among old families of animal miRNAs, indicating that moRs form a distinct functional class of miRNA-like small RNAs. QDE-2-interacting small RNAs (qiRNAs) were identified in the filamentous fungus Neurospora crassa and represent a new type of sRNA induced by DNA damage [148]. QiRNAs have an average length of about 20e21 nt with a strong preference for 50 uridine and originate mostly from the ribosomal DNA locus. DNA damage induces the production of pre-siRNAs which are subsequently matured/converted into qiRNAs by consecutive action of a RdRP (QDE-1), a helicase (QDE-3) and dicer proteins. It is believed that qiRNAs function in DNA damage response by interfering with translation. 4. Perspective Considering the complex relationships between small RNAs, the RNA silencing machinery and their impact on the stable maintenance of the cell, it is evident that identification and characterization of all small RNAs involved in regulatory processes and the discovery of so far unknown modes of regulation by small RNAs is of high biological impact. Many laboratories demonstrate the

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