Steroid receptor RNA activator: Biologic function and role in disease

Clinica Chimica Acta 459 (2016) 137–146

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Review

Steroid receptor RNA activator: Biologic function and role in disease Chan Liu a,b,1, Hong-Tao Wu c,1, Neng Zhu d, Ya-Ning Shi a, Zheng Liu a,b, Bao-Xue Ao a,b, Duan-Fang Liao a, Xi-Long Zheng a,e, Li Qin a,⁎ a

School of Pharmacy, Hunan University of Chinese Medicine, Changsha, Hunan, China Institute of Pharmacy and Pharmacology, University of South China, Hengyang, Hunan, China The Second Xiangya Hospital, Central South University, Changsha, China d The First Hospital of Hunan University of Chinese Medicine, Changsha, China e Department of Biochemistry & Molecular Biology, Libin Cardiovascular Institute of Alberta, Cumming School of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada b c

a r t i c l e

i n f o

a b s t r a c t

Article history: Received 2 February 2016 Received in revised form 5 June 2016 Accepted 5 June 2016 Available online 06 June 2016

Steroid receptor RNA activator (SRA) is a type of long noncoding RNA (lncRNA) which coordinates the functions of various transcription factors, enhances steroid receptor-dependent gene expression, and also serves as a distinct scaffold. The novel, profound and expanded roles of SRA are emerging in critical aspects of coactivation of nuclear receptors (NRs). As a nuclear receptor coactivator, SRA can coactivate androgen receptor (AR), estrogen receptor α (ERα), ERβ, progesterone receptor (PR), glucocorticoid receptor (GR), thyroid hormone receptor and retinoic acid receptor (RAR). Although SRA is one of the least well-understood molecules, increasing studies have revealed that SRA plays a key role in both biological processes, such as myogenesis and steroidogenesis, and pathological changes, including obesity, cardiomyopathy, and tumorigenesis. Furthermore, the SRA-related signaling pathways, such as the mitogen-activated protein kinase (p38 MAPK), Notch and tumor necrosis factor α (TNFα) pathways, play critical roles in the pathogenesis of estrogen-dependent breast cancers. In addition, the most recent data demonstrates that SRA expression may serve as a new prognostic marker in patients with ER-positive breast cancer. Thus, elucidating the molecular mechanisms underlying SRA-mediated functions is important to develop proper novel strategies to target SRA in the diagnosis and treatment of human diseases. © 2016 Elsevier B.V. All rights reserved.

Keywords: SRA Long noncoding RNAs Nuclear receptor Transcriptional coactivator

Contents 1. 2. 3. 4. 5. 6.

Introduction . . . . . . . . . . . . . Structures of SRA . . . . . . . . . . . Protein partners of SRA . . . . . . . . RNA modifications on SRA . . . . . . . SRA may influence histone modifications Biological functions . . . . . . . . . . 6.1. Adipocyte differentiation . . . . 6.2. Myoblast differentiation . . . . 6.3. Proliferation and apoptosis . . . 6.4. Stem cell . . . . . . . . . . .

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Abbreviations: AF-2, activation function-2; AR, androgen receptor; C/EBPα, ccaat-enhancer-binding protein α; CTCF, CCCTC-binding factor; Ddx5, DEAD box protein 5; ERα, estrogen receptor α; GR, glucocorticoid receptor; Hes1, hairy and enhancer of split-1; HP1γ, heterochromatin protein 1γ; JNK, c-Jun N-terminal kinases; lncRNA, long noncoding RNA; MyoD, myogenic differentiation 1; NANOG, homeobox protein NANOG; NR, nuclear receptor(s); p38 MAPK, mitogen-activated protein kinase; PACT, protein kinase RNA activator; PCOS, polycystic ovary syndrome; PKR, protein kinase RNA-dependent; PPARγ, Peroxisome Proliferator-Activated Receptor gamma; PR, progesterone receptor; PRC2, polycomb repressive complex 2; preTCRα, pre T-cell antigen receptor alpha; Pus1p, pseudouridine synthesis protein; RAR, retinoic acid receptor; RBP-J, recombination signal-binding protein J; RRM, RNA recognition motif; SF-1, steroidogenic factor 1; SHARP, retinoid and thyroid hormone receptors (SMRT)/HDAC1-associated repressor protein; SLIRP, stem-loop loop-interacting RNA RNA-binding protein; SRA, Steroid receptor RNA activator; SRAP, steroid receptor RNA activator protein; SRC-1, steroid receptor coactivator-1; STR, stem-loop structure; TNFα, tumor necrosis factor α; TRBP, transactivation response RNA-binding protein; TrxG, trithorax group. ⁎ Corresponding author at: School of Pharmacy, Hunan University of Chinese Medicine, 300 Xueshi Road, Hanpu Science and Education District, Changsha, Hunan 410208, China. E-mail address: [email protected] (L. Qin). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.cca.2016.06.004 0009-8981/© 2016 Elsevier B.V. All rights reserved.

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6.5.

SRA acts as a nuclear receptor coregulator. . . . . . . . . . 6.5.1. SRA acts as a coactivator of ERs . . . . . . . . . . 6.5.2. SRA acts as a coregulator of progesterone receptor. . 6.5.3. SRA acts as a coactivator of glucocorticoid receptor . 6.5.4. SRA acts as a coactivator of retinoic acid receptor . . 6.5.5. SRA acts as a coactivator of thyroid hormone receptor 6.5.6. SRA acts as a coactivator of PPARγ . . . . . . . . . 7. SRA-related signaling pathways . . . . . . . . . . . . . . . . . 7.1. Nuclear receptor signaling pathway . . . . . . . . . . . . 7.2. MAPK pathway. . . . . . . . . . . . . . . . . . . . . . 7.3. Notch signaling pathway . . . . . . . . . . . . . . . . . 7.4. TNFα signaling pathway . . . . . . . . . . . . . . . . . 8. SRA and diseases . . . . . . . . . . . . . . . . . . . . . . . . 8.1. SRA and tumors . . . . . . . . . . . . . . . . . . . . . 8.2. SRA and obesity . . . . . . . . . . . . . . . . . . . . . 8.3. SRA and cardiovascular diseases . . . . . . . . . . . . . . 8.4. SRA and polycystic ovary syndrome . . . . . . . . . . . . 9. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction LncRNAs, initially discovered in the 1990s are more than 200 nucleotides in length. The expression of lncRNAs is usually low [1] and like transcriptional noise. In the past decade, an increasing number of noncoding RNAs with regulatory functions have been reported [2]. Increasing studies demonstrate that lncRNAs play a significant role in diverse important biological processes [3,4], including transcription, splicing [5], stem cell pluripotency, embryogenesis, translation, protein localization, maintenance of cellular structural integrity, and cell division. Particularly, lncRNAs act as scaffolds for binding proteins responsible for modifying chromatin and mediating their deposition at specific genomic locations [6,7], whereas small interfering noncoding RNAs are mostly non-functional [8,9]. SRA, a type of lncRNA, has a large number of isoforms, most of which share a central core region [10]. SRA can enhance the transcriptional activity of steroid receptors on target genes and serves as a distinct scaffold [11–13]. Vicent et al. found that depletion of SRA1 unsettles a repressive complex and brings together different proteins that required

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for the repressive function, thus supporting its role as a scaffold in breast cancer cells [14]. SRA1 is a bifunctional gene, actives as an RNA and encodes a conserved steroid receptor RNA activator protein (SRAP) [15, 16], both of which can modulate gene transcription in cell- and system-specific manners. Gain-loss-of-function experiments have demonstrated that the products of SRA1 play a critical role in altering breast cancer invasion and are indispensable for harmonious zebrafish heart development [17], whereas SRAP prevents the transcriptional regulatory activity of SRA1 gene by binding a specific SRA stem-loop [18]. Furthermore, some functional motifs, with predicted secondary structures are required for SRA1 function [19]. Hube and co-workers constructed four SRA mutants and revealed that the interaction between SRA1 and SRAP requires the stem-loop structure (STR) [18], which is important for SRA function [20]. However, McKay et al. did not reveal a specific interaction between SRAP and SRA both in vitro and in cultured cells [21]. Moreover, evidence suggests that SRA1 levels may affect some biological functions. For example, SRA1 is much higher in polycystic ovary syndrome (PCOS) women than those in the healthy population [22]. Nevertheless, mounting evidence shows that SRA plays diverse roles in both

Fig. 1. Historical view of SRA. SRA was first described in 1999 [28] as a noncoding RNA present in a complex with the steroid receptor coactivator-1 (SRC-1). In 2002, Lanz et al. [19] provided a solid foundation for a coherent model of SRA function. In 2003, Kawashima et al. [17] reported the endogenous SRAP for the first time in human breast cells. In 2010, Xu et al. [29] found multiple roles for the noncoding RNA SRA in the regulation of adipogenesis and insulin sensitivity. In 2012, Redfern et al. [30] reported three new SRA-binding partners. The first experimentally derived SRA secondary structure was discovered; this RNA substructure was found to affect all SRA coactivator functions [31]. Further research conducted in 2014 revealed the functions of SRA in the first SRA knock-out mouse [32].

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normal biological processes, such as myogenesis [18,23] and steroidogenesis [24], and pathological changes, including cardiomyopathy [16] and tumorigenesis [25–27] (Fig. 1). 2. Structures of SRA RNA substructures of SRA are important to its function as coactivators [19]. Therefore, exploring the structure of SRA is of great significance. Three SRA sequences (I, II and III) were previously described, which share a common central core sequence and have different 5′ and 3′ extremities [28]. In addition, the SRA core sequence is necessary for its function as a coactivator [28]. Lanz et al. identified six secondary structural motifs (STR-1, -9, -10, -7, -11 and -12) through mutation assays and genetic deletion analysis [19]. Novikova et al. defined the actual secondary structure of SRA by chemical and enzymatic approaches [31]. These SRA motifs are crucial for its coactivation [19]. Several STRs, including SRA-STR1 and SRA-STR7, contribute to the transactivation of nuclear receptors [19]. A uridine at position 206, which is located in the small hairpin structure STR5 in the conserved core sequence, is a key pseudouridylation target [33]. The N-terminal domains of ERα and AR lack an RNA-binding motif, yet directly bind to SRA [34]. Moreover, the RNA recognition motif (RRM) 3/RRM4 platform is vital for the formation of a stable complex with the H12–H13 region of SRA RNA [35]. In addition, a specific region containing the H12–H13 substructure of SRA RNA is sufficient to mediate the binding pattern [20]. These observations not only emphasize the functional importance of the structural characteristics of SRA but also indicate their potential roles in modulating the ability of SRA to interact with other molecules. In summary, these data provide a framework or identification of SRA ligands and the construction of a coherent molecular model that can be used in further investigations of the functions of SRA [19]. It would be of great interest to further propose the interactions between SRA and its binding partners, and find more binding domains in SRA secondary structure (Fig. 2). 3. Protein partners of SRA SRA can be recruited to DNA through interactions with proteins that bind either directly or indirectly to and serve as a natural organizer of protein–protein interactions [11,36]. To investigate its binding partners will greatly enhance our understanding of the functions of SRA. To date, the partners that have been found to bind directly to SRA are P72, pseudouridine synthesis protein (Pus1p), Pus3p, Peroxisome Proliferator-Activated Receptor gamma (PPARγ), steroidogenic factor 1 (SF-1), silencing mediator of retinoid and thyroid hormone receptors (SMRT)/HDAC1-associated repressor protein (SHARP), thyroid hormone receptors, stem-loop loop-interacting RNA RNA-binding protein (SLIRP), trithorax group (TrxG), polycomb repressive complex 2 (PRC2) and homeobox protein NANOG (NANOG) [37]. The partners that form a complex with SRA include AR, ERα, PR, P68, RAR, SRC-1, SRC-2, myogenic differentiation 1 (MyoD), CCCTC-binding factor (CTCF), DAX-1, Dicer, transactivation response RNA-binding protein (TRBP), protein kinase RNA activator (PACT), protein kinase RNA-

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dependent (PKR). In addition, the only PR was found to interact with SRA in yeast 2-hybrid assay [28]. The proteins that directly or indirectly bind to SRA are summarized in Fig. 3. The identification of additional binding partners requires more in-depth investigations in the future. All of the identified partners work together through an array of biological mechanisms, ranging from nuclear receptor coactivation to repression, to exert positive or negative effects on adipogenesis, myogenesis, gene insulation, adrenal gland gene regulation and pseudouridylation. SRA functions as a part of ribonucleoprotein complex containing SRC1 that is recruited by the steroid receptor to target genes in the presence of hormones [28]. This characteristic distinguishes SRA from the other coactivators. SHARP, an important regulator of various transcriptional processes, such as nuclear receptor-mediated responses and Notch-mediated transcriptional activation [38], was the first transcriptional repressor found to interact with SRA through its three RRMs [39]. SHARP also acts as a co-regulator of nuclear receptors and binds to SRA to modulate ER transactivation, and attenuates the steroid response of SRA and initiates repression by SMRT [39]. Arieti et al. discovered that the RNA structural context is crucial to the SHARP-SRA interaction [35]. For instance, this type of binding is dependent on both single- and double-stranded RNA sequences. In contrast, SRA has also been reported to have a repressive function through its interaction with the SLIRP [20]. SLIRP, which is mainly located in the mitochondria, is actively recruited to the promoters of nuclear receptors. Additionally, SLIRP overexpression represses a broad range of nuclear receptors including ER, glucocorticoid receptor, AR, PPAR, thyroid hormone receptor and vitamin D receptor, whereas siRNA-mediated SLIRP depletion may result in nuclear receptors reporter activation and endogenous target genes expression. The SLIRP-SRA association is thought to enhance the recruitment of the nuclear receptor corepressor promoter. In addition, SRA binds to SLIRP and regulates its downstream target genes, including TMEM65 [40], a mitochondrial inner-membrane protein [41]. Decreased SRA expression is associated with attenuated TMEM65 mRNA expression [40]. Furthermore, both SHARP and SLIRP can directly bind to STR7 of SRA via an intrinsic RNA-binding domain [20,39], whereas Pus1p binds directly to STR5 [12,33].The most recent study revealed that SRA/TrxG/PRC2 complexes interact with several transcription factors and activate or repress histone modifications, which are important for regulation of gene expression [37]. However, the specific binding sites of SRA with its partner molecules have not yet been fully elucidated (Table 1). STR5 specifically inhibits the ERα- and AR-dependent transactivation of target genes in steroidsensitive carcinoma cells [34]. For instance, STR5 may serve as a novel class of RNA inhibitor of ERα and AR signaling pathway by interfering with Pus1p-mediated SRA pseudouridylation [34]. SRA has also been shown to bind the DEAD-box protein p68/p72, which can promote amino-terminal activation and integrate SRC/p160-mediated activation function-2 (AF-2) coactivator functions [23]. The transactivation ability of p68/p72 is further enhanced by the recruitment of SRA to a group of genes regulated by two different types of transcription factors: the nuclear hormone receptors and MyoD [23]. Furthermore, SRA binds directly to the amino-terminal of PPARγ to regulate adipogenesis and enhance insulin sensitivity [29]. These findings support a general

Fig. 2. Primary structures of SRA. Three SRA sequences are identical in the “core” region of 687 bp, but divergent in length and sequence in 5′ and 3′ extremities. One sequence has been registered with the National Center for Biotechnology Information nucleotide database (AF092038). There are other human SRA cDNA sequences deposited in NCBI (e.g., NM_001035235.3, NM_001253764.1, NR_045586.1, NR_045587.1, AF293024.1, and DQ286291.1). The hSRA gene comprises five exons within a region of approximately 6.5 kb at 5q31.3. Introns and exons are represented by black lines and yellow boxes, respectively. Blue boxes represent untranslated exons while yellow represent coding exons. Two putative translation initiation codons in exon 2, leading to the translation of a protein of 224 or 236 amino acids, respectively, are indicated by vertical lines, and the proposed termination codon of the putative open reading frame is indicated by a star.

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Fig. 3. Interactions of SRA with known binding partners and their biological functions. The direct and indirect (dotted arrows) bindings of SRA to various proteins produce an array of consequences, ranging from nuclear receptor coactivation to repression, which exert positive and negative effects on adipogenesis, myogenesis, gene insulation, adrenal gland gene regulation and pseudouridylation. The proteins depicted in the same color participle in the similar biological process. SLIRP and SHARP both directly bind to STR7 of SRA; PUS1 and PUS3 directly bind to STR5.

function of SRA rather than its role as a steroid receptor coactivator. Further detection of interactions between SRA and its binding partners would be of great importance to figure out SRA function.

4. RNA modifications on SRA Pseudouridylation of SRA occurs at one mapped uridine, but other sites likely occur. It remains unclear whether these modifications may be in proximity to each other in the three-dimensional structure of SRA. In human SRA, at least one modification resides in a predicted stem-loop structure in the conserved “core” (exons 2–5) region, but other positions still wait for recognition. The mPus1p-dependent pseudouridylation of SRA represents an additional type of posttranscriptional modification of a NR-coactivator complex [12].

5. SRA may influence histone modifications Some individual lncRNAs have been shown to interact with either TrxG or PRC2 protein complexes, which deliver histone modifications associated respectively with transcriptionally active or inactive chromatin [3]. Wongtrakoongate et al. confirmed that SRA distinctively forms complexes with both TrxG and PRC2 by using the RNA pull-down assay [37]. So, SRA may be involved in the delivery of histone modifications associated with either activation or silencing of gene expression, and even deliver both. For example, some SRA binding sites in human pluripotent stem cells overlap with bivalent domains, which carry both kinds of histone modifications. Besides, SRA is related to H3K4 trimethylation [37]. In human pluripotent stem cell NTERA2, SRA binding sites are significantly enriched for H3K4 trimethylation. Also, SRA may undergo other modifications such as m6A and m5C, which are found in lncRNAs.

Table 1 Direct and indirect SRA RNA-interacting proteins. Protein

Interaction process

RNA-binding domain

Related signaling pathway

Reference

p72 Pus1p PPARγ Pus3p SF-1/NR5A1 SHARP SLIRP TrxG PRC2 NANOG TRα1/2-β1 AR CTCF DAX-1 Dicer ERα MyoD p68 PACT PKR PR RAR SRAP SRC-1 SRC-2/TIF2 TRBP

Direct interaction Direct interaction Direct interaction Direct interaction Direct interaction Direct interaction Direct interaction Direct interaction Direct interaction Direct interaction Direct interaction Complex formation Complex formation Complex formation Complex formation Complex formation Complex formation Complex formation Complex formation Complex formation Complex formation Complex formation Complex formation Complex formation Complex formation Complex formation

DEAD box None Amino-terminal None FTZ-F1 box-containing region RRM (x3) RRM None None None Unique, ssRNA affinity None None N3R domain None None DEAD box DEAD box None None None None RNP-2 motif None None None

None NR None NR None NR, Notch NR None None None Hormone Hormone None None NR MAPK, Hormone None None NR NR Hormone Hormone None None None NR

[23] [12] [29] [33] [24] [20,39] [20] [37] [37] [37] [13,28] [28] [42] [24] [30] [23] [23] [23,42] [30] [30] [28] [12] [18] [20,28] [42] [30]

For each protein, the table indicates whether the protein interacts with SRA through complex formation or direct binding and provides the respective RNA-binding domains, related signaling pathways, and references.

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6. Biological functions The structure features and molecular functions of SRA are gradually emerging, but little is known regarding the physiological roles of these coactivators. As previously noted, SRA and its coactivators play a significant role in the regulation of adipogenesis, lipid metabolism, glucose homeostasis and target gene expression. SRA was cloned as an AF1-dependent coactivator, which enhances the transcriptional activity of selected steroid receptors [28,43]. Because SRA exists in a ribonucleoprotein complex, it may serve as a scaffold to recruit coactivator proteins to target genes [11,36,44]. Xu et al. found four intersection genes associated with inflammation, including zinc finger protein 36, tolllike receptor 4, haptoglobin and N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1 that are negatively regulated by SRA [29]. The role of SRA as a coactivator is mediated through an RNA transcript rather than another peptide product [28]. However, the mechanism underlying transcriptional coactivation by SRA remains unclear. Consequently, the functional regions of SRA are not limited to a single, discrete domain, but rather comprise several sections distributed throughout the core sequence [19]. Almost 470 genes are regulated by hormones in a SRA-dependent manner, and 60% of these genes remain silent in the absence of hormones and are up-regulated by knocking down SRA in T47D breast cancer cells [14,40,45,46]. Foulds et al. did SRA knockdown in HeLa cervical or MCF-7 breast cancer cells and found the majority of genes were downregulated, suggesting SRA mainly functions as a coactivator [40]. Also, when SRA was knockdown in MDA-MB-231 breast cancer cells, these authors found invasive potential reduced. Caretti et al. did SRA knockdown in C2C12 cells and found genes which are important for muscle cell differentiation downregulated [23]. Finally, Yan et al. did SRA knockdown in MDA-MB-231 cells and found migration pathways affected [46]. SRA increases the estrogen-induced activity of both fulllength ER subtypes in different cell models [47,48]. Reduced SRA expression in MCF-7 cells [40] inhibits ERα-dependent activity [47]. Mouse mammary tumor virus-long terminal repeat-driven SRA expression in transgenic mice results in extensive epithelial hyperplasia. Elucidation of the underlying mechanism showed that SRA has proliferationpromoting activities that are involved in enhancing ER and PR activity [49]. Thus, SRA may have diverse biological functions such as anti-inflammatory functions in adipose tissue. 6.1. Adipocyte differentiation Adipogenesis is a complex process driven by the synergistic effect of numerous transcription factors and signaling molecules, including PPARγ [50], wingless proteins [51], and cell cycle regulatory proteins. SRA enhances adipogenesis and adipocyte functions through a variety of mechanisms, including the coactivation of PPARγ, the promotion of S-phase entry during mitotic clonal expansion, the phosphorylation of Akt/protein kinase B and forkhead box protein O1 in response to insulin, and the regulation of inflammatory gene expression and signal transduction in response to insulin and TNFα. The distinctive ability of SRA RNA to coactivate nuclear receptors and PPARγ [20] has led to a recent exploration of its role in adipocyte functions [29]. It was recently revealed that SRA is expressed at higher levels in adipose tissue than in other tissues [32]. The livers of SRA-knockout mice possess fewer lipid droplets after high-fat diet administration. Moreover, adipogenesis-associated and inflammatory-associated gene expression is decreased, whereas insulin sensitivity is markedly improved [32]. Insulin sensitivity or resistance constitutes a key step in the development of metabolic diseases. A previous study conducted by Liu et al. showed that SRA overexpression promotes adipogenesis partly through stimulation of insulin-like growth factor-1 signaling pathway [52]. P38 MAPK activation inhibits adipogenesis [53], and SRA overexpression also inhibits MAPK and c-Jun N-terminal kinases (JNK) phosphorylation in early ST2 mesenchymal precursor cell differentiation [52]. These findings suggest a pivotal role of SRA in the

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biological processes of adipose tissue in vivo [32]. Moreover, SRA also functions as a transcriptional coactivator of PPARγ which promotes adipocyte differentiation in vitro. Therefore, adipogenesis mediated by SRA is complicated, and SRA is likely a therapeutic target for the treatment of metabolic diseases. 6.2. Myoblast differentiation The SRA1 is expressed at its highest levels in skeletal muscle, heart and liver but at a lower level in the brain. Accordingly, SRA is capable of regulating muscle differentiation through MyoD, which is involved in skeletal myogenesis. Moreover, SRA can coactivate MyoD-dependent transcription, which is required for skeletal muscle cell differentiation [23]. To confirm the ability of individual SRA in enhancing MyoD transcriptional activity in vivo, Hube et al. performed gain-loss-of-function experiments on SRA. As a result, the conversion of MyoD-dependent myogenic from non-muscle cells is enhanced by SRA [18]. Consistent with the above findings, SRA is associated with endogenous p68 or MyoD in skeletal muscle cells by differentiated myotubes experiment. Further investigations identified that the SRAP could prevent SRA RNA-mediated activation of MyoD, thus the ratio between noncoding and coding SRA isoform is key for cells to differentiate into muscle fibers [18]. It is well known that activation of p38 MAPK is essential for the initiation of myogenic differentiation in myoblasts and embryo [54], while overexpression of SRA inhibits MAPK and p38 phosphorylation [52]. In summary, evidence suggests a crucial role of SRA RNA in the control of myoblast differentiation. 6.3. Proliferation and apoptosis As previously reported SRA coactivates steroid-dependent transcription and then generates a proliferative response. The proliferative feature of SRA is closely related to NR signaling coactivation. Through a BrdU immunohistochemistry assay on abdominal mammary gland sections, SRA was found to promote mitotic and apoptotic activities. Although transgenic mice that overexpress SRA display increased proliferation, these mice did not show any increase in tumor incidence, suggesting that SRA overexpression alone was insufficient to induce tumorigenesis. The SRA also affects proliferation and apoptosis in vivo, and SRA depletion compromises hormone-dependent proliferation and inhibits apoptosis in T47D breast cancer cells [14,49]. 6.4. Stem cell Dax1 is a transcriptional repressor that binds to the NR SF-1 and inhibits SF-1 dependent steroidogenic gene transcription. A previous study revealed that Dax1 overexpression caused mES cell differentiation [55]. RNA-immunoprecipitation experiments using a Myc-tagged Dax1 revealed a significant enrichment of SRA on Dax1 [56], showing that Dax1 likely interacts with SRA in mES cells. The interaction of Dax1 with SRA in steroidogenic cells switches the role of Dax1 to an SF-1 coactivator in a dose-dependent manner [24]. How SRA exactly coactivates steroid receptors remains to be elucidated. Most recently, Wongtrakoongate et al. showed that SRA is crucial for maintaining the stem cell state and helping human fibroblasts to achieve the pluripotent state [37]. 6.5. SRA acts as a nuclear receptor coregulator SRA is not only an RNA molecule but also a coactivator that can selectively enhance the AF-1 activity of class I nuclear receptors AR, ERα, PR, and GR. Parallel results have demonstrated that SRA coactivates a range of nuclear receptors, including ERα, ERβ, PR and GR, in a ligand-dependent manner by directly interacting with other co-regulatory proteins, such as SRC-1, SHARP, and SLIRP [20,28,39,43]. Thus, SRA acts as an RNA steroid receptor coactivator. Additionally, SRA is an AF-1-

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dependent coactivator, thereby SRA mediates transactivation via the activation function of its amino terminus [28] and enhances the transcriptional activity of selected steroid receptors [28]. Meanwhile, SRA was reported to affect the activity of non-steroid receptors such as thyroid hormone receptor and RAR [12,13].

SRA functions as a thyroid hormone receptor coactivator via direct physical interaction [12,13], thus enhances thyroid hormone receptor function. The SRA binding domain of thyroid hormone receptor is a 41-amino-acid region located between the second zinc finger and the ligand-binding domain.

6.5.1. SRA acts as a coactivator of ERs SRA potentiates the estrogen-induced transcriptional activity of both ERα and ERβ. Because SRA was shown to be part of a large complex containing SRC-1 [28], Deblois and Giguère then showed that SRC-1 and SRA co-expression can increase the effect of SRA on ERα-mediated transactivation [43]. SRA may be recruited to ERs via SRC-1, which interacts with AF-2 of the receptor in a ligand-dependent manner [28]. Moreover, SRA mediates ERα coactivation via multiple molecular mechanisms. Most significantly, SRA augments ERα activity through MAPK signaling and an intact serine residue at position 118 phosphorylation in ERα via receptor AF-1 activity [43]. The related signaling pathways of SRA play a key role in the pathogenic process of estrogen-dependent breast cancer [28].

6.5.6. SRA acts as a coactivator of PPARγ PPARγ is a master transcriptional regulator of adipogenesis [50,57]. Xu et al. revealed that SRA binds to PPARγ in 3T3-L1 adipocytes, and enhances PPARγ transcriptional activity [29]. Thiazolidinediones are adipogenic ligands for PPARγ that induce the expression of a multitude of genes in adipocytes [60]. Like Thiazolidinediones, SRA may facilitate the increased expression of PPARγ, ccaat-enhancer-binding protein α (C/EBPα), and other adipocyte genes. More recently, Liu et al. illustrated that SRA binds to PPARγ to coactivate PPARγ-dependent reporter gene expression in a ligand-dependent manner, which improves insulinstimulated glucose uptake in adipocytes in vitro [52].

6.5.2. SRA acts as a coregulator of progesterone receptor SRA was originally identified in a human B-lymphocyte library using PR AF-1 as the bait in a yeast two-hybrid assay [28]. Through in vitro binding assays, researchers identified the direct interaction of PR with SRA [14]; SRA coactivates PR in an open reading frame-independent manner [28]. SRA is involved in the regulation of hormone-dependent genes. In breast cancer cells, PR binds to genomic sites and then targets a novel and repressive complex containing heterochromatin protein 1γ (HP1γ), lysine-specific demethylase 1, HDAC1/2 and SRA, among other factors, to 20% of hormone-inducible genes, supporting the role of SRA as a scaffold. Vicent et al. reported that the unliganded PR, SRA and HP1γ protein participate in properly anchoring the repressive complex to chromatin [14]. Moreover, the unliganded PR SRA silences a subset of hormone-inducible genes by mediating this RNA-containing repressive complex. These results indicate that coactivation of SRA and unliganded PR play a vital role in hormone-regulated breast cancer cells.

Recent studies have elucidated the activities and effects of SRA, and also revealed its critical roles in various signal transduction pathways. The in-depth understanding of these signaling molecular pathways related to SRA may help develop new types of disease therapeutics (Fig. 4).

6.5.3. SRA acts as a coactivator of glucocorticoid receptor GR plays a pivotal role in regulating early adipogenesis [57]. SRA coactivates GR as part of a ribonucleoprotein complex with p160 coactivators, rather than through direct physical interaction. In addition, SRA promotes GR-mediated transactivation in a ligand-dependent manner [28]. Global knockout of SRA protects against diet-induced obesity and improves whole-body glucose homeostasis in animals [32]. Consistent with this finding, SMRT regulates the metabolic functions of GR in adipocytes in vivo. Modulating GR-SMRT interactions in adipocytes represents a novel approach to regulate glucocorticoid actions and affecting the metabolic function of adipocytes [58]. 6.5.4. SRA acts as a coactivator of retinoic acid receptor RARs are members of the NR superfamily and represent a type of transcriptional modulator [59]. mPus1p binds to the first zinc finger of mRAR, the association between mPus1p/RARγ and mPus1p/SRA has been confirmed in vitro [12]. The mPus1p/mRARγ/SRA complex has been observed in the nucleus as a retinoid-independent, promoter-bound complex [12]. In addition, SRA has been shown to be a substrate of mPus1p. Accordingly, SRA acts as a coactivator of mPus1p. Furthermore, SRA and mPus1p, when co-expressed, can cooperatively enhance mRAR-mediated transcription [12]. The mPus1p-dependent pseudouridylation of SRA constitutes one posttranscriptional modification of an NR-coactivator complex that is involved in NR signaling. 6.5.5. SRA acts as a coactivator of thyroid hormone receptor SRA plays a crucial role in brain development, differentiation and metabolic homeostasis via binding to nuclear thyroid hormone receptors. Xu et al. performed cotransfection experiments and showed that

7. SRA-related signaling pathways

7.1. Nuclear receptor signaling pathway The interactions between SRA and various proteins play a pivotal role in NR activities [11,36,44], but the molecular mechanisms and biological functions of these interactions remain unclear. The SHARP/SRA complex is thought to regulate ER transactivation partially when SRA is separated from the complex [39]. SRA is an ERα AF-1-specific coactivator that can also associate with the N-terminal domain of AR and ER [48]. STR5, a new class of RNA inhibitor, reduces steroid receptor signaling in hormone-sensitive cancer cells by preventing Pus1p-dependent pseudouridylation and SRA activation [34]. Additionally, SRA pseudouridylated by Pus3p adjusts a functional switch that regulates NR signaling. The ligand-activated signaling pathway induced by nuclear receptors plays a key role in the promotion of human tumorigenesis. 7.2. MAPK pathway JNK and MAPK family members integrate signals that affect proliferation, differentiation, survival and migration [61]. An intact serine residue at position 118 in ERα AF-1 is a target of MAPK-induced phosphorylation in the absence of ligand binding [62]. In addition, SRA enhances the MAPK-mediated activation of ERα AF-1 [43], which requires the integrity of an intact serine residue at position 118 in ERα. Phosphorylation of an intact serine residue at position 118 may help recruit the helices of p68 RNA. Aberrant MAPK activation has been reported in human breast tumors [63], and the MAPK pathway promotes ERα AF-1 activity in breast tumors [63]. Recent findings have revealed that the inflammatory cytokine TNFα is a key activator of p38 MAPK during myogenesis in an autocrine/paracrine manner [64]. 7.3. Notch signaling pathway The Notch signaling pathway belongs to an evolutionarily conserved signal transduction system and plays critical roles during development through regulation of cell proliferation, differentiation, and apoptosis, and many other processes. SRA and DEAD box protein 5 (Ddx5) function together as coactivators of Notch signaling pathway [65]. Ddx5 localizes to recombination signal-binding protein J (RBP-J)-binding sites within the Notch target genes pre T-cell antigen receptor alpha (preTCRα), hairy and enhancer of split-1 (Hes1) and CD25. SRA

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Fig. 4. Signaling pathways involving SRA. SRA increases PPARγ expression and induces a multitude of adipocyte PPARγ target genes, including ERK1/2. The overexpression of SRA inhibits p38 phosphorylation. Activated p38 further activates NF-κB, which induces cell apoptosis. In addition, the interaction of SRA with insulin receptor increases the phosphorylation of Akt and forkhead box protein O1, which is a downstream target of Akt signaling, and subsequently activates Fas. Furthermore, activated Fas promotes adipogenesis. In contrast, the absence of JNK1 enhances insulin receptor signaling and adipocyte differentiation. Activated JNK1 then activates c-Jun. Moreover, SRA opposes TNFα signaling and inhibits TNFα-induced JNK phosphorylation.

decreases preTCRα and Hes1 expression. Many studies have demonstrated the role of SHARP in NR-mediated transcriptional responses and a novel component of the Notch/RBP-Jκ signaling pathway [38,39]. 7.4. TNFα signaling pathway TNFα is a major inflammatory factor that is induced in response to injury and contributes to the normal regulatory processes of bone resorption [66]. In addition, TNFα may lead to new therapies to augment recovery and reduce the incidence of complications in fracture healing [67]. Pro-inflammatory cytokines, including TNFα, are produced in adipose tissue [68]. TNFα can activate JNK, which is a negative regulator of insulin signaling, and cause insulin resistance [69,70]. A microarray analysis revealed hundreds of SRA-responsive genes in adipocytes, including genes involved in the cell cycle, and the insulin and TNFα signaling pathways [29]. SRA overexpression promotes adipocyte precursor ST2 cell differentiation [29]. Consistent with these findings, the results from knockdown of endogenous SRA in mature 3T3-L1 cells also suggest that SRA increases insulin sensitivity, and inhibits TNFα signaling [29]. 8. SRA and diseases In recent years, with the development of genomics and bioinformatics, especially high-throughput sequencing technologies, researchers found that a growing number of diseases are associated with SRA mutation or abnormal expression. For example, SRA is up-regulated in tumors of steroid hormone responsive tissues, such as breast, uterus, and ovary compared with matched normal tissue [27]. The therapeutic

drug targeting of transcription factors has been proposed as a frontier in medicine, whereas SRA can modulate the activity of transcription factors [71]. Cooper et al. confirmed that antisense oligonucleotides targeting SRA work well in transfected cells, such as T5 breast cancer cells [26]. Therefore, the SRA-related diseases must be elucidated. 8.1. SRA and tumors Cancer remains a major cause of human morbidity and mortality due to the lack of effective therapies. SRA plays an important role in nuclear receptor-mediated, hormone-dependent cancers [11,44]. Previous studies have shown that ERβ mRNA expression is reduced in tumor tissues compared with normal tissues in various estrogen-regulated cancers, particularly ovarian, breast and prostate cancers [72–74], suggesting that the loss of ERβ expression may be involved in carcinogenesis. SRA is expressed in both normal and malignant human mammary tissues [27], and its expression varies during breast tumor progression [75]. Elevated SRA levels have been found in breast cancer compared with those in adjacent normal tissues [49]. Reduced ER mRNA expression has also been found in tumor tissues in various estrogen-dependent cancers, such as ovarian, breast, and prostate cancers, suggesting that the loss of ER expression is closely related to carcinogenesis. SRA transcripts may be involved in breast tumorigenesis and tumor progression through regulation of the expression of specific genes [26,27,40,46]. Researchers suggest that depletion of SRA decreases MDA-MB-231 breast cancer cell migration and invasion [40, 46]. Mammary gland development is critically sensitive to steroid hormones, and transcriptional activity in mammary glands is regulated by

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coactivators and corepressors. SRA is thought to participate in breast carcinogenesis via the aberrant stimulation of ER activity [19,27]. Moreover, SRAP physically interacts with multiple transcription factors, including ERα and ERβ, and the resulting complexes are recruited to specific promoter regions to reduce transcription. SRAP may be involved in tumorigenesis and tumor progression [25]. Yan et al. clarify that SRAP, not RNA, may promote cancer cell migration [46]. Therefore, SRAP can affect patients' survival rates [76]. Hube et al. also found that SRAP overexpression represses ER activity in breast cancer [10]. Breast cancer patients with high SRA expression have a significantly worse survival rate than those with low SRA expression. Therefore, SRA expression may serve as a new prognostic marker in patients with ERpositive breast cancers [76]. Importantly, the ratio of SRA to SRAP expression differs in breast cancer tumor cells, and better outcomes are obtained in patients with SRAP-overexpressing tumors [77]. These observations indicate that SRA RNA is a promising target for cancer therapy [27,49,75]. Additionally, SRC-3 overexpression is associated with tamoxifen resistance in breast cancer patients. However, no significant differences in the relative expression of SRA have been found between tamoxifen-sensitive patients and de novo tamoxifen-resistant patients [78]. Increasing evidence indicates that estrogens promote ovarian carcinogenesis through their receptors (ERα and ERβ) and thus play a role in the pathogenesis of ovarian cancer [79,80]. The well-characterized SRC family, which is also known as the p160 family of coactivators, is potentially a paramount determinant of ovarian cells in response to estrogens. SRA was shown to coactivate AR activity in prostate cell lines in which the SRAP peptide has been deleted through the use of constructs expressing either the full-length protein or the amino terminus alone [17]. Additionally, continuous AR activation with androgen ablation and anti-androgen therapy appears to play a pivotal role in the hormone-refractory progression of prostate cancer. Further studies are required to reveal the association between hSRA and hormoneindependent prostate cancer progression. 8.2. SRA and obesity Chronic tissue inflammation is a crucial cause of obesity-induced insulin resistance [81]. Evidence suggests that inhibition of the inflammatory signaling pathway may contribute to the enhancement of insulin sensitivity in SRA-knockout mice [32]. SRA is expressed at higher levels in adipose tissue than other organs. Liu et al. found that SRA may improve insulin sensitivity in an animal model fed a high-fat diet. The data indicates that SRA is an important regulator of adiposity in diet-induced obesity. The use of a tissue-specific knockout animal model in future research may help clarify the underlying mechanisms associated with insulin sensitivity and likely shed further light on the biological functions of SRA as a potential target for the prevention and treatment of obesity and type 2 diabetes [32]. 8.3. SRA and cardiovascular diseases Human dilated cardiomyopathy is a myocardial disease characterized by dilatation and impaired systolic function of the ventricles that leads to heart failure [82]. The genetic characteristics of human dilated cardiomyopathy involve heterogeneous, monogenic and multifactorial factors, which contribute to these diseases. Increasing evidence suggests that inflammation and autoimmunity may play a role in this idiopathic disease. Cardiac function in zebrafish is impaired when SRA expression is reduced through the use of morpholino antisense reagents [16]. 8.4. SRA and polycystic ovary syndrome PCOS has been recognized as a common reproductive and endocrine disorder [83]. Compelling evidence indicates that SRA affects multiple biological processes such as steroid receptor signaling, steroidogenesis,

adipogenesis and insulin sensitivity [32,52,84], and all of these functions may be involved in the pathogenesis of PCOS. SRA expression is markedly increased in women with PCOS. Jakimiuk et al. [85] reported that the ERβ mRNA and protein levels are lower in both granulosa and theca cells from PCOS follicles than in those from control follicles and that SRA can affect ERβ. Thus, it was speculated that SRA may play an important role in the pathogenesis of PCOS [86]. Furthermore, obesity is positively associated with the high SRA expression detected in women with PCOS [87]. Taken together, these results suggest that SRA expression is potentially associated with PCOS and correlated with obesity in PCOS [22]. 9. Perspectives Since SRA was first defined as an lncRNA coactivator of steroid receptors, the functions of SRA have been extended beyond NR signaling. In fact, SRA is now known to be associated with several diseases, including obesity, cardiovascular diseases, PCOS, and breast cancer. Furthermore, studies of SRA-binding co-regulatory proteins have provided important insights into the mechanisms of SRA effects and new opportunities for targeting the NR signaling pathways as therapeutic strategies. Most importantly, the biological functions of SRA may serve as potential targets to control obesity and type 2 diabetes, once the molecular mechanisms underlying the association between insulin sensitivity and SRA are better understood [32]. Moreover, investigations of the inflammatory genetic background in human dilated cardiomyopathy patients represent a promising means for identifying new susceptibility genes and providing new insights into the pathogenesis of the disease. However, the interactions between SRA and its binding partners have not been fully elucidated. Identifying additional binding domains in the secondary structures that specifically interact with certain partners is of great interest, and verifying the mechanism through which SRA serves as a scaffold is crucial. Conflicts of interest The authors declare no conflict of interest. Acknowledgements The authors gratefully acknowledge the financial support from the National Natural Sciences Foundation of China (No. 81000946, No. 81270359, and No. 81173047), key projects of Hunan Provincial Education Department (No. 15A138) and the Natural Science Foundation of Guangxi Province (No. 2015GXNSFEA139003). References [1] T. Derrien, R. Johnson, G. Bussotti, et al., The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression, Genome Res. 22 (2012) 1775–1789. [2] J.S. Mattick, I.V. Makunin, Small regulatory RNAs in mammals, Hum. Mol. Genet. 14 (2005) R121–R132 (Spec No 1). [3] J.L. Rinn, H.Y. Chang, Genome regulation by long noncoding RNAs, Annu. Rev. Biochem. 81 (2012) 145–166. [4] X. Wang, X. Song, C.K. Glass, M.G. Rosenfeld, The long arm of long noncoding RNAs: roles as sensors regulating gene transcriptional programs, Cold Spring Harb. Perspect. Biol. 3 (2011) a003756. [5] V. Tripathi, J.D. Ellis, Z. Shen, et al., The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation, Mol. Cell 39 (2010) 925–938. [6] J.T. Lee, Lessons from X-chromosome inactivation: long ncRNA as guides and tethers to the epigenome, Genes Dev. 23 (2009) 1831–1842. [7] D.P. Caley, R.C. Pink, D. Trujillano, D.R. Carter, Long noncoding RNAs, chromatin, and development, TheScientificWorldJournal 10 (2010) 90–102. [8] P.P. Amaral, M.B. Clark, D.K. Gascoigne, M.E. Dinger, J.S. Mattick, lncRNAdb: a reference database for long noncoding RNAs, Nucleic Acids Res. 39 (2011) D146–D151. [9] C.A. Brosnan, O. Voinnet, The long and the short of noncoding RNAs, Curr. Opin. Cell Biol. 21 (2009) 416–425. [10] F. Hube, J. Guo, S. Chooniedass-Kothari, et al., Alternative splicing of the first intron of the steroid receptor RNA activator (SRA) participates in the generation of coding

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