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Steroid Receptor RNA Activator – A nuclear receptor coregulator with multiple partners: Insights and challenges
Biochimie 93 (2011) 1966e1972
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Biochimie journal homepage: www.elsevier.com/locate/biochi
Mini-review
Steroid Receptor RNA Activator e A nuclear receptor coregulator with multiple partners: Insights and challenges Shane M. Colley a, Peter J. Leedman a, b, * a b
Laboratory for Cancer Medicine, University of Western Australia Centre for Medical Research, Western Australian Institute for Medical Research, Perth, WA 6000, Australia School of Medicine and Pharmacology, University of Western Australia, Perth, WA 6009, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 May 2011 Accepted 4 July 2011 Available online 27 July 2011
Steroid Receptor RNA Activator (SRA) occupies a unique and enigmatic position within the nuclear receptor (NR) field and more broadly in transcriptional regulation. This is as a result of its transcripts having both coding and non-coding coactivator activities along with its protein product SRAP performing mixed coactivator/repressor functions. Recent publications have provided greater understanding of SRA gene product activities and how they affect not only NR function, but now more broadly, signalling pathways involved in differentiation and metabolism. This review will discuss the isolation of SRA, its gene products, regulation of transcription along with its in vitro and in vivo activities with a particular focus on its actions as an RNA and its binding partners. Ó 2011 Published by Elsevier Masson SAS.
Keywords: SRA Nuclear receptor coregulator SLIRP SRAP Hormone action Cancer Metabolism
1. Isolation of SRA The activities of the NR superfamily of ligand-inducible transcription factors are instrumental in the regulation of reproduction, development and metabolism of mammalian and other species [1]. The transcriptional activity of NRs is modulated by an even larger group of functionally diverse molecules that either augment (coactivators) or reduce (corepressors) the actions of the NRs [2]. The great majority of these coregulators are proteins, however, while screening for novel modulators of the Progesterone Receptor (PR), the RNA coactivator Steroid Receptor RNA Activator (SRA) was uncovered [3]. Curiously, in spite of a yeast two-hybrid assay being performed, the initial SRA clone isolated was found to function as a non-coding RNA (ncRNA) thereby identifying a novel mode of NR modulation. Subsequent studies have now found the products of the SRA gene include coding and ncRNAs along with the Steroid Receptor RNA Activator Protein (SRAP), each of which modulate gene transcription in a cell- and system-specific manner [4,5]. As SRAP is the subject of a comprehensive review in this issue [18], this
* Corresponding author. Laboratory for Cancer Medicine, University of Western Australia Centre for Medical Research, Western Australian Institute for Medical Research, Level 6, MRF Building, Rear 50 Murray St., Perth, WA 6000, Australia. Tel.: þ61 8 9224 0333; fax: þ61 8 9224 0322. E-mail address: [email protected] (P.J. Leedman). 0300-9084/$ e see front matter Ó 2011 Published by Elsevier Masson SAS. doi:10.1016/j.biochi.2011.07.004
article will focus on SRA gene products, their isoforms, prevalence and biological functions with a particular focus on the transcripts and interacting molecules.
2. The SRA gene and its products The SRA gene is well conserved between species [6] with human (5q31.3), rat (18p11) and mouse (18B2) homologues each having 5 exons. Studies by a number of groups have revealed the existence of multiple RNA isoforms transcribed from the SRA gene with the most recent contribution, involving EST data base searching, 50 RACE validation and sequencing, bringing the total up to 20 different types [7]. SRA transcripts may be placed in either coding or non-coding groups accounting for 39% and 61% of the clones, respectively. Coding transcripts, contain exons 1e5 within which are two methionine codons defining 236 and 224 amino acid (aa) SRAP proteins. Doublet proteins of approximately 31 and 32 kDa have been detected in human cancer cell lines [5,8] and breast cancer patient samples [9] confirming the utilisation of both initiating methionines. Three coding mRNA isoforms, SRA1e3, have been reported [5]. These contain essentially identical coding sequences except that SRA2 has a C / T (theonine / isoleucine) alteration at nt 338 and a silent A / C change at nt 348 (as listed in AF293025) while SRA3 has a combined substitution/insertion of G / CGAC at position 520 (AF293026) resulting in the insertion of
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an arginine and valine / leucine substitution at aas 109 and 110. The presence of these isoforms correlates with the genomic sequence of the tissues from which they were sourced indicating they represent individual alleles. SRA ncRNAs result from differential splicing of intron 1, the retention of which disrupts the SRAP reading frame, or by the absence of exon 3. The initial SRA clones isolated by the O’Malley group [3] contained a 687 nt sequence “core” sequence coded for by exons 2e5 that contains a 162 aa open reading frame. These clones have variable 50 and 30 regions flanking the core sequence however they each lack the subsequently identified initiation codons present in exon 1 [5]. A more recent study found exons 4 and 5 were present in >99.7% of SRA1 sequences although the 30 extremity of exon 5 appeared to vary between isoforms [7]. Exon 2 was present in 86% of transcripts and intron 1 sequences in 33%. Northern analysis of human tissue found SRA RNA in all tissues although at higher levels in liver, skeletal muscle and heart [3]. Remarkably, in spite of being an NR coregulator, lower levels of SRA were detected in breast, prostate, ovary and uterus [10]. The predominant SRA transcript in normal tissue is approximately 0.7e0.85 kb while less abundant, larger transcripts of 1.31.5 kb have been identified. Notably, the larger form was more prevalent in human cell lines from a range of tissue types. When matched with sequencing results, Northern data (generated using the core sequence as a probe) suggest that transcripts including exons 2e5 and a polyadenylation signal 393 nts into the last exon would have a minimum size of 831 nt plus a poly A tail. Given the variable inclusion of exon 1, 3 and intron 1 sequences identified by sequencing, this suggests the smaller transcripts are predominantly ncRNAs. Inclusion of exon 1 in coding transcripts would add a further 288 or 250 nt depending upon the initiation sequence utilised which would be consistent with the larger transcripts observed. The SRA isoforms identified have different biological activities and associations with normal and disease states as outlined below. 3. Establishing the non-coding nature of SRA and identification of functional stem-loop structures The isolation of SRA and its recognition as a ncRNA coactivator was a unique and highly significant finding given its novel mechanism of NR regulation. Initially, the ability of the core SRA RNA and derivatives with stop, frame shift and deletion mutations that disrupted its 162 aa ORF to coactivate NR activity were compared [3]. While larger deletion and inverted sequence mutants failed to transactivate reporter gene activity, the mutant, ncRNAs each augmented transcription. In a subsequent study, phylogenetic and thermodynamic approaches were used to identify topologically conserved SRA RNA domains [11]. Eleven structures (STRs) were identified within the core domain, with evidence of covariation of human and mouse sequences conserving the predicted topological structures. For a subset of these loops, mutations which preserved the protein coding sequence but altered their intrastrand binding/structure compromised their ability to coactivate in NR reporter assays. Disruption of a single loop did not cause complete loss of activity indicating that multiple structures are involved in SRA coactivation [11]. Coactivation was also observed in the presence of translational inhibition further supporting the conclusion that the RNA itself was facilitating transcription. In more recent studies, the actions of full length, coding and mutated non-coding sequences have been compared using three different estrogen response elements (EREs) in HeLa cells [12]. This study found that, in general, full-length (fl)-SRA transcripts coding for wild type (wt) protein coactivated estrogen receptor
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(ER) activity while initiating codon-mutated transcripts showed reduced coactivation. In contrast to studies using the core region performed in COS and HeLa cells to examine PR activity described above [3,11], wt protein coding but RNA loop disrupting fl-SRA transcripts also coactivated ER activity relative to empty vector controls. Coactivation was observed with the vitellogenin ERE with all fl-SRA constructs used, independent of whether they expressed nc or coding RNA or if the RNA was wt or silent mutated. Similar effects were observed with the PR-ERE however only wt protein encoding RNAs coactivated the pS2-ERE. Notably, non-coding SRA core RNA had both coactivator and repressor like activities and variable regulatory actions dependent upon the reporter element, presence or absence of ligand and type of exogenously transfected ER assessed in CHO cells [13]. In HeLa and prostate cancer cell lines, no difference between the ability of full-length SRA1, 2 and 3 isoforms to coactivate NR transcription was observed [8]. The variation of responses in these studies highlights the need to carefully control for cell background and reporter/assay used when comparing the effects of SRA gene products on NR activity.
4. SRA RNA isoforms and disease Aberrant SRA transcription has been observed in a range of human tumours [10,14e16]. SRA levels were elevated in the majority of ovarian and uterine tumour samples tested in one study [10] and in breast tumour samples compared with normal adjacent tissue [15]. Within breast cancer samples, SRA expression may correlate with proliferation but it does not appear to be linked to tumour type or grade [10]. Exon 3 deleted SRA transcripts have been detected in primary breast tumours but with similar frequency in paired normal samples suggesting its expression is not tumour specific [15]. Exon 3 deleted transcripts have also been detected in ovarian tumours but at low frequencies [16]. Differences between SRA1e3 isoform activities in these tumours have not been reported. Alternative splicing results in the generation of coding and non-coding SRA isoforms in breast cancer samples [17]. The balance between fully spliced SRA and intron 1-containing RNAs varies between breast tumours with alterations to the relative proportions of the RNA types affecting cancer cell growth [18]. Although no difference in ncRNA transcript levels was observed when high and low ER expressing tumours were compared, it was elevated in high relative to low PR breast cancer samples [18]. Oligonucleotide mediated increases in the ratio of non-coding to coding SRA RNA in T5 breast cancer cells resulted in elevated expression of genes associated with invasion and fewer viable cells [18]. Regarding the latter, it was not established whether cell proliferation was decreased, cell death promoted or a combination of the two. In contrast, SRA depletion in MDA-MD-231 (ER negative) cells resulted in reduced invasiveness and expression of genes associated with this process [19]. This same group also observed that SRA depletion only affected a subset of direct ERa target genes. Also, ODN targeting of SRA in MCF-7 cells did not affect estrogen-induced proliferation while similar targeting of SRC-1 and TIF2 did [20]. Taken together, these data indicate that the effects of SRA on breast cancer may be less ER dependent than initially thought. Tissue microarray studies comparing SRAP expression in breast cancer patients with ER, PR, age and lymph node status showed that while overall SRAP was not a predictor of outcome, its elevated expression in younger, node negative, ER positive patients was a poor prognostic indicator [21]. While such a study does not discriminate between SRA RNA and protein effects, it is consistent
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with SRA gene products coactivating ER activity, a prime driver in breast cancer proliferation [12].
[25]. Further studies are therefore needed in a range of cell types to validate what may be a key role for SRA in metabolism (see Fig. 1).
4.1. SRA and prostate cancer 4.3. SRA and myoblast differentiation SRA has been shown to coactivate AR activity in prostate lines using constructs expressing an amino-terminal deleted SRAP peptide [4] and full-length protein [8]. Experiments performed in LNCaP (ARþ) and DU145 (AR) prostate lines showed proliferation with SRA knock down but not for PC-3 (AR) cells [22]. This study also demonstrated that loss of the AR coactivator SRC-3 resulted in decreased PSA, TMPRSS2 and PMEPA1 mRNA in prostate lines but loss of SRA only affected TMPRSS2. In addition, down regulation of SRA caused reduced LNCaP and DU145 proliferation in the presence of ligand but PC-3 cells were unaffected. It is notable that similar siRNA targeting of SRA in MCF-7 breast cancer cells showed that although the ER target genes GREB1 and TFF were induced by ligand, they were not affected by loss of SRA [19]. Also as discussed above, SRA knock down in MCF-7 cells did not affect ligand-induced proliferation [20]. Thus, data from both breast and prostate cancer cell lines studies raises questions regarding the NR dependency of SRA’s activities. 4.2. SRA promotes differentiation of adipocytes The ability of SRA RNA to coactivate NRs in general and the adipocyte transcriptional regulator PPARg in particular [3,4,23], has led to the recent examination of its role in adipocyte function [24]. Interestingly, the amino-terminal of PPARg has been found to bind directly to SRA RNA (Table 1). Transfection of marrow derived mesenchymal ST2 cells with pSCT-SRA, a ncRNA, promotes adipogenesis while its depletion inhibits differentiation of 3T3-L1 preadipocytes [24]. SRA also enhances insulin signalling and glucose uptake in differentiated adipocytes in vitro [24]. In agreement with these findings, SRA knock down in HeLa cells results in decreased transcription of genes associated with glucose and lipid metabolism including GLUT3, SLC2A3, ABCA1 and INSIG-1 [19]. Further, SRA transgenic mice have perturbed fat pad morphology in their breast tissue [10]. It was also noted that in addition to promoting adipogenesis, SRA ncRNA coactivated GATA3 activity [19]. This finding is at odds with a report that promotion of GATA3 activity inhibits adipocyte differentiation
SRA influences muscle differentiation through its interactions with the DEAD box containing, RNA helicase coregulators p68, p72 and MyoD transcription factor (Table 1) [26]. Over expression of SRA potentiates MyoD activity while its depletion reduces myoblast differentiation and muscle specific gene expression. Increased p68 and p72 expression also augments MyoD function while p68 knock down inhibited both myoblast [26] and adipocyte differentiation [27]. It has recently been reported that the proportion of SRA ncRNA increases during myogenic differentiation [7]. Opposing this, SRAP inhibits muscle differentiation via its binding of substructure STR7 of SRA, a loop bound by other coregulators including SRA Loop Interacting RNA-binding Protein (SLIRP) [23,28]. These data lead to a potential regulatory model in which coding SRA is initially transcribed binding its RNA and inhibiting myoblast differentiation then, through differential splicing events, SRA ncRNA transcripts are favoured that coactivate MyoD and promote myoblast differentiation [29]. Studies of other differentiation models will be informative as to whether this is a muscle specific SRA mechanism. 4.4. SRA is an essential component of p68/CTCF gene insulator complexes Adding to interactions between p68 and SRA, as a complex, these molecules have been demonstrated to participate in transcriptional insulation complexes [30]. CCCTC-binding factor (CTCF) is a DNA-binding protein involved in chromatin organisation that recruits the cohesion complex to insulator sites. Mass spectrophotometry of CTFC immunoprecipitates identified p68 in these conjugates and demonstrated SRA dependency by IP-RT-PCR. Depletion of SRA RNA reduced p68/CTCF association and reduced target gene binding thereby decreasing insulator function. 5. In vivo effects of SRA To investigate the physiological roles of SRA, transgenic mouse and Zebra fish knock down studies have been performed. The
Table 1 Direct and indirect SRA RNA interacting proteins. IP-RT-PCR, immunoprecipitation-reverse transcription-polymerase chain reaction; REMSA, RNA electrophoretic mobility shift assay; Y3H, yeast three hybrid assay; UVXL, ultraviolet light cross-linking assay. Protein
Direct interaction
SHARP p72 PUS1 PUS3 TRa & b SLIRP SF-1/NR5A1 PPARg DAX-1 p68 SRAP SRC-1 AR PR SRC-2/TIF2 ERa RAR Myo D CTCF
O O O O O O O O O
Complex formation
O O O O O O O O O O
Method of detecting interaction
RNA-binding domain
Reference
In vitro binding assays, IP-RT-PCR, REMSA REMSA, IP-RT-PCR Pseudouridylation, In vitro pull down-RT-PCR Pseudouridylation In vitro binding assays, IP-RT-PCR Y3H, UVXL, REMSA, IP-RT-PCR In vitro binding, IP-RT-PCR In vitro binding In vitro binding, IP-RT-PCR IP-RT-PCR IP-RT-PCR Co-purification, IP-RT-PCR Co-purification, IP-RT-PCR Co-purification IP-RT-PCR IP-RT-PCR IP-RT-PCR IP-RT-PCR Mass Spectrophotometry, IP-RT-PCR
RRM (x3) DEAD Box None None Unique, ssRNA affinity RRM FTZ-F1 box containing region Amino-terminal N3R domain DEAD Box RNP-2 motif None None None None None None None None
[23,33] [35] [37] [38] [3,53] [23] [56] [24] [56,57] [26,35] [7] [3,23] [3] [3] [35] [35] [37] [26] [24]
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Fig. 1. SRA and its protein binding partners. Direct (black arrows) and indirect (grey) binding of SRA by various proteins produces an array of consequences ranging from nuclear receptor coactivation and repression, positive and negative effects on adipogenesis and myogenesis, gene insulation, adrenal gland gene regulation and pseudouridinylation. SLIRP and SHARP bind directly to STR7 of SRA, while indirect interactions have been shown for SRAP. Binding sites for the remainder of the molecules depicted have not been determined.
MMTV-SRA mouse bears a human, non-coding, 1262 nt SRA 2 sequence under the control of the mouse mammary tumour virus (MMTV) promoter which directs transgene transcription in mammary tissue and uterus of females and the urogenital system of males [10]. Early mammary duct development in transgenic mice is comparable with the wt however, mature virgin mice showed aberrant mammary gland development characterised by ductal ectasia, acinar hyperplasia and intraductal proliferation of luminal epithelial cells with increased apoptosis in the stratified epithelia. Precocious ductalealveolar development during pregnancy was observed in transgenic mice implicating heightened PR activity. This was supported by elevated expression of the NR in transgenic mammary tissue consistent with SRA coactivating ER activity. Disturbances were also seen in male tissues with their seminal vesicles having a “fist” like gross morphology compared with the lobular, horn shape of the wt structure. Given the coactivating nature of SRA ncRNA on NR activity, particularly those associated with fertility, it was not surprising that both male and female transgenic mice have reduced fertility. The ability of SRA to coactivate the ER, a prime proliferative driver in breast cancer, and the dysplasia in the MMTV-SRA mammary tissue led to its closer examination. Histological assessment of mammary tissue from these mice revealed increased mitotic and apoptotic indices, however, while they displayed sporadic preneoplastic lesions such as excessive proliferation of branching tubules, frank malignancies were not reported. Reasoning that elevated SRA transcription was not enough to provoke metastasis, the MMTV-SRA mouse was crossed with MMTV-ras mice. Curiously, the resultant progeny had a lower incidence of mammary neoplasia than the monogenic carriers [10]. This unexpected result suggested that rather than SRA promoting breast cancer development, it appeared to oppose it. Further in vivo studies involving enforced SRA expression in all tissues may give
a clearer indication of its activities particularly given its influence on adipose, muscle and uritogenital tissues. Following linkage disequilibrium studies aimed at identifying genes associated with dilated cardiac myopathy in humans, SRA was highlighted as a candidate [31]. To investigate this possibility, a morpholino antisense approach was utilised to deplete SRA in Zebra fish from the 1e2 cell stage of development. Remarkably, impaired contractility, predominantly in the ventricular chambers and pericardial oedema was observed in treated animals [31]. Loss of SRA therefore results in disturbed cardiac development, a finding consistent with the well defined requirement of NR activity, particularly the thyroid hormone and mineralocorticoid receptors, for normal cardiac function [32]. 6. SRA mechanism of action SRA coding and ncRNAs along with SRAP act to modulate transcription but, by virtue of their different biochemical compositions, do so by different mechanisms. Numerous studies have shown that SRA RNA can coactivate NRs in a ligand-dependent manner [3,11,23,24,33,34]. As described above, stem-loop structures in the core domain of SRA RNA were predicted by in silico analysis and their involvement in coactivation assessed by cell based NR reporter assays [3,11]. It was found that while mutation of individual loops compromised coactivation by SRA, loss of individual sub-structures resulted in only partial loss of activity. As multiple molecules including transcription factors, NR coactivators, RNA helicases, gene insulator molecules (Table 1) bind either directly or indirectly to SRA at different loops, it suggests it may function as a scaffold to bring together multiple factors that regulate gene expression. AF-1 domain involvement in SRA coactivation of NR activity has been reported by multiple groups [3,34e36]. Ligand-independent
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coactivation of ERa involving MAPK and S118 of the receptor’s AF-1 domain has also been demonstrated [34]. Mammalian two-hybrid assays comparing mutant and wt ER AF-1 domains however showed alteration to S118 caused only a slight reduction in coactivation by SRA and that mutations to additional residues in region were required [36]. SRA does coactivate ERb but only in the presence of ligand and thus in an AF-2 dependent manner leading to the suggestion that its activities may involve separate AF-1 and AF-2 activities [36]. 6.1. Regulation of SRA RNA activity by pseudouridinylation An additional twist to the regulation of SRA RNA activity is provided by the actions of pseudouridine synthases (PUS) 1 and 3 [37,38]. These enzymes pseudouridinylate SRA RNA potentiating its coactivation of NR activity. PUS1 augments RARg, TR, ER, GR, AR and PR-B activity while PUS3 has similar effects but does not increase sex steroid receptor activity. PUS molecules also differ in their SRA target sites and cause different patterns of modification which may influence coactivator binding. It has been noted that a U206A mutation results in hyperpseudouridinylation of SRA causing it to become a transcriptional repressor leading to the suggestion that PUS proteins may influence SRA coactivator/ repressor switching [38]. Mutation to PUS1 has been associated with mitochondrial myopathy and sideroblastic anaemia suggesting a defect in SRA processing could contribute to these diseases [39e41]. 6.2. SRAP binds its own RNA (SRA) and acts as a transcriptional repressor It has now been shown that SRAP binds its own RNA [7]. SRA RNA was detected by RT-PCR following immunoprecipitation with anti-SRAP antibodies from both human and mouse muscle samples. This interaction was dependent on 1) the presence of STR7 in the SRA transcripts and 2) an RNP-2 like motif, similar to that in RRMRNA-binding motifs, in SRAP. Although this domain in SRAP is well conserved across species, further binding studies are required to confirm this is a direct interaction between these molecules. In order to further understand SRAP’s functional role, ChIPeChip arrays have been used to identify regions of the genome with which SRAP associates. The absence of a defined DBD in SRAP suggests it does not bind DNA directly and so requires binding partners with such domains. Studies in MCF-7 breast cancer cells stably expressing tagged SRA protein have identified a long list of such molecules [42]. As may be predicted, the most highly represented are those associated with NR target genes, specifically for NR subfamily 2 factors, GR and PPARs. In addition, GATA and AP1 sites were also identified. Subsequent in vitro two-hybrid assays showed recruitment of SRAP to the GAL4-RE reduced reporter gene activity and that HDAC2 was immunoprecipitated with SRAP. Further detailed discussion of SRAP and its interactions may be found in [18]. 7. Other SRA-binding partners 7.1. SLIRP binds SRA and is a potent NR corepressor Reasoning that SRA-binding molecules may themselves be NR coregulators, our laboratory sought to identify novel SRA interactors by yeast 3-hybrid (Y3H) analysis. Loop structures that had previously been demonstrated to participate in coactivation [11] were used to screen a primary breast cancer tissue derived library [23]. Both STR1 and STR7 were effective baits and were bound by different molecules, the majority of which contained RNA-binding domains. One molecule however, now referred to as
SLIRP, had not been previously characterised so its properties were investigated further. SLIRP codes for a 109 aa human protein that is well conserved between species. Its amino-terminal codes for a putative mitochondrial signal while its core region contains an RNA recognition motif (RRM) RNA-binding domain. Over expression of SLIRP represses the activities of NRs as assessed by both reporter assays and changes in endogenous gene transcription. Conversely, potentiation of NR target gene transcription results from SLIRP knock down. ChIP studies confirm SLIRP’s presence in conjugates at NR binding sites in the absence of ligand which diminishes with hormone treatment. The binding of SLIRP to the STR7 sub-structure of SRA has been demonstrated by Y3H, RNA electrophoretic mobility shift assay and immunoprecipitation-reverse transcriptase-PCR assays [23]. Mutation to SLIRP’s RRM domain abrogated its ability to repress SRA-mediated NR coactivation. Further, SLIRP did not repress NR activity following cotransfection with a STR7 mutant SRA consistent with it binding this loop. It is intriguing that SLIRP, SHARP and SRAP each target STR7 and act as repressors. The affinity of each repressor for a common domain may point to SRA playing a general role in coactivation with the differential expression of these repressors regulating its activity in a tissue/temporal specific manner. Although ChIP studies confirmed the presence of SLIRP in the nucleus, immunofluorescence show the majority of this protein is in the mitochondria [23]. While unexpected, this result is consistent with SLIRP having a mitochondrial localisation signal in its amino-terminal, epitope capping of which results in its altered localisation. Finding SLIRP in the mitochondria is consistent with the detection in the same organelle of multiple NRs including Nur77/TR3 [43], ER [44], GR [45], and TR [46] along with NR coregulators NCOA6 [47] PGC-1a [48]. Examples of the importation of nuclear encoded RNAs into the mitochondria are limited but have been identified in yeast, plants and mammals including humans [49] and references cited therein. As both NRs and coregulators have been found in the mitochondria, it is exciting to speculate that SRA RNA may also be imported and modulate transcription within this organelle. In this context, its interesting to note two recent reports in which SLIRP is implicated in mitochondrial biology. In the first, SLIRP was shown to regulate the expression of key mitochondrial mRNAs (mtRNAs) [50]. This group showed that depletion of SLIRP reduced oxidative phosphorylation activity and mtRNAs coding for complex I and IV proteins. In a second report investigating the biology of the Leucine-Rich Pentatrico-Repeat Containing protein (LRPPRC), mutation of which leads to mitochondrial dysfunction, SLIRP was shown to regulate mitochondrial gene expression [51]. Taken together these data suggest SLIRP may play an important role in mitochondrial biology, potentially regulating energy metabolism and apoptosis, especially in high energydemand tissues. 7.2. SHARP, a SRA-binding NR corepressor SHARP (SMRT/HDAC1 associated repressor protein) is an NR corepressor that interacts with SRA in vitro and contains three RRMs required to repress SRA-augmented E2-induced transactivation [33]. In the initial description of SHARP as an NR corepressor, the nature of the interaction with SRA was not defined. However, the identification of high aa sequence homology between SHARP and SLIRP, especially in their RRM domains, prompted evaluation of SHARP’s capacity to target SRA STR7. We found that recombinant SHARP bound this region of SRA avidly [23], which suggested a functional interaction may exist between these molecules in vivo. In functional studies with an E2-responsive reporter, we were also able to show that when SLIRP was cotransfected with
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SHARP and SRA, an additional 2-fold repression of SRA-augmented coactivation was observed [23]. Thus, SHARP and SLIRP appear to act in an additive fashion to enhance repression of NR activity, based on their capacity to target a specific stem-loop sub-structure of SRA, STR7.
examples of the breadth of SRA’s effects, and its capacity to interact with “classical” NRs as well as “orphan” NRs to mediate key events at the transcriptional level.
7.3. Thyroid hormone receptors bind SRA
The continuing characterisation of SRA is yielding greater understanding of its activities. While initially defined as a ncRNA coactivator of steroid activity, it is clear that its functions now extend beyond NR signalling and it is involved in the regulation of non-NR activities. The involvement of SRA in regulating metabolism, adipogenesis, myogenesis and chromatin organisation adds to its repertoire of pathways influenced and tissues affected. Identification of multiple coding and ncRNAs, the proportion of which appears to influence differentiation and potentially neoplastic pathways, requires further investigation as to the factors that regulate their production. The discovery of SLIRP, a novel RRMcontaining SRA-binding protein that regulates signalling by multiple NRs, and also has a role in the regulation of mitochondrial function, has provided new insight into the multiple roles that some NR coregulators can be engaged in. Understanding the interactions between the three known SRA STR7-binding proteins and how they regulate SRA-mediated transactivation in various tissues is an important goal. Similarly, investigation of SRAP, and in particular its binding partners, will greatly enhance our understanding of its regulation of transcription and regulation of its own expression. Furthermore, further assessment of SRA RNA and protein expression in tumour samples is warranted, as they may prove to be useful in clinical prognosis. In sum, the SRA gene retains its unique position amongst the NR coregulators with its RNA and protein products both influencing transcription the clinical impact of which we are only just beginning to realise.
The TRa and TRb NRs are encoded by separate genes and produce several proteins via alternate splicing and promoter usage [52]. The amino-terminal 370 aas of TRa1 and 2 proteins are identical but the carboxy-terminal 40 aas of TRa1 differ from the last 122 aa of TRa2. TRa2 does not bind triiodothyronine (T3) and transactivate TR target gene expression. However, all three forms can bind SRA, via a 41 aa region between their second zinc finger and ligand-binding domains [53,54]. Interestingly, phosphorylation of TRa2 at CK2 kinase sites abrogates SRA binding and redistributes the majority of TRa2 from being predominantly nuclear to mainly cytoplasmic. However, phosphorylation of TRa1 has little impact on its ability to bind SRA or alter its nuclear localisation. These data support a model whereby SRA is bound by the transcriptionally silent non-phosphorylated TRa2 in the nucleus, acting as a repressor by sequestering SRA in inactive NR complexes. However, following phosphorylation, TRa2 would lose its ability to bind SRA and exit the nucleus thereby releasing SRA for participation in coactivator complexes. Further studies are required to validate this intriguing model. 7.4. PPARs and SRA The PPAR NRs (a, g, d) heterodimerize with retinoid X receptors (RXRs) and once bound by low affinity ligands (from dietary fat or intracellular metabolism) activate transcription of a range of genes involved in lipid metabolism, transport and storage in specific tissues [55]. There is compelling evidence for roles of each of the PPARs in metabolism in key target organs, including the liver, muscle, adipose tissue, macrophages and more recently the b-cells of the pancreas. Interestingly, SRA has recently been shown to regulate adipogenesis and enhance insulin sensitivity, in part via binding to PPARg [24]. As a ncRNA, SRA promoted differentiation of mesenchymal cells into preadipocytes, and regulated key genes in the insulin and TNFa signalling pathways. Similarly, in HeLa cells, SRA has been shown to regulate multiple metabolic genes, including INSIG-1, ABCA1 and a glucose transporter [19]. Notably, SRA is expressed widely in mammals, particularly in high energydemand tissues such as liver, heart and muscle. These novel interactions between SRA and key regulators of metabolic pathways suggest a previously unrecognised role for SRA in metabolism acting via direct interactions with the PPARg. 7.5. Dax-1, SF-1 and SRA Through its interactions with the orphan NRs Steroidogenic factor 1 (SF-1) and Dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (Dax1) SRA influences adrenal function [56]. SF-1 and Dax-1 regulate adrenal gland function and sexual development. Dax-1 binds to SF1 and repressing its target gene expression. SF-1 and Dax-1 can both bind to SRA, and coactivation by Dax-1 is abolished with SRA knock down, resulting in significant changes in expression of downstream adrenal genes [56]. These findings provided an unrealised pivotal role for SRA in steroidogenesis and adrenal function. Additional data from the same group shows that Dax-1 augmentation of Liver homologue receptor 1 (LRH-1)-mediated Oct4 activation is SRA-dependent [57]. These data provide further
8. Future directions
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Mini-review
Steroid Receptor RNA Activator e A nuclear receptor coregulator with multiple partners: Insights and challenges Shane M. Colley a, Peter J. Leedman a, b, * a b
Laboratory for Cancer Medicine, University of Western Australia Centre for Medical Research, Western Australian Institute for Medical Research, Perth, WA 6000, Australia School of Medicine and Pharmacology, University of Western Australia, Perth, WA 6009, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 May 2011 Accepted 4 July 2011 Available online 27 July 2011
Steroid Receptor RNA Activator (SRA) occupies a unique and enigmatic position within the nuclear receptor (NR) field and more broadly in transcriptional regulation. This is as a result of its transcripts having both coding and non-coding coactivator activities along with its protein product SRAP performing mixed coactivator/repressor functions. Recent publications have provided greater understanding of SRA gene product activities and how they affect not only NR function, but now more broadly, signalling pathways involved in differentiation and metabolism. This review will discuss the isolation of SRA, its gene products, regulation of transcription along with its in vitro and in vivo activities with a particular focus on its actions as an RNA and its binding partners. Ó 2011 Published by Elsevier Masson SAS.
Keywords: SRA Nuclear receptor coregulator SLIRP SRAP Hormone action Cancer Metabolism
1. Isolation of SRA The activities of the NR superfamily of ligand-inducible transcription factors are instrumental in the regulation of reproduction, development and metabolism of mammalian and other species [1]. The transcriptional activity of NRs is modulated by an even larger group of functionally diverse molecules that either augment (coactivators) or reduce (corepressors) the actions of the NRs [2]. The great majority of these coregulators are proteins, however, while screening for novel modulators of the Progesterone Receptor (PR), the RNA coactivator Steroid Receptor RNA Activator (SRA) was uncovered [3]. Curiously, in spite of a yeast two-hybrid assay being performed, the initial SRA clone isolated was found to function as a non-coding RNA (ncRNA) thereby identifying a novel mode of NR modulation. Subsequent studies have now found the products of the SRA gene include coding and ncRNAs along with the Steroid Receptor RNA Activator Protein (SRAP), each of which modulate gene transcription in a cell- and system-specific manner [4,5]. As SRAP is the subject of a comprehensive review in this issue [18], this
* Corresponding author. Laboratory for Cancer Medicine, University of Western Australia Centre for Medical Research, Western Australian Institute for Medical Research, Level 6, MRF Building, Rear 50 Murray St., Perth, WA 6000, Australia. Tel.: þ61 8 9224 0333; fax: þ61 8 9224 0322. E-mail address: [email protected] (P.J. Leedman). 0300-9084/$ e see front matter Ó 2011 Published by Elsevier Masson SAS. doi:10.1016/j.biochi.2011.07.004
article will focus on SRA gene products, their isoforms, prevalence and biological functions with a particular focus on the transcripts and interacting molecules.
2. The SRA gene and its products The SRA gene is well conserved between species [6] with human (5q31.3), rat (18p11) and mouse (18B2) homologues each having 5 exons. Studies by a number of groups have revealed the existence of multiple RNA isoforms transcribed from the SRA gene with the most recent contribution, involving EST data base searching, 50 RACE validation and sequencing, bringing the total up to 20 different types [7]. SRA transcripts may be placed in either coding or non-coding groups accounting for 39% and 61% of the clones, respectively. Coding transcripts, contain exons 1e5 within which are two methionine codons defining 236 and 224 amino acid (aa) SRAP proteins. Doublet proteins of approximately 31 and 32 kDa have been detected in human cancer cell lines [5,8] and breast cancer patient samples [9] confirming the utilisation of both initiating methionines. Three coding mRNA isoforms, SRA1e3, have been reported [5]. These contain essentially identical coding sequences except that SRA2 has a C / T (theonine / isoleucine) alteration at nt 338 and a silent A / C change at nt 348 (as listed in AF293025) while SRA3 has a combined substitution/insertion of G / CGAC at position 520 (AF293026) resulting in the insertion of
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an arginine and valine / leucine substitution at aas 109 and 110. The presence of these isoforms correlates with the genomic sequence of the tissues from which they were sourced indicating they represent individual alleles. SRA ncRNAs result from differential splicing of intron 1, the retention of which disrupts the SRAP reading frame, or by the absence of exon 3. The initial SRA clones isolated by the O’Malley group [3] contained a 687 nt sequence “core” sequence coded for by exons 2e5 that contains a 162 aa open reading frame. These clones have variable 50 and 30 regions flanking the core sequence however they each lack the subsequently identified initiation codons present in exon 1 [5]. A more recent study found exons 4 and 5 were present in >99.7% of SRA1 sequences although the 30 extremity of exon 5 appeared to vary between isoforms [7]. Exon 2 was present in 86% of transcripts and intron 1 sequences in 33%. Northern analysis of human tissue found SRA RNA in all tissues although at higher levels in liver, skeletal muscle and heart [3]. Remarkably, in spite of being an NR coregulator, lower levels of SRA were detected in breast, prostate, ovary and uterus [10]. The predominant SRA transcript in normal tissue is approximately 0.7e0.85 kb while less abundant, larger transcripts of 1.31.5 kb have been identified. Notably, the larger form was more prevalent in human cell lines from a range of tissue types. When matched with sequencing results, Northern data (generated using the core sequence as a probe) suggest that transcripts including exons 2e5 and a polyadenylation signal 393 nts into the last exon would have a minimum size of 831 nt plus a poly A tail. Given the variable inclusion of exon 1, 3 and intron 1 sequences identified by sequencing, this suggests the smaller transcripts are predominantly ncRNAs. Inclusion of exon 1 in coding transcripts would add a further 288 or 250 nt depending upon the initiation sequence utilised which would be consistent with the larger transcripts observed. The SRA isoforms identified have different biological activities and associations with normal and disease states as outlined below. 3. Establishing the non-coding nature of SRA and identification of functional stem-loop structures The isolation of SRA and its recognition as a ncRNA coactivator was a unique and highly significant finding given its novel mechanism of NR regulation. Initially, the ability of the core SRA RNA and derivatives with stop, frame shift and deletion mutations that disrupted its 162 aa ORF to coactivate NR activity were compared [3]. While larger deletion and inverted sequence mutants failed to transactivate reporter gene activity, the mutant, ncRNAs each augmented transcription. In a subsequent study, phylogenetic and thermodynamic approaches were used to identify topologically conserved SRA RNA domains [11]. Eleven structures (STRs) were identified within the core domain, with evidence of covariation of human and mouse sequences conserving the predicted topological structures. For a subset of these loops, mutations which preserved the protein coding sequence but altered their intrastrand binding/structure compromised their ability to coactivate in NR reporter assays. Disruption of a single loop did not cause complete loss of activity indicating that multiple structures are involved in SRA coactivation [11]. Coactivation was also observed in the presence of translational inhibition further supporting the conclusion that the RNA itself was facilitating transcription. In more recent studies, the actions of full length, coding and mutated non-coding sequences have been compared using three different estrogen response elements (EREs) in HeLa cells [12]. This study found that, in general, full-length (fl)-SRA transcripts coding for wild type (wt) protein coactivated estrogen receptor
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(ER) activity while initiating codon-mutated transcripts showed reduced coactivation. In contrast to studies using the core region performed in COS and HeLa cells to examine PR activity described above [3,11], wt protein coding but RNA loop disrupting fl-SRA transcripts also coactivated ER activity relative to empty vector controls. Coactivation was observed with the vitellogenin ERE with all fl-SRA constructs used, independent of whether they expressed nc or coding RNA or if the RNA was wt or silent mutated. Similar effects were observed with the PR-ERE however only wt protein encoding RNAs coactivated the pS2-ERE. Notably, non-coding SRA core RNA had both coactivator and repressor like activities and variable regulatory actions dependent upon the reporter element, presence or absence of ligand and type of exogenously transfected ER assessed in CHO cells [13]. In HeLa and prostate cancer cell lines, no difference between the ability of full-length SRA1, 2 and 3 isoforms to coactivate NR transcription was observed [8]. The variation of responses in these studies highlights the need to carefully control for cell background and reporter/assay used when comparing the effects of SRA gene products on NR activity.
4. SRA RNA isoforms and disease Aberrant SRA transcription has been observed in a range of human tumours [10,14e16]. SRA levels were elevated in the majority of ovarian and uterine tumour samples tested in one study [10] and in breast tumour samples compared with normal adjacent tissue [15]. Within breast cancer samples, SRA expression may correlate with proliferation but it does not appear to be linked to tumour type or grade [10]. Exon 3 deleted SRA transcripts have been detected in primary breast tumours but with similar frequency in paired normal samples suggesting its expression is not tumour specific [15]. Exon 3 deleted transcripts have also been detected in ovarian tumours but at low frequencies [16]. Differences between SRA1e3 isoform activities in these tumours have not been reported. Alternative splicing results in the generation of coding and non-coding SRA isoforms in breast cancer samples [17]. The balance between fully spliced SRA and intron 1-containing RNAs varies between breast tumours with alterations to the relative proportions of the RNA types affecting cancer cell growth [18]. Although no difference in ncRNA transcript levels was observed when high and low ER expressing tumours were compared, it was elevated in high relative to low PR breast cancer samples [18]. Oligonucleotide mediated increases in the ratio of non-coding to coding SRA RNA in T5 breast cancer cells resulted in elevated expression of genes associated with invasion and fewer viable cells [18]. Regarding the latter, it was not established whether cell proliferation was decreased, cell death promoted or a combination of the two. In contrast, SRA depletion in MDA-MD-231 (ER negative) cells resulted in reduced invasiveness and expression of genes associated with this process [19]. This same group also observed that SRA depletion only affected a subset of direct ERa target genes. Also, ODN targeting of SRA in MCF-7 cells did not affect estrogen-induced proliferation while similar targeting of SRC-1 and TIF2 did [20]. Taken together, these data indicate that the effects of SRA on breast cancer may be less ER dependent than initially thought. Tissue microarray studies comparing SRAP expression in breast cancer patients with ER, PR, age and lymph node status showed that while overall SRAP was not a predictor of outcome, its elevated expression in younger, node negative, ER positive patients was a poor prognostic indicator [21]. While such a study does not discriminate between SRA RNA and protein effects, it is consistent
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with SRA gene products coactivating ER activity, a prime driver in breast cancer proliferation [12].
[25]. Further studies are therefore needed in a range of cell types to validate what may be a key role for SRA in metabolism (see Fig. 1).
4.1. SRA and prostate cancer 4.3. SRA and myoblast differentiation SRA has been shown to coactivate AR activity in prostate lines using constructs expressing an amino-terminal deleted SRAP peptide [4] and full-length protein [8]. Experiments performed in LNCaP (ARþ) and DU145 (AR) prostate lines showed proliferation with SRA knock down but not for PC-3 (AR) cells [22]. This study also demonstrated that loss of the AR coactivator SRC-3 resulted in decreased PSA, TMPRSS2 and PMEPA1 mRNA in prostate lines but loss of SRA only affected TMPRSS2. In addition, down regulation of SRA caused reduced LNCaP and DU145 proliferation in the presence of ligand but PC-3 cells were unaffected. It is notable that similar siRNA targeting of SRA in MCF-7 breast cancer cells showed that although the ER target genes GREB1 and TFF were induced by ligand, they were not affected by loss of SRA [19]. Also as discussed above, SRA knock down in MCF-7 cells did not affect ligand-induced proliferation [20]. Thus, data from both breast and prostate cancer cell lines studies raises questions regarding the NR dependency of SRA’s activities. 4.2. SRA promotes differentiation of adipocytes The ability of SRA RNA to coactivate NRs in general and the adipocyte transcriptional regulator PPARg in particular [3,4,23], has led to the recent examination of its role in adipocyte function [24]. Interestingly, the amino-terminal of PPARg has been found to bind directly to SRA RNA (Table 1). Transfection of marrow derived mesenchymal ST2 cells with pSCT-SRA, a ncRNA, promotes adipogenesis while its depletion inhibits differentiation of 3T3-L1 preadipocytes [24]. SRA also enhances insulin signalling and glucose uptake in differentiated adipocytes in vitro [24]. In agreement with these findings, SRA knock down in HeLa cells results in decreased transcription of genes associated with glucose and lipid metabolism including GLUT3, SLC2A3, ABCA1 and INSIG-1 [19]. Further, SRA transgenic mice have perturbed fat pad morphology in their breast tissue [10]. It was also noted that in addition to promoting adipogenesis, SRA ncRNA coactivated GATA3 activity [19]. This finding is at odds with a report that promotion of GATA3 activity inhibits adipocyte differentiation
SRA influences muscle differentiation through its interactions with the DEAD box containing, RNA helicase coregulators p68, p72 and MyoD transcription factor (Table 1) [26]. Over expression of SRA potentiates MyoD activity while its depletion reduces myoblast differentiation and muscle specific gene expression. Increased p68 and p72 expression also augments MyoD function while p68 knock down inhibited both myoblast [26] and adipocyte differentiation [27]. It has recently been reported that the proportion of SRA ncRNA increases during myogenic differentiation [7]. Opposing this, SRAP inhibits muscle differentiation via its binding of substructure STR7 of SRA, a loop bound by other coregulators including SRA Loop Interacting RNA-binding Protein (SLIRP) [23,28]. These data lead to a potential regulatory model in which coding SRA is initially transcribed binding its RNA and inhibiting myoblast differentiation then, through differential splicing events, SRA ncRNA transcripts are favoured that coactivate MyoD and promote myoblast differentiation [29]. Studies of other differentiation models will be informative as to whether this is a muscle specific SRA mechanism. 4.4. SRA is an essential component of p68/CTCF gene insulator complexes Adding to interactions between p68 and SRA, as a complex, these molecules have been demonstrated to participate in transcriptional insulation complexes [30]. CCCTC-binding factor (CTCF) is a DNA-binding protein involved in chromatin organisation that recruits the cohesion complex to insulator sites. Mass spectrophotometry of CTFC immunoprecipitates identified p68 in these conjugates and demonstrated SRA dependency by IP-RT-PCR. Depletion of SRA RNA reduced p68/CTCF association and reduced target gene binding thereby decreasing insulator function. 5. In vivo effects of SRA To investigate the physiological roles of SRA, transgenic mouse and Zebra fish knock down studies have been performed. The
Table 1 Direct and indirect SRA RNA interacting proteins. IP-RT-PCR, immunoprecipitation-reverse transcription-polymerase chain reaction; REMSA, RNA electrophoretic mobility shift assay; Y3H, yeast three hybrid assay; UVXL, ultraviolet light cross-linking assay. Protein
Direct interaction
SHARP p72 PUS1 PUS3 TRa & b SLIRP SF-1/NR5A1 PPARg DAX-1 p68 SRAP SRC-1 AR PR SRC-2/TIF2 ERa RAR Myo D CTCF
O O O O O O O O O
Complex formation
O O O O O O O O O O
Method of detecting interaction
RNA-binding domain
Reference
In vitro binding assays, IP-RT-PCR, REMSA REMSA, IP-RT-PCR Pseudouridylation, In vitro pull down-RT-PCR Pseudouridylation In vitro binding assays, IP-RT-PCR Y3H, UVXL, REMSA, IP-RT-PCR In vitro binding, IP-RT-PCR In vitro binding In vitro binding, IP-RT-PCR IP-RT-PCR IP-RT-PCR Co-purification, IP-RT-PCR Co-purification, IP-RT-PCR Co-purification IP-RT-PCR IP-RT-PCR IP-RT-PCR IP-RT-PCR Mass Spectrophotometry, IP-RT-PCR
RRM (x3) DEAD Box None None Unique, ssRNA affinity RRM FTZ-F1 box containing region Amino-terminal N3R domain DEAD Box RNP-2 motif None None None None None None None None
[23,33] [35] [37] [38] [3,53] [23] [56] [24] [56,57] [26,35] [7] [3,23] [3] [3] [35] [35] [37] [26] [24]
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Fig. 1. SRA and its protein binding partners. Direct (black arrows) and indirect (grey) binding of SRA by various proteins produces an array of consequences ranging from nuclear receptor coactivation and repression, positive and negative effects on adipogenesis and myogenesis, gene insulation, adrenal gland gene regulation and pseudouridinylation. SLIRP and SHARP bind directly to STR7 of SRA, while indirect interactions have been shown for SRAP. Binding sites for the remainder of the molecules depicted have not been determined.
MMTV-SRA mouse bears a human, non-coding, 1262 nt SRA 2 sequence under the control of the mouse mammary tumour virus (MMTV) promoter which directs transgene transcription in mammary tissue and uterus of females and the urogenital system of males [10]. Early mammary duct development in transgenic mice is comparable with the wt however, mature virgin mice showed aberrant mammary gland development characterised by ductal ectasia, acinar hyperplasia and intraductal proliferation of luminal epithelial cells with increased apoptosis in the stratified epithelia. Precocious ductalealveolar development during pregnancy was observed in transgenic mice implicating heightened PR activity. This was supported by elevated expression of the NR in transgenic mammary tissue consistent with SRA coactivating ER activity. Disturbances were also seen in male tissues with their seminal vesicles having a “fist” like gross morphology compared with the lobular, horn shape of the wt structure. Given the coactivating nature of SRA ncRNA on NR activity, particularly those associated with fertility, it was not surprising that both male and female transgenic mice have reduced fertility. The ability of SRA to coactivate the ER, a prime proliferative driver in breast cancer, and the dysplasia in the MMTV-SRA mammary tissue led to its closer examination. Histological assessment of mammary tissue from these mice revealed increased mitotic and apoptotic indices, however, while they displayed sporadic preneoplastic lesions such as excessive proliferation of branching tubules, frank malignancies were not reported. Reasoning that elevated SRA transcription was not enough to provoke metastasis, the MMTV-SRA mouse was crossed with MMTV-ras mice. Curiously, the resultant progeny had a lower incidence of mammary neoplasia than the monogenic carriers [10]. This unexpected result suggested that rather than SRA promoting breast cancer development, it appeared to oppose it. Further in vivo studies involving enforced SRA expression in all tissues may give
a clearer indication of its activities particularly given its influence on adipose, muscle and uritogenital tissues. Following linkage disequilibrium studies aimed at identifying genes associated with dilated cardiac myopathy in humans, SRA was highlighted as a candidate [31]. To investigate this possibility, a morpholino antisense approach was utilised to deplete SRA in Zebra fish from the 1e2 cell stage of development. Remarkably, impaired contractility, predominantly in the ventricular chambers and pericardial oedema was observed in treated animals [31]. Loss of SRA therefore results in disturbed cardiac development, a finding consistent with the well defined requirement of NR activity, particularly the thyroid hormone and mineralocorticoid receptors, for normal cardiac function [32]. 6. SRA mechanism of action SRA coding and ncRNAs along with SRAP act to modulate transcription but, by virtue of their different biochemical compositions, do so by different mechanisms. Numerous studies have shown that SRA RNA can coactivate NRs in a ligand-dependent manner [3,11,23,24,33,34]. As described above, stem-loop structures in the core domain of SRA RNA were predicted by in silico analysis and their involvement in coactivation assessed by cell based NR reporter assays [3,11]. It was found that while mutation of individual loops compromised coactivation by SRA, loss of individual sub-structures resulted in only partial loss of activity. As multiple molecules including transcription factors, NR coactivators, RNA helicases, gene insulator molecules (Table 1) bind either directly or indirectly to SRA at different loops, it suggests it may function as a scaffold to bring together multiple factors that regulate gene expression. AF-1 domain involvement in SRA coactivation of NR activity has been reported by multiple groups [3,34e36]. Ligand-independent
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coactivation of ERa involving MAPK and S118 of the receptor’s AF-1 domain has also been demonstrated [34]. Mammalian two-hybrid assays comparing mutant and wt ER AF-1 domains however showed alteration to S118 caused only a slight reduction in coactivation by SRA and that mutations to additional residues in region were required [36]. SRA does coactivate ERb but only in the presence of ligand and thus in an AF-2 dependent manner leading to the suggestion that its activities may involve separate AF-1 and AF-2 activities [36]. 6.1. Regulation of SRA RNA activity by pseudouridinylation An additional twist to the regulation of SRA RNA activity is provided by the actions of pseudouridine synthases (PUS) 1 and 3 [37,38]. These enzymes pseudouridinylate SRA RNA potentiating its coactivation of NR activity. PUS1 augments RARg, TR, ER, GR, AR and PR-B activity while PUS3 has similar effects but does not increase sex steroid receptor activity. PUS molecules also differ in their SRA target sites and cause different patterns of modification which may influence coactivator binding. It has been noted that a U206A mutation results in hyperpseudouridinylation of SRA causing it to become a transcriptional repressor leading to the suggestion that PUS proteins may influence SRA coactivator/ repressor switching [38]. Mutation to PUS1 has been associated with mitochondrial myopathy and sideroblastic anaemia suggesting a defect in SRA processing could contribute to these diseases [39e41]. 6.2. SRAP binds its own RNA (SRA) and acts as a transcriptional repressor It has now been shown that SRAP binds its own RNA [7]. SRA RNA was detected by RT-PCR following immunoprecipitation with anti-SRAP antibodies from both human and mouse muscle samples. This interaction was dependent on 1) the presence of STR7 in the SRA transcripts and 2) an RNP-2 like motif, similar to that in RRMRNA-binding motifs, in SRAP. Although this domain in SRAP is well conserved across species, further binding studies are required to confirm this is a direct interaction between these molecules. In order to further understand SRAP’s functional role, ChIPeChip arrays have been used to identify regions of the genome with which SRAP associates. The absence of a defined DBD in SRAP suggests it does not bind DNA directly and so requires binding partners with such domains. Studies in MCF-7 breast cancer cells stably expressing tagged SRA protein have identified a long list of such molecules [42]. As may be predicted, the most highly represented are those associated with NR target genes, specifically for NR subfamily 2 factors, GR and PPARs. In addition, GATA and AP1 sites were also identified. Subsequent in vitro two-hybrid assays showed recruitment of SRAP to the GAL4-RE reduced reporter gene activity and that HDAC2 was immunoprecipitated with SRAP. Further detailed discussion of SRAP and its interactions may be found in [18]. 7. Other SRA-binding partners 7.1. SLIRP binds SRA and is a potent NR corepressor Reasoning that SRA-binding molecules may themselves be NR coregulators, our laboratory sought to identify novel SRA interactors by yeast 3-hybrid (Y3H) analysis. Loop structures that had previously been demonstrated to participate in coactivation [11] were used to screen a primary breast cancer tissue derived library [23]. Both STR1 and STR7 were effective baits and were bound by different molecules, the majority of which contained RNA-binding domains. One molecule however, now referred to as
SLIRP, had not been previously characterised so its properties were investigated further. SLIRP codes for a 109 aa human protein that is well conserved between species. Its amino-terminal codes for a putative mitochondrial signal while its core region contains an RNA recognition motif (RRM) RNA-binding domain. Over expression of SLIRP represses the activities of NRs as assessed by both reporter assays and changes in endogenous gene transcription. Conversely, potentiation of NR target gene transcription results from SLIRP knock down. ChIP studies confirm SLIRP’s presence in conjugates at NR binding sites in the absence of ligand which diminishes with hormone treatment. The binding of SLIRP to the STR7 sub-structure of SRA has been demonstrated by Y3H, RNA electrophoretic mobility shift assay and immunoprecipitation-reverse transcriptase-PCR assays [23]. Mutation to SLIRP’s RRM domain abrogated its ability to repress SRA-mediated NR coactivation. Further, SLIRP did not repress NR activity following cotransfection with a STR7 mutant SRA consistent with it binding this loop. It is intriguing that SLIRP, SHARP and SRAP each target STR7 and act as repressors. The affinity of each repressor for a common domain may point to SRA playing a general role in coactivation with the differential expression of these repressors regulating its activity in a tissue/temporal specific manner. Although ChIP studies confirmed the presence of SLIRP in the nucleus, immunofluorescence show the majority of this protein is in the mitochondria [23]. While unexpected, this result is consistent with SLIRP having a mitochondrial localisation signal in its amino-terminal, epitope capping of which results in its altered localisation. Finding SLIRP in the mitochondria is consistent with the detection in the same organelle of multiple NRs including Nur77/TR3 [43], ER [44], GR [45], and TR [46] along with NR coregulators NCOA6 [47] PGC-1a [48]. Examples of the importation of nuclear encoded RNAs into the mitochondria are limited but have been identified in yeast, plants and mammals including humans [49] and references cited therein. As both NRs and coregulators have been found in the mitochondria, it is exciting to speculate that SRA RNA may also be imported and modulate transcription within this organelle. In this context, its interesting to note two recent reports in which SLIRP is implicated in mitochondrial biology. In the first, SLIRP was shown to regulate the expression of key mitochondrial mRNAs (mtRNAs) [50]. This group showed that depletion of SLIRP reduced oxidative phosphorylation activity and mtRNAs coding for complex I and IV proteins. In a second report investigating the biology of the Leucine-Rich Pentatrico-Repeat Containing protein (LRPPRC), mutation of which leads to mitochondrial dysfunction, SLIRP was shown to regulate mitochondrial gene expression [51]. Taken together these data suggest SLIRP may play an important role in mitochondrial biology, potentially regulating energy metabolism and apoptosis, especially in high energydemand tissues. 7.2. SHARP, a SRA-binding NR corepressor SHARP (SMRT/HDAC1 associated repressor protein) is an NR corepressor that interacts with SRA in vitro and contains three RRMs required to repress SRA-augmented E2-induced transactivation [33]. In the initial description of SHARP as an NR corepressor, the nature of the interaction with SRA was not defined. However, the identification of high aa sequence homology between SHARP and SLIRP, especially in their RRM domains, prompted evaluation of SHARP’s capacity to target SRA STR7. We found that recombinant SHARP bound this region of SRA avidly [23], which suggested a functional interaction may exist between these molecules in vivo. In functional studies with an E2-responsive reporter, we were also able to show that when SLIRP was cotransfected with
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SHARP and SRA, an additional 2-fold repression of SRA-augmented coactivation was observed [23]. Thus, SHARP and SLIRP appear to act in an additive fashion to enhance repression of NR activity, based on their capacity to target a specific stem-loop sub-structure of SRA, STR7.
examples of the breadth of SRA’s effects, and its capacity to interact with “classical” NRs as well as “orphan” NRs to mediate key events at the transcriptional level.
7.3. Thyroid hormone receptors bind SRA
The continuing characterisation of SRA is yielding greater understanding of its activities. While initially defined as a ncRNA coactivator of steroid activity, it is clear that its functions now extend beyond NR signalling and it is involved in the regulation of non-NR activities. The involvement of SRA in regulating metabolism, adipogenesis, myogenesis and chromatin organisation adds to its repertoire of pathways influenced and tissues affected. Identification of multiple coding and ncRNAs, the proportion of which appears to influence differentiation and potentially neoplastic pathways, requires further investigation as to the factors that regulate their production. The discovery of SLIRP, a novel RRMcontaining SRA-binding protein that regulates signalling by multiple NRs, and also has a role in the regulation of mitochondrial function, has provided new insight into the multiple roles that some NR coregulators can be engaged in. Understanding the interactions between the three known SRA STR7-binding proteins and how they regulate SRA-mediated transactivation in various tissues is an important goal. Similarly, investigation of SRAP, and in particular its binding partners, will greatly enhance our understanding of its regulation of transcription and regulation of its own expression. Furthermore, further assessment of SRA RNA and protein expression in tumour samples is warranted, as they may prove to be useful in clinical prognosis. In sum, the SRA gene retains its unique position amongst the NR coregulators with its RNA and protein products both influencing transcription the clinical impact of which we are only just beginning to realise.
The TRa and TRb NRs are encoded by separate genes and produce several proteins via alternate splicing and promoter usage [52]. The amino-terminal 370 aas of TRa1 and 2 proteins are identical but the carboxy-terminal 40 aas of TRa1 differ from the last 122 aa of TRa2. TRa2 does not bind triiodothyronine (T3) and transactivate TR target gene expression. However, all three forms can bind SRA, via a 41 aa region between their second zinc finger and ligand-binding domains [53,54]. Interestingly, phosphorylation of TRa2 at CK2 kinase sites abrogates SRA binding and redistributes the majority of TRa2 from being predominantly nuclear to mainly cytoplasmic. However, phosphorylation of TRa1 has little impact on its ability to bind SRA or alter its nuclear localisation. These data support a model whereby SRA is bound by the transcriptionally silent non-phosphorylated TRa2 in the nucleus, acting as a repressor by sequestering SRA in inactive NR complexes. However, following phosphorylation, TRa2 would lose its ability to bind SRA and exit the nucleus thereby releasing SRA for participation in coactivator complexes. Further studies are required to validate this intriguing model. 7.4. PPARs and SRA The PPAR NRs (a, g, d) heterodimerize with retinoid X receptors (RXRs) and once bound by low affinity ligands (from dietary fat or intracellular metabolism) activate transcription of a range of genes involved in lipid metabolism, transport and storage in specific tissues [55]. There is compelling evidence for roles of each of the PPARs in metabolism in key target organs, including the liver, muscle, adipose tissue, macrophages and more recently the b-cells of the pancreas. Interestingly, SRA has recently been shown to regulate adipogenesis and enhance insulin sensitivity, in part via binding to PPARg [24]. As a ncRNA, SRA promoted differentiation of mesenchymal cells into preadipocytes, and regulated key genes in the insulin and TNFa signalling pathways. Similarly, in HeLa cells, SRA has been shown to regulate multiple metabolic genes, including INSIG-1, ABCA1 and a glucose transporter [19]. Notably, SRA is expressed widely in mammals, particularly in high energydemand tissues such as liver, heart and muscle. These novel interactions between SRA and key regulators of metabolic pathways suggest a previously unrecognised role for SRA in metabolism acting via direct interactions with the PPARg. 7.5. Dax-1, SF-1 and SRA Through its interactions with the orphan NRs Steroidogenic factor 1 (SF-1) and Dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (Dax1) SRA influences adrenal function [56]. SF-1 and Dax-1 regulate adrenal gland function and sexual development. Dax-1 binds to SF1 and repressing its target gene expression. SF-1 and Dax-1 can both bind to SRA, and coactivation by Dax-1 is abolished with SRA knock down, resulting in significant changes in expression of downstream adrenal genes [56]. These findings provided an unrealised pivotal role for SRA in steroidogenesis and adrenal function. Additional data from the same group shows that Dax-1 augmentation of Liver homologue receptor 1 (LRH-1)-mediated Oct4 activation is SRA-dependent [57]. These data provide further
8. Future directions
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