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Ingrid Ehrmann,
Caroline Dalgliesh,
Yilei Liu,
Marina Danilenko,
Moira Crosier,
Lynn Overman,
Helen M Arthur,
Susan Lindsay,
Gavin J Clowry,
Julian P Venables,
Philippe Fort, David J Elliott
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ABSTRACT: The RNA binding protein T-STAR was created following a gene triplication 520-610 million years ago, which also produced its two parologs Sam68 and SLM-1. Here we have created a T-STAR null mouse to identify the endogenous functions of this RNA binding protein. Mice null for T-STAR developed normally and were fertile, surprisingly, given the high expression of T-STAR in the testis and the brain, and the known infertility and pleiotropic defects of Sam68 null mice. Using a transcriptome-wide search for splicing targets in the adult brain, we identified T-STAR protein as a potent splicing repressor of the alternatively spliced segment 4 (AS4) exons from each of the Neurexin1-3 genes, and exon 23 of the Stxbp5l gene. T-STAR protein was most highly concentrated in forebrain-derived structures like the hippocampus, which also showed maximal Neurexin1-3 AS4 splicing repression. In the absence of endogenous T-STAR protein, Nrxn1-3 AS4 splicing repression dramatically decreased, despite physiological co-expression of Sam68. In transfected cells Neurexin3 AS4 alternative splicing was regulated by either T-STAR or Sam68 proteins. In contrast, Neurexin2 AS4 splicing was only regulated by T-STAR, through a UWAA-rich response element immediately downstream of the regulated exon conserved since the radiation of bony vertebrates. The AS4 exons in the Nrxn1 and Nrxn3 genes were also associated with distinct patterns of conserved UWAA repeats. Consistent with an ancient mechanism of splicing control, human T-STAR protein was able to repress splicing inclusion of the zebrafish Nrxn3 AS4 exon. Although Neurexin1-3 and Stxbp5l encode critical synaptic proteins, T-STAR null mice had no detectable spatial memory deficits, despite an almost complete absence of AS4 splicing repression in the hippocampus. Our work identifies T-STAR as an ancient and potent tissue-specific splicing regulator that uses a concentration-dependent mechanism to co-ordinately regulate regional splicing patterns of the Neurexin1-3 AS4 exons in the mouse brain.
PLoS Genetics 04/2013; 9(4):e1003474. · 8.69 Impact Factor
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ABSTRACT: The splicing regulator protein Tra2β is conserved between humans and insects and is essential for mouse development. Recent identification of physiological RNA targets has started to uncover molecular targets and mechanisms of action of Tra2β. At a transcriptome-wide level, Tra2β protein binds a matrix of AGAA-rich sequences mapping frequently to exons. Particular tissue-specific alternatively spliced exons contain high concentrations of high scoring Tra2β-binding sites and bind Tra2β strongly in vitro. These top exons were also activated for splicing inclusion in cellulo by co-expression of Tra2β protein and were significantly down-regulated after genetic depletion of Tra2β. Tra2β itself seems to be fairly evenly expressed across several different mouse tissues. In the present paper, we review the properties of Tra2β and its regulated target exons, and mechanisms through which this fairly evenly expressed alternative splicing regulator might drive tissue-specific splicing patterns.
Biochemical Society Transactions 08/2012; 40(4):784-8. · 3.71 Impact Factor
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Sushma Grellscheid,
Caroline Dalgliesh,
Markus Storbeck,
Andrew Best,
Yilei Liu,
Miriam Jakubik,
Ylva Mende,
Ingrid Ehrmann,
Tomaz Curk,
Kristina Rossbach,
Cyril F Bourgeois,
James Stévenin,
David Grellscheid,
Michael S Jackson,
Brunhilde Wirth, David J Elliott
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ABSTRACT: Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2β (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2β is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2β binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10(fl/fl); Nestin-Cre(tg/+)). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2β regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2β binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2β protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2β. Versions of Tra2β lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2β protein.
PLoS Genetics 12/2011; 7(12):e1002390. · 8.69 Impact Factor
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Haya H Al-Balool,
David Weber,
Yilei Liu,
Mark Wade,
Kamlesh Guleria,
Pitsien Lang Ping Nam,
Jake Clayton,
William Rowe,
Jonathan Coxhead,
Julie Irving, David J Elliott,
Andrew G Hall,
Mauro Santibanez-Koref,
Michael S Jackson
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ABSTRACT: In silico analyses have established that transcripts from some genes can be processed into RNAs with rearranged exon order relative to genomic structure (post-transcriptional exon shuffling, or PTES). Although known to contribute to transcriptome diversity in some species, to date the structure, distribution, abundance, and functional significance of human PTES transcripts remains largely unknown. Here, using high-throughput transcriptome sequencing, we identify 205 putative human PTES products from 176 genes. We validate 72 out of 112 products analyzed using RT-PCR, and identify additional PTES products structurally related to 61% of validated targets. Sequencing of these additional products reveals GT-AG dinucleotides at >95% of the splice junctions, confirming that they are processed by the spliceosome. We show that most PTES transcripts are expressed in a wide variety of human tissues, that they can be polyadenylated, and that some are conserved in mouse. We also show that they can extend into 5' and 3' UTRs, consistent with formation via trans-splicing of independent pre-mRNA molecules. Finally, we use real-time PCR to compare the abundance of PTES exon junctions relative to canonical exon junctions within the transcripts from seven genes. PTES exon junctions are present at <0.01% to >90% of the levels of canonical junctions, with transcripts from MAN1A2, PHC3, TLE4, and CDK13 exhibiting the highest levels. This is the first systematic experimental analysis of PTES in human, and it suggests both that the phenomenon is much more widespread than previously thought and that some PTES transcripts could be functional.
Genome Research 09/2011; 21(11):1788-99. · 13.61 Impact Factor
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ABSTRACT: Tra2β regulates a number of splicing switches including activation of the human testis-specific exon HIPK3-T in the Homeodomain Interacting Protein Kinase 3 gene. By testing HIPK3-T exons of different intrinsic strengths, we found Tra2β most efficiently activated splicing inclusion of intrinsically weak exons, although these were spliced at a lower overall level. Both the RRM and N-terminal RS-rich region of Tra2β were required for splicing activation. Bioinformatic searches for splicing enhancers and repressors mapped four physically distinct exonic splicing enhancers (ESEs) within HIPK3-T, each containing the known Tra2β AGAA-rich binding site. Surprisingly disruption of each single ESE prevented Tra2β-mediated activation, although single mutated exons could still bind Tra2β protein by gel shifts and functional splicing analyses. Titration experiments indicate an additive model of HIPK3-T splicing activation, requiring availability of an array of four distinct ESEs to enable splicing activation. To enable this efficient Tra2β-mediated splicing switch to operate, a closely adjacent downstream and potentially competitive stronger 5'-splice site is actively repressed. Our data indicate that a novel arrangement of multiple mono-specific AGAA-rich ESEs coupled to a weak 5'-splice site functions as a responsive gauge. This gauge monitors changes in the specific nuclear concentration of the RNA binding protein Tra2β, and co-ordinately regulates HIPK3-T exon splicing inclusion.
Nucleic Acids Research 06/2011; 39(18):8092-104. · 8.03 Impact Factor
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Prabhakar Rajan,
Caroline Dalgliesh,
Phillippa J Carling,
Thomas Buist,
Chaolin Zhang,
Sushma N Grellscheid,
Kelly Armstrong,
Jacqueline Stockley,
Cedric Simillion,
Luke Gaughan,
Gabriela Kalna,
Michael Q Zhang,
Craig N Robson,
Hing Y Leung, David J Elliott
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ABSTRACT: Androgens drive the onset and progression of prostate cancer (PCa) by modulating androgen receptor (AR) transcriptional activity. Although several microarray-based studies have identified androgen-regulated genes, here we identify in-parallel global androgen-dependent changes in both gene and alternative mRNA isoform expression by exon-level analyses of the LNCaP transcriptome. While genome-wide gene expression changes correlated well with previously-published studies, we additionally uncovered a subset of 226 novel androgen-regulated genes. Gene expression pathway analysis of this subset revealed gene clusters associated with, and including the tyrosine kinase LYN, as well as components of the mTOR (mammalian target of rapamycin) pathway, which is commonly dysregulated in cancer. We also identified 1279 putative androgen-regulated alternative events, of which 325 (∼25%) mapped to known alternative splicing events or alternative first/last exons. We selected 30 androgen-dependent alternative events for RT-PCR validation, including mRNAs derived from genes encoding tumour suppressors and cell cycle regulators. Of seven positively-validating events (∼23%), five events involved transcripts derived from alternative promoters of known AR gene targets. In particular, we found a novel androgen-dependent mRNA isoform derived from an alternative internal promoter within the TSC2 tumour suppressor gene, which is predicted to encode a protein lacking an interaction domain required for mTOR inhibition. We confirmed that expression of this alternative TSC2 mRNA isoform was directly regulated by androgens, and chromatin immunoprecipitation indicated recruitment of AR to the alternative promoter region at early timepoints following androgen stimulation, which correlated with expression of alternative transcripts. Together, our data suggest that alternative mRNA isoform expression might mediate the cellular response to androgens, and may have roles in clinical PCa.
PLoS ONE 01/2011; 6(12):e29088. · 4.09 Impact Factor
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ABSTRACT: The RNA binding protein Sam68 (Src-associated in mitosis 68 kD) is implicated in cell signalling, transcriptional regulation, pre-mRNA splicing, and is overexpressed and/or hyperphosphorylated in breast, prostate, and renal cancers. Sam68 has roles in normal breast development; however, a study by Song et al published in this issue of The Journal of Pathology reports overexpression of nuclear and cytoplasmic Sam68 protein in a large cohort of clinical breast tumours, implicating Sam68 as a potential prognostic indicator and target for therapy. In breast cancer cells, nuclear Sam68 protein might affect the expression of cancer-relevant genes and/or modulate exon splicing patterns in a dose-dependent manner. Sam68-regulated expression of alternative transcripts may help drive mammary tumourigenesis. The high levels of cytoplasmic Sam68 protein observed in breast cancer cells could also impact on cellular signalling pathways important for mammary tumour cell biology.
The Journal of Pathology 11/2010; 222(3):223-6. · 6.32 Impact Factor
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ABSTRACT: Nuclear RNA processing is a critical stage in eukaryotic gene expression, and is controlled in part by the expression and concentration of nuclear RNA-binding proteins. Different nuclear RNA-binding proteins are differentially expressed in different cells, helping the spliceosome to decode pre-mRNAs into alternatively spliced mRNAs. Recent post-genomic technology has exposed the complexity of nuclear RNA processing, and is starting to reveal the mechanisms and rules through which networks of RNA-binding proteins can regulate multiple parallel pathways. Identification of multiple parallel processing pathways regulated by nuclear RNA-binding proteins is leading to a systems-wide understanding of the rules and consequences of alternative nuclear RNA processing.
Biochemical Society Transactions 02/2010; 38(Pt 1):237-41. · 3.71 Impact Factor
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Chantal Sellier,
Frédérique Rau,
Yilei Liu,
Flora Tassone,
Renate K Hukema,
Renata Gattoni,
Anne Schneider,
Stéphane Richard,
Rob Willemsen, David J Elliott,
Paul J Hagerman,
Nicolas Charlet-Berguerand
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ABSTRACT: Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder caused by expansion of 55-200 CGG repeats in the 5'-UTR of the FMR1 gene. FXTAS is characterized by action tremor, gait ataxia and impaired executive cognitive functioning. It has been proposed that FXTAS is caused by titration of RNA-binding proteins by the expanded CGG repeats. Sam68 is an RNA-binding protein involved in alternative splicing regulation and its ablation in mouse leads to motor coordination defects. Here, we report that mRNAs containing expanded CGG repeats form large and dynamic intranuclear RNA aggregates that recruit several RNA-binding proteins sequentially, first Sam68, then hnRNP-G and MBNL1. Importantly, Sam68 is sequestered by expanded CGG repeats and thereby loses its splicing-regulatory function. Consequently, Sam68-responsive splicing is altered in FXTAS patients. Finally, we found that regulation of Sam68 tyrosine phosphorylation modulates its localization within CGG aggregates and that tautomycin prevents both Sam68 and CGG RNA aggregate formation. Overall, these data support an RNA gain-of-function mechanism for FXTAS neuropathology, and suggest possible target routes for treatment options.
The EMBO Journal 02/2010; 29(7):1248-61. · 9.20 Impact Factor
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ABSTRACT: RBMY is a male germline RNA binding protein and potential alternative splicing regulator, but the lack of a convenient biological system has made its cellular functions elusive. We found that human RBMY fused to green fluorescent protein was strictly nuclear in transfected cells, but spatially enriched in areas around nuclear speckles with some components of the exon junction complex (EJC). Human RBMY (hRBMY) and the EJC components Magoh and Y14 also physically interacted but, unlike these two proteins, hRBMY protein did not shuttle to the cytoplasm. In addition, it relocalised into nucleolar caps after inhibition of RNA polymerase II transcription. Protein interactions were also detected between RBMY and splicing factors 9G8 and transformer-2 protein homolog beta (Tra2-beta), mediated by multiple regions of the RBMY protein that contain serine/arginine-rich dipeptides, but not by the single region lacking such dipeptides. These interactions modulated the splicing of several pre-mRNAs regulated by 9G8 and Tra2-beta. Importantly, ectopic expression of hRBMY stimulated the inclusion of a testis-enriched exon from the Acinus gene, whereas 9G8 and Tra2-beta repressed this exon. We propose that hRBMY associates with regions of the nucleus enriched in nascent RNA and participates in the regulation of specific splicing events in the germline by modulating the activity of constitutively expressed splicing factors.
Journal of Cell Science 01/2010; 123(Pt 1):40-50. · 6.11 Impact Factor
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ABSTRACT: Spermatogenesis is one of the few major developmental pathways which are still ongoing in the adult. In this chapter we review the properties of Sam68 and T-STAR, which are the STAR proteins functionally implicated in mammalian spermatogenesis. Sam68 is a ubiquitously expressed member of the STAR family, but has an essential role in spermatogenesis. Sam68 null mice are male infertile and at least in part this is due to a failure in important translational controls that operate during and after meiosis. The homologous T-STAR protein has a much more restricted anatomic expression pattern than Sam68, with highest levels seen in the testis and the developing brain. The focus of this chapter is the functional role of Sam68 and T-STAR proteins in male germ cell development. Since these proteins are known to have many cellular functions we extrapolate from other cell types and tissues to speculate on each of their likely functions within male germ cells, including control of alternative pre-mRNA splicing patterns in male germ cells.
Advances in experimental medicine and biology 01/2010; 693:67-81. · 1.09 Impact Factor
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ABSTRACT: The human testis has almost as high a frequency of alternative splicing events as brain. While not as extensively studied as brain, a few candidate testis-specific splicing regulator proteins have been identified, including the nuclear RNA binding proteins RBMY and hnRNP G-T, which are germ cell-specific versions of the somatically expressed hnRNP G protein and are highly conserved in mammals. The splicing activator protein Tra2beta is also highly expressed in the testis and physically interacts with these hnRNP G family proteins. In this study, we identified a novel testis-specific cassette exon TLE4-T within intron 6 of the human transducing-like enhancer of split 4 (TLE4) gene which makes a more transcriptionally repressive TLE4 protein isoform. TLE4-T splicing is normally repressed in somatic cells because of a weak 5' splice site and surrounding splicing-repressive intronic regions. TLE4-T RNA pulls down Tra2beta and hnRNP G proteins which activate its inclusion. The germ cell-specific RBMY and hnRNP G-T proteins were more efficient in stimulating TLE4-T incorporation than somatically expressed hnRNP G protein. Tra2b bound moderately to TLE4-T RNA, but more strongly to upstream sites to potently activate an alternative 3' splice site normally weakly selected in the testis. Co-expression of Tra2beta with either hnRNP G-T or RBMY re-established the normal testis physiological splicing pattern of this exon. Although they can directly bind pre-mRNA sequences around the TLE4-T exon, RBMY and hnRNP G-T function as efficient germ cell-specific splicing co-activators of TLE4-T. Our study indicates a delicate balance between the activity of positive and negative splicing regulators combinatorially controls physiological splicing inclusion of exon TLE4-T and leads to modulation of signalling pathways in the testis. In addition, we identified a high-affinity binding site for hnRNP G-T protein, showing it is also a sequence-specific RNA binding protein.
PLoS Genetics 11/2009; 5(11):e1000707. · 8.69 Impact Factor
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ABSTRACT: The biological diversity of prostate cancer confounds standardization of therapy. Advances in molecular profiling suggest that differences in the genetic composition of tumors significantly contribute to the complexity of the disease. Alternative pre-mRNA splicing is a key genetic process underlying biological diversity. During alternative splicing, coding and noncoding regions of a single gene are rearranged to generate several messenger RNA transcripts yielding distinct protein isoforms with differing biological functions. Misregulation of the splicing machinery and mutations in key regulatory elements affect splicing of cancer-relevant genes. In prostate cancer, aberrant and alternative splicing generates proteins that influence cell phenotypes and survival of patients. Splicing events may be exploited for clinical benefit, and technological advances are beginning to uncover novel biomarkers and therapeutic targets. Since splicing mediates information transfer from the genome to the proteome, it adds an important dimension to '-omics'-based molecular signatures used to individualize care of patients.
Nature Reviews Urology 09/2009; 6(8):454-60. · 4.41 Impact Factor
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ABSTRACT: Prostate cancer is the most common cancer seen in aging males in the Western world, and is a major clinical challenge in uro-oncology due to biological heterogeneity. Recent advances in molecular medicine suggest that the genetic composition of a prostate tumor contributes significantly to the complexity of the disease. An important genetic mechanism underlying biological diversity is alternative pre-mRNA splicing, which is thought to affect approximately 95% of transcripts derived from protein-encoding genes. During alternative splicing, coding (exons) and non-coding (introns) regions of pre-messenger RNA (pre-mRNA) transcripts derived from a single gene are rearranged to generate several mRNAs species, which are translated into distinct protein isoforms with differing biological functions. Recent emerging evidence suggests that prostate cancer-specific aberrant and alternative splicing may contribute to the biological heterogeneity of the disease. Furthermore, identification of prostate cancer-specific splice variants may yield novel biomarkers and targets for therapy to improve patient care and clinical outcome.
Discovery medicine 08/2009; 8(41):74-80.
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ABSTRACT: Medulloblastoma is the most common malignant brain tumour of childhood. The identification of critical genes involved in its pathogenesis will be central to advances in our understanding of its molecular basis, and the development of improved therapeutic approaches.
We performed a SNP-array based genome-wide copy number analysis in medulloblastoma cell lines, to identify regions of genomic amplification and homozygous deletion, which may harbour critical disease genes. A series of novel and established medulloblastoma defects were detected (MYC amplification (n = 4), 17q21.31 high-level gain (n = 1); 9p21.1-p21.3 (n = 1) and 6q23.1 (n = 1) homozygous deletion). Most notably, a novel recurrent region of genomic amplification at 8q24.22-q24.23 was identified (n = 2), and selected for further investigation. Additional analysis by interphase fluorescence in situ hybridisation (iFISH), PCR-based mapping and SNP-array revealed this novel amplification at 8q24.22-q24.23 is independent of MYC amplification at 8q24.21, and is unique to medulloblastoma in over 800 cancer cell lines assessed from different tumour types, suggesting it contains key genes specifically involved in medulloblastoma development. Detailed mapping identified a 3Mb common minimal region of amplification harbouring 3 coding genes (ZFAT1, LOC286094, KHDRBS3) and two genes encoding micro-RNAs (hsa-miR-30b, hsa-miR-30d). Of these, only expression of hsa-miR-30b, hsa-miR-30d and KHDRBS3 correlated with copy number status, and all three of these transcripts also displayed evidence of elevated expression in sub-sets of primary medulloblastomas, measured relative to the normal cerebellum.
These data implicate hsa-miR-30b, hsa-miR-30d and KHDRBS3 as putative oncogenic target(s) of a novel recurrent medulloblastoma amplicon at 8q24.22-q24.23. Our findings suggest critical roles for these genes in medulloblastoma development, and further support the contribution of micro-RNA species to medulloblastoma pathogenesis.
PLoS ONE 02/2009; 4(7):e6159. · 4.09 Impact Factor
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ABSTRACT: The human testis has almost as high a frequency of alternative splicing events as brain. While not as extensively studied as brain, a few candidate testis-specific splicing regulator proteins have been identified, including the nuclear RNA binding proteins RBMY and hnRNP G-T, which are germ cell-specific versions of the somatically expressed hnRNP G protein and are highly conserved in mammals. The splicing activator protein Tra2b is also highly expressed in the testis and physically interacts with these hnRNP G family proteins. In this study, we identified a novel testis-specific cassette exon TLE4-T within intron 6 of the human transducing-like enhancer of split 4 (TLE4) gene which makes a more transcriptionally repressive TLE4 protein isoform. TLE4-T splicing is normally repressed in somatic cells because of a weak 59 splice site and surrounding splicing-repressive intronic regions. TLE4-T RNA pulls down Tra2b and hnRNP G proteins which activate its inclusion. The germ cell-specific RBMY and hnRNP G-T proteins were more efficient in stimulating TLE4-T incorporation than somatically expressed hnRNP G protein. Tra2b bound moderately to TLE4-T RNA, but more strongly to upstream sites to potently activate an alternative 39 splice site normally weakly selected in the testis. Co-expression of Tra2b with either hnRNP G-T or RBMY re-established the normal testis physiological splicing pattern of this exon. Although they can directly bind pre-mRNA sequences around the TLE4-T exon, RBMY and hnRNP G-T function as efficient germ cell-specific splicing co-activators of TLE4-T. Our study indicates a delicate balance between the activity of positive and negative splicing regulators combinatorially controls physiological splicing inclusion of exon TLE4-T and leads to modulation of signalling pathways in the testis. In addition, we identified a high-affinity binding site for hnRNP G-T protein, showing it is also a sequence-specific RNA binding protein. Copyright: ß 2009 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the BBSRC (grant number BB/D013917/1) and the Wellcome Trust (grant number WT080368MA) awarded to DJE, a Royal Society Joint International Grant awarded to DJE and JS, and the EURASNET NoE (European Commission FP6) to JS. YL was partly supported by an ORSAS studentship. SP and YHS were supported by NSFC (30771100) and 973 project (2010CB126306). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
PLoS Genet. 01/2009; 5.
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Emma L Clark,
Anne Coulson,
Caroline Dalgliesh,
Prabhakar Rajan,
Samantha M Nicol,
Stewart Fleming,
Rakesh Heer,
Luke Gaughan,
Hing Y Leung, David J Elliott,
Frances V Fuller-Pace,
Craig N Robson
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ABSTRACT: The androgen receptor (AR) is a member of the nuclear steroid hormone receptor family and is thought to play an important role in the development of both androgen-dependent and androgen-independent prostatic malignancy. Elucidating roles by which cofactors regulate AR transcriptional activity may provide therapeutic advancement for prostate cancer (PCa). The DEAD box RNA helicase p68 (Ddx5) was identified as a novel AR-interacting protein by yeast two-hybrid screening, and we sought to examine the involvement of p68 in AR signaling and PCa. The p68-AR interaction was verified by colocalization of overexpressed protein by immunofluorescence and confirmed in vivo by coimmunoprecipitation in the PCa LNCaP cell line. Chromatin immunoprecipitation in the same cell line showed AR and p68 recruitment to the promoter region of the androgen-responsive prostate-specific antigen (PSA) gene. Luciferase reporter, minigene splicing assays, and RNA interference (RNAi) were used to examine a functional role of p68 in AR-regulated gene expression, whereby p68 targeted RNAi reduced AR-regulated PSA expression, and p68 enhanced AR-regulated repression of CD44 splicing (P = 0.008). Tyrosine phosphorylation of p68 was found to enhance coactivation of ligand-dependent transcription of AR-regulated luciferase reporters independent of ATP-binding. Finally, we observe increased frequency and expression of p68 in PCa compared with benign tissue using a comprehensive prostate tissue microarray (P = 0.003; P = 0.008). These findings implicate p68 as a novel AR transcriptional coactivator that is significantly overexpressed in PCa with a possible role in progression to hormone-refractory disease.
Cancer Research 11/2008; 68(19):7938-46. · 7.86 Impact Factor
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ABSTRACT: The mechanisms involved in the transition from androgen-dependent to androgen-independent PCa (prostate cancer) remain largely undefined. The AR (androgen receptor) is an androgen-dependent transcription factor and is thought to play an important role in the development of both androgen-dependent and -independent prostatic malignancy. AR-mediated transcription is regulated by the binding of various cofactor proteins to the AR that facilitate transcriptional initiation and elongation. Elucidating the mechanisms by which cofactors regulate AR transcriptional activity may reveal the therapeutic potential of cofactors in PCa. Current models of gene expression indicate that transcription and RNA processing are tightly coupled. In this review, we discuss how the ATP-dependent DEAD box RNA helicase p68, which has established roles in transcription and RNA processing, may function as an 'adaptor' or coupling protein to facilitate cross-talk between transcription and RNA processing in AR-regulated genes by controlling the rate of transcriptional initiation/elongation.
Biochemical Society Transactions 07/2008; 36(Pt 3):546-7. · 3.71 Impact Factor
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ABSTRACT: Sam68 (Src-associated in mitosis 68 kDa) is the prototypical member of the STAR (signal transducer and activator of RNA) family of RNA-binding proteins. Sam68 is implicated in a number of cellular processes including signal transduction, transcription, RNA metabolism, cell cycle regulation and apoptosis. In the present review, we summarize the functions of Sam68 as a transcriptional and post-transcriptional regulator of gene expression, with particular relevance to cancer.
Biochemical Society Transactions 07/2008; 36(Pt 3):505-7. · 3.71 Impact Factor
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Ingrid Ehrmann,
Caroline Dalgliesh,
Aikaterini Tsaousi,
Maria Paola Paronetto,
Bettina Heinrich,
Ralf Kist,
Paul Cairns,
Weiping Li,
Christian Mueller,
Michael Jackson,
Heiko Peters,
Karim Nayernia,
Philippa Saunders,
Michael Mitchell,
Stefan Stamm,
Claudio Sette, David J Elliott
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ABSTRACT: Human HNRNPGT, encoding the protein hnRNP G-T, is one of several autosomal retrogenes derived from RBMX. It has been suggested that HNRNPGT functionally replaces the sex-linked RBMX and RBMY genes during male meiosis. We show here that during normal mouse germ cell development, hnRNP G-T protein is strongly expressed during and after meiosis when proteins expressed from Rbmx or Rbmx-like genes are absent. Amongst these Rbmx-like genes, DNA sequence analyses indicate that two other mouse autosomal Rbmx-derived retrogenes have evolved recently in rodents and one already shows signs of degenerating into a non-expressed pseudogene. In contrast, orthologues of Hnrnpgt are present in all four major groups of placental mammals. The sequence of Hnrnpgt is under considerable positive selection suggesting it performs an important germ cell function in eutherians. To test this, we inactivated Hnrnpgt in ES cells and studied its function during spermatogenesis in chimaeric mice. Although germ cells heterozygous for this targeted allele could produce sperm, they did not contribute to the next generation. Chimaeric mice with a high level of mutant germ cells were infertile with low sperm counts and a high frequency of degenerate seminiferous tubules and abnormal sperm. Chimaeras made from a 1:1 mix of targeted and wild-type ES cell clones transmitted wild-type germ cells only. Our data show that haploinsufficiency of Hnrnpgt results in abnormal sperm production in the mouse. Genetic defects resulting in reduced levels of HNRNPGT could, therefore, be a cause of male infertility in humans.
Human Molecular Genetics 07/2008; 17(18):2803-18. · 7.64 Impact Factor
