RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing.

Histone deacetylases (HDACs) and lysine acetyltransferases (KATs) catalyze dynamic histone acetylation at regulatory and coding regions of transcribed genes. Highly phosphorylated HDAC2 is recruited within corepressor complexes to regulatory regions, while the nonphosphorylated form is associated with the gene body. In this study, we characterized the nonphosphorylated HDAC2 complexes recruited to the transcribed gene body and explored the function of HDAC-complex-mediated dynamic histone acetylation. HDAC1 and 2 were coimmunoprecipitated with several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1) which has roles in alternative splicing. The co-chromatin immunoprecipitation of HDAC1/2 and SRSF1 to the gene body was RNA-dependent. Inhibition of HDAC activity and knockdown of HDAC1, HDAC2 or SRSF1 showed that these proteins were involved in alternative splicing of MCL1. HDAC1/2 and KAT2B were associated with nascent pre-mRNA in general and with MCL1 pre-mRNA specifically. Inhibition of HDAC activity increased the occupancy of KAT2B and acetylation of H3 and H4 of the H3K4 methylated alternative MCL1 exon 2 nucleosome. Thus, nonphosphorylated HDAC1/2 is recruited to pre-mRNA by splicing factors to act at the RNA level with KAT2B and other KATs to catalyze dynamic histone acetylation of the MCL1 alternative exon and alter the splicing of MCL1 pre-mRNA.

Increasing the relative expression of endogenous non-coding Steroid Receptor RNA Activator (SRA) in human breast cancer cells using modified oligonucleotides.

Products of the Steroid Receptor RNA Activator gene (SRA1) have the unusual property to modulate the activity of steroid receptors and other transcription factors both at the RNA (SRA) and the protein (SRAP) level. Balance between these two genetically linked entities is controlled by alternative splicing of intron-1, whose retention alters SRAP reading frame. We have previously found that both fully-spliced SRAP-coding and intron-1-containing non-coding SRA RNAs co-exist in breast cancer cell lines. Herein, we report a significant (Student's t-test, P < 0.003) higher SRA-intron-1 relative expression in breast tumors with higher progesterone receptor contents. Using an antisense oligoribonucleotide, we have successfully reprogrammed endogenous SRA splicing and increased SRA RNA-intron-1 relative level in T5 breast cancer cells. This increase is paralleled by significant changes in the expression of genes such as plasminogen urokinase activator and estrogen receptor beta. Estrogen regulation of other genes, including the anti-metastatic NME1 gene, is also altered. Overall, our results suggest that the balance coding/non-coding SRA transcripts not only characterizes particular tumor phenotypes but might also, through regulating the expression of specific genes, be involved in breast tumorigenesis and tumor progression.

Steroid Receptor RNA Activator Protein (SRAP): a potential new prognostic marker for estrogen receptor-positive/node-negative/younger breast cancer patients.

INTRODUCTION: The steroid receptor RNA activator is a functional RNA suspected to participate in the mechanisms underlying breast tumor progression. This RNA is also able to encode for a protein, Steroid Receptor RNA Activator Protein (SRAP), whose exact function remains to be determined. Our aim was to assess, in a large breast cancer cohort, whether levels of this protein could be associated with outcome or established clinical parameters. METHODS: Following antibody validation, SRAP expression was assessed by tissue-microarray (TMA) analysis of 372 breast tumors. Clinical follow-up and parameters such as steroid receptor and node status were available for all the corresponding cases. Immunohistochemical scores were independently determined by three investigators and averaged. Statistical analyses were performed using standard univariate and multivariate tests. RESULTS: SRAP levels were significantly (Mann-Whitney rank sum test, P < 0.05) higher in estrogen receptor-alpha positive (ER+, n = 271), in progesterone receptor positive (PR+, n = 257) and in older patients (age > 64 years, n = 182). When considering ER+ tumors, PR+ tumors, or younger patients (< or = 64 years), cases with high SRAP expression had a significantly (Mantel-Cox test, P < 0.05) worse breast cancer specific survival (BCSS) than those with low SRAP levels. SRAP also appeared as a very powerful indicator of poor prognostic for BCSS in the subset of ER+, node negative and young breast cancer patients (Cox regression analysis, n = 60, BCSS Hazard Ratio = 8.61, P < 0.006). CONCLUSIONS: Our data suggest that SRAP levels might provide additional information on potential risk of recurrence and negative outcome in a specific set of patients with otherwise good prognosis when considering only estrogen receptor and nodal status.

Alternative splicing of the first intron of the steroid receptor RNA activator (SRA) participates in the generation of coding and noncoding RNA isoforms in breast cancer cell lines.

The Steroid Receptor RNA Activator 1 (SRA1) has originally been described as a noncoding RNA specifically activating steroid receptor transcriptional activity. We have, however, identified, in human breast tissue, exon- 1 extended SRA1 isoforms containing two initiating AUG codons and encoding a protein we called SRAP. We recently reported a decreased estrogen receptor activity in breast cancer cells overexpressing SRAP, suggesting antagonist roles played by SRA1 RNA and SRAP. SRA1 appears to be the first example of a molecule active both at the RNA and at the protein level. No data are currently available regarding the mechanisms possibly involved in the generation of coding and noncoding functional SRA1 RNAs. Using 5'-Rapid Amplification of cDNA Extremities (5'-RACE), we have herein identified several putative transcription initiation sites surrounding the second methionine codon and used to generate coding SRA1 transcripts. In the process, we also identified an alternatively spliced noncoding SRA1 transcript still containing an intron-1 sequence. Using targeted RT-PCR approaches, we confirmed the presence in breast cancer cell lines of SRA1 RNAs containing a full as well as a partial intron-1 sequence and established that the relative proportion of these RNAs varied within breast cancer cell lines. Using a "minigene" strategy, we also showed that artificial RNAs containing the SRA1 intron-1 sequence are alternatively spliced in breast cancer cell lines. Interestingly, the splicing pattern of the minigene products parallels the one of the endogenous SRA1 transcripts. Altogether, our data suggest that the primary genomic sequence in and around intron-1 is sufficient to lead to a differential splicing of this intron. We propose that alternative splicing of intron-1 is one mechanism used by breast cancer cells to regulate the balance between coding and functional noncoding SRA1 RNAs.

Idiopathic Hypogonadotropic Hypogonadism Caused by Inactivating Mutations in SRA1.

OBJECTIVE: What initiates the pubertal process in humans and other mammals is still unknown. We hypothesized that gene(s) taking roles in triggering human puberty may be identified by studying a cohort of idiopathic hypogonadotropic hypogonadism (IHH). METHODS: A cohort of IHH cases was studied based on autozygosity mapping coupled with whole exome sequencing. RESULTS: Our studies revealed three independent families in which IHH/delayed puberty is associated with inactivating SRA1 variants. SRA1 was the first gene to be identified to function through its protein as well as noncoding functional ribonucleic acid products. These products act as co-regulators of nuclear receptors including sex steroid receptors as well as SF-1 and LRH-1, the master regulators of steroidogenesis. Functional studies with a mutant SRA1 construct showed a reduced co-activation of ligand-dependent activity of the estrogen receptor alpha, as assessed by luciferase reporter assay in HeLa cells. CONCLUSION: Our findings strongly suggest that SRA1 gene function is required for initiation of puberty in humans. Furthermore, SRA1 with its alternative products and functionality may provide a potential explanation for the versatility and complexity of the pubertal process.

Intermittent hypoxia induces proteasome-dependent down-regulation of estrogen receptor alpha in human breast carcinoma.

PURPOSE: Hypoxia may influence gene expression to promote malignancy, and acute hypoxia has been shown to transiently repress estrogen receptor (ER)-alpha expression in breast cell lines. However, the effect of intermittent hypoxia, which is likely more prevalent in breast cancers, remains to be determined. EXPERIMENTAL DESIGN: ER-alpha expression was assessed by Western blot and immunohistochemistry in a selected cohort of 51 ER-alpha-positive breast carcinomas, in relation to markers of hypoxia. The effect of acute and intermittent hypoxia on ER-alpha expression was also determined in MCF7 and ZR-75 breast cell lines, together with the role of proteasome function with the proteasome inhibitor bortezomib. RESULTS: Regional loss of ER-alpha expression occurs in breast tumors and is consistently present in hypoxic regions defined by the proximity of necrosis and induction of hypoxia-induced genes carbonic anhydrase IX (CA-IX) and glucose transporter 1 (Glut-1), in both in situ (n = 29; P < 0.0001) and invasive (n = 20; P = 0.0001) carcinomas. In MCF7 and ZR-75 cells, ER-alpha is transiently down-regulated by acute hypoxia and rapidly restored by reoxygenation. However, intermittent, acute hypoxia can cause a similar down-regulation of ER-alpha that is not attributable to decreased mRNA and persists in MCF7 cells despite reoxygenation for up to 14 days. This effect occurs with no change in cell viability but a corresponding reduction in growth response to estradiol. However, ER-alpha expression can be restored by bortezomib. CONCLUSIONS: Intermittent hypoxia can cause persistent changes in proteasome function that may contribute to reduced ER-alpha expression in breast tumors and consequently to diminished response and development of resistance to endocrine therapy.

The steroid receptor RNA activator protein (SRAP) controls cancer cell migration/motility.

The steroid receptor RNA activator gene (SRA1) produces both a functional RNA (SRA) and a protein (SRAP), whose exact physiological roles remain unknown. To identify cellular processes regulated by SRAP we compared the transcriptome of Hela and MDA-MB-231 cancer cells upon depletion of the SRA/SRAP transcripts or overexpression of the SRAP protein. RNA-seq and Ontology analyses pinpointed cellular movement as potentially regulated by SRAP. Using live cell imaging, we found that SRA/SRAP depletion and SRAP overexpression lead respectively to a decrease and increase in cancer cell motility. Our results highlight for the first time a link existing between SRA1 gene expression and cell motility.

Steroid receptor RNA activator protein (SRAP) expression as a prognostic factor in ER+ human breast tumors.

BACKGROUND: The steroid receptor RNA activator protein (SRAP) is a newly described protein modulating the activity of multiple transcription factors including the estrogen receptor (ER). We have recently reported the immunodetection by Western blot of multiple SRAP peptides in breast tissue. High expression of these peptides, assessed by tissue micro-array (TMA) analysis, was associated with poor prognosis in patients whose primary tumors were ER positive (ER+). In such studies, it is recognized that intensity as well as specificity of the signal detected directly depends upon the antibody used as well as the position of the epitope recognized. To confirm the potential relevance of SRAP as a new prognostic factor, it is critical to establish whether similar results are obtained with independent antibodies. METHODS: Two commercial anti-SRAP antibodies (742A and 743A), respectively, recognizing the N- and C-terminal extremity of the protein, were first used to analyze by Western blot SRAP expression in protein extracts from frozen breast tumor tissue sections. These antibodies were further used to investigate by immunohistochemistry (IHC) SRAP location in paraffin-embedded breast tumors. Comparative TMA analysis of 170 ER+ tumors was eventually performed in order to establish the potential associations existing between SRAP expression and clinical outcome. RESULTS: Multiple SRAP peptides were differentially detected by Western blot. Both antibodies led to similar nuclear and cytoplasmic staining in breast tissue section. A solid correlation was found (Spearman r = 0.46, P < 0.001) between 742A and 743A IHC scores. Results from both antibodies independently showed that dividing expression levels into lower 25 percentile, 26-75 percentile, and highest 25 percentile demonstrated a hazard ratio (HR) of 1.82 (P = 0.0042) for 742A antibody and 1.35 (P = 0.14) for 743A antibody. When both scores are combined, double high expressor (by 742A and 743A) was associated with a poor prognosis of breast-cancer-specific survival (Mantel-Cox: P = 0.005, HR = 2.24). CONCLUSION: Overall, our data suggest the existence in breast tumor tissue of multiple SRAP-like peptides. Assessing their expression in primary breast tumors can predict clinical outcome in ER+ breast cancer patients.

Steroid Receptor RNA Activator bi-faceted genetic system: Heads or Tails?

The Steroid Receptor RNA Activator (SRA) was first identified by Lanz et al. in 1999 as a functional non-coding RNA able to co-activate steroid nuclear receptors. Since this incipient study, our understanding of SRA as a broader co-regulator of nuclear receptors as well as other transcription factors has greatly expanded. Accumulated data has now revealed the diverse roles played by this transcript in both normal biological processes such as myogenesis and adipogenesis, as well as in mechanisms underlying diseases including cardio-myopathies and cancers. Remarkably, as early as 2000, SRA isoforms were identified that were also able to code for a protein now referred to as the Steroid Receptor RNA Activator Protein (SRAP). SRA and SRAP now define a very intriguing bi-faceted genetic system, where both RNA and protein products of the same gene play specific and sometime overlapping roles in cell biology. Due to its initial molecular characterization as an RNA, most reports have in the last ten years focused on the non-coding part of this twosome. As such, only a handful of laboratories have investigated the molecular and biological roles played by SRAP. The scope of this review is to summarize and discuss our current knowledge of the molecular features and functions specifically attributable to the coding nature of the bi-faceted products of the SRA1 gene.

The protein encoded by the functional steroid receptor RNA activator is a new modulator of ER alpha transcriptional activity.

The steroid receptor RNA activator gene (SRA1) encodes for a functional RNA (SRA) as well as a protein (SRAP). While several groups reported on SRA-RNA mechanism of action, SRAP exact function remains to be elucidated, mainly due to a lack of studies investigating the function of the protein independently of its RNA counterpart. Using two independent models to examine its specific functions, SRAP was found to enhance estrogen receptor alpha activity in a ligand and response-element dependent manner. Our data therefore suggest that both transcript and protein products of the SRA1 gene co-modulate the transcriptional activity of steroid receptors.

The steroid receptor RNA activator protein is recruited to promoter regions and acts as a transcriptional repressor.

Products of the steroid receptor RNA activator (SRA1) gene have the unusual property to function both at the RNA and the protein levels. SRA-RNA has long been known to increase the activity of multiple nuclear receptors. It has more recently been proposed than steroid receptor RNA activator protein (SRAP) also modulates steroid receptors activity. Herein, we show for the first time that SRAP physically interacts with multiple transcription factors and is recruited to specific promoter regions. Artificially recruiting SRAP to the promoter of a luciferase reporter gene under the control of the strong transcriptional activator VP16 leads to a decrease in transcription. Altogether we propose that SRAP could be a new transcriptional regulator, able to function as a repressor through direct association with promoters.