Mohammad K. Hamedani

The steroid receptor RNA activator is the first functional RNA encoding a protein

The steroid receptor RNA activator (SRA) has previously been characterized as belonging to the growing family of functional non‐coding RNAs. However, we recently reported the Western blot detection of a putative endogenous SRA protein (SRAP) in breast cancer cells. Herein, we successfully suppressed the expression of this protein through specific RNA interference assay, unequivocally confirming its existence. Moreover, using database searches and Western blot analysis, we also showed that SRAP is highly conserved among chordata. Overall, our results suggest that SRA is the first example of a new class of functional RNAs also able to encode a protein.

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 (Students 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.

The steroid receptor RNA activator protein is expressed in breast tumor tissues

The steroid receptor RNA activator (SRA) was originally described as the first functional noncoding RNA able to specifically coactivate the activity of steroid receptors. We previously demonstrated the existence in breast cancer cell lines of new SRA isoforms that, as opposed to the first cloned SRA RNA, encode for a 236–amino acid protein, SRAP. To investigate the possible implications of the coding SRA RNA and SRAP expression on breast cancer progression, we examined by Western blot analysis 74 primary breast tumors of patients subsequently treated with tamoxifen. Patients whose primary tumors were positive for SRAP expression (n = 24) had a significantly (Kaplan‐Meier survival curve p = 0.047) lower likelihood of dying from recurrent disease than SRAP‐negative patients (n = 50). We generated 2 cell lines, SRAP‐V5‐High.A and SRAP‐V5‐High.B, by stably overexpressing SRAP in the estrogen receptor‐positive MCF‐7 breast cancer cell line. Transient transfection experiments, performed using a luciferase reporter gene under the control of an estrogen‐responsive element, revealed decreased sensitivity to estradiol but no additional sensitivity to tamoxifen in SRAP‐overexpressing cells. Overall, our data suggest that the presence of both coding SRA RNA and its corresponding SRAP modifies the activity of estrogen receptor in breast cancer cells and that SRAP could be a new clinical marker for breast cancer. Further studies are needed to define the respective mechanisms of action and the roles of SRA RNA and protein in breast tumorigenesis and tumor progression.

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

MINT‐7761068: SRAP (uniprotkb:Q9HD15) physically interacts (MI:0915) with HDAC2 (uniprotkb:Q92769) by anti bait coimmunoprecipitation (MI:0006)

Relationship Between Mammaglobin Expression and Estrogen Receptor Status in Breast Tumors

Mammaglobin (SCGB2A2) is a breast-specific member of the secretoglobin (SCGB) gene family. SCGB2A2 has previously been found overexpressed in breast tumors but possible associations between its expression and established prognostic tumor characteristics such as the levels of estrogen and progesterone receptors have not yet been investigated. We evaluated SCGB2A2 expression at the mRNA and at the protein level by reverse-transcription polymerase chain reaction and immunocytochemistry in 52 and 32 breast tumors, respectively. Both SCGB2A2 mRNA and protein expression were significantly higher in estrogen-receptor-positive compared to estrogen-receptor-negative tumors (Mann-Whitney rank sum test, p=0.04; chi-square test, p=0.01; respectively). In contrast, SCGB2A2 expression did not correlate with progesterone receptor levels or Nottingham grade. As estrogen and antiestrogen treatment of estrogen-positive breast cancer cell lines does not modify SCGB2A2 expression we suggest that SCGB2A2 may be a new independent breast cancer prognostic marker.

PDK2-mediated alternative splicing switches Bnip3 from cell death to cell survival

In hypoxia, the survival property of cancer cells mediated by the glycolytic enzyme PDK2 is obligatorily linked to alternative splicing and generation of a novel isoform of death gene Bnip3, which suppresses mitochondrial injury and promotes survival.

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.

Dynamic Histone Acetylation of H3K4me3 Nucleosome Regulates MCL1 Pre-mRNA Splicing.

Pre‐mRNA splicing is a cotranscriptional process affected by the chromatin architecture along the body of coding genes. Recruited to the pre‐mRNA by splicing factors, histone deacetylases (HDACs) and K‐acetyltransferases (KATs) catalyze dynamic histone acetylation along the gene. In colon carcinoma HCT 116 cells, HDAC inhibition specifically increased KAT2B occupancy as well as H3 and H4 acetylation of the H3K4 trimethylated (H3K4me3) nucleosome positioned over alternative exon 2 of the MCL1 gene, an event paralleled with the exclusion of exon 2. These results were reproduced in MDA‐MB‐231, but not in MCF7 breast adenocarcinoma cells. These later cells have much higher levels of demethylase KDM5B than either HCT 116 or MDA‐MB‐231 cells. We show that H3K4me3 steady‐state levels and H3K4me3 occupancy at the end of exon 1 and over exon 2 of the MCL1 gene were lower in MCF7 than in MDA‐MB‐231 cells. Furthermore, in MCF7 cells, there was minimal effect of HDAC inhibition on H3/H4 acetylation and H3K4me3 levels along the MCL1 gene and no change in pre‐mRNA splicing choice. These results show that, upon HDAC inhibition, the H3K4me3 mark plays a critical role in the exclusion of exon 2 from the MCL1 pre‐mRNA. J. Cell. Physiol. 231: 2196–2204, 2016.

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

Our results highlight for the first time a link existing between SRA1 gene expression and cell motility.