Structure and Function of Steroid Receptor RNA Activator Protein, the Proposed Partner of SRA Noncoding RNA

Abstract

In a widely accepted model, the steroid receptor RNA activator protein (SRA protein; SRAP) modulates the transcriptional regulatory activity of SRA RNA by binding a specific stem-loop of SRA. We first confirmed that SRAP is present in the nucleus as well as the cytoplasm of MCF-7 breast cancer cells, where it is expressed at the level of about 105 molecules/cell. However, our SRAP-RNA binding experiments, both in vitro with recombinant protein and in cultured cells with plasmid-expressed protein and RNA, did not reveal a specific interaction between SRAP and SRA. We determined the crystal structure of the carboxy-terminal domain of human SRAP and found that it does not have the postulated RRM (RNA recognition motif). The structure is a five-helix bundle that is distinct from known RNA-binding motifs and instead is similar to the carboxy-terminal domain of the yeast spliceosome protein PRP18, which stabilizes specific protein-protein interactions within a multisubunit mRNA splicing complex. SRA binding experiments with this domain gave negative results. Transcriptional regulation by SRA/SRAP was examined with siRNA knockdown. Effects on both specific estrogen-responsive genes and genes identified by RNA-seq as candidates for regulation were examined in MCF-7 cells. Only a small effect (~20% change) on one gene resulting from depletion of SRA/SRAP could be confirmed. We conclude that the current model for SRAP function must be re-evaluated; we suggest SRAP may function in a different context to stabilize specific intermolecular interactions in the nucleus.

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

“…As such, the forementioned changes in gene expression are mostly very small. Interestingly, McKay et al concluded in their recent study, using a cut-off of two fold changes, that not a single gene appeared to be significantly regulated upon silencing SRA1 expression with individual siRNAs [25]. Our results corroborate their conclusion and suggest that SRAP does not act as a major transcriptional regulator.…”

“…This suggests that SRAP could be a potential prognostic marker and/or contribute to increasing tumor aggressiveness. Even though several groups have now tried to identify SRAP cellular functions, the exact roles played by this protein remain to be identified [5,21,22,25,26].…”

Non-coding RNAs: long non-coding RNAs and microRNAs in endocrine-related cancers

“…The SRA1 gene also encodes a protein steroid receptor coactivator protein (SRAP) (reviewed in (Leygue 2007)) that interacts with AR and is involved in PCa (Kawashima, et al 2003). Notably, SRA1 and SRAP do not interact (McKay, et al 2014). …”

CncRNAs: RNAs with both coding and non-coding roles in development

“…A recent study reporting the crystal structure of human SRAP suggests that the protein does not harbor a predicted RNArecognition motif (RRM) but rather resembles the spliceosome complex protein PRP18 (also known as PRPF18) (McKay et al, 2014). Biochemical binding assays performed in vitro did not find a specific interaction between SRAP and SRA, nor was a specific response on estrogen receptor targets observed in cell culture assays.…”

“…Biochemical binding assays performed in vitro did not find a specific interaction between SRAP and SRA, nor was a specific response on estrogen receptor targets observed in cell culture assays. These observations have led to the proposal of an alternative model wherein SRAP is thought to stabilize intermolecular interactions within a nuclear splicing complex, rather than directly binding and regulating SRA RNA (McKay et al, 2014). Genetic analysis of the sra locus with mutations that disrupt specific SRA RNA versus SRAP domains, which could resolve the roles and mechanisms by which the RNA and protein function during development, differentiation and homeostasis, is currently lacking.…”