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

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• 作者：David B. McKay¹ ; ² ; ³ ; ^&dagger ; ; Linghe Xi¹ ; ⁴ ; ^&dagger ; ; Kristen K.B. Barthel¹ ; ⁴ ; Thomas R. Cech¹ ; ² ; ³ ; ⁴ ; ^{Thomas.Cech@Colorado.edu" class="auth_mail}
• 关键词：DMEM ; Dulbecco's modified Eagle's medium ; EDTA ; ethylenediaminetetraacetic acid ; EMSA ; electrophoretic mobility shift assay ; ER ; estrogen receptor ; ERE ; estrogen-responsive element ; hTERT ; human telomerase reverse transcriptase ; hTR ; human telomerase RNA ; MBP ; maltose binding protein ; PDB ; Protein Data Bank ; PR ; progesterone receptor ; GREB1 ; growth regulation by estrogen in breast cancer 1 ; RRM ; RNA recognition motif ; RT-qPCR ; reverse transcription&ndash ; quantitative polymerase chain reaction ; S
• 刊名：Journal of Molecular Biology
• 出版年：17 April, 2014
• 年：2014
• 卷：426
• 期：8
• 页码：1766-1785
• 全文大小：1495 K

文摘

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 per 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 reevaluated; we suggest that SRAP may function in a different context to stabilize specific intermolecular interactions in the nucleus.