BioFrontiers Institute, University of Colorado, Boulder, CO 80309, USA.
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA.
J Mol Biol. 2014 Apr 17;426(8):1766-1785. doi: 10.1016/j.jmb.2014.01.006. Epub 2014 Jan 30.
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 10(5) 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.
在一个被广泛接受的模型中，类固醇受体 RNA 激活蛋白（SRA 蛋白；SRAP）通过结合 SRA 的特定茎环结构来调节 SRA RNA 的转录调控活性。我们首先证实，SRAP 存在于 MCF-7 乳腺癌细胞的核内和细胞质中，其表达水平约为每个细胞 10^5 个分子。然而，我们的 SRAP-RNA 结合实验，无论是使用重组蛋白进行的体外实验，还是使用质粒表达蛋白和 RNA 的培养细胞进行的实验，都没有揭示 SRAP 和 SRA 之间的特异性相互作用。我们确定了人源 SRAP 羧基末端结构域的晶体结构，发现它没有假定的 RRM（RNA 识别基序）。该结构是一个五螺旋束，与已知的 RNA 结合基序不同，而是类似于酵母剪接体蛋白 PRP18 的羧基末端结构域，该结构域稳定多亚基 mRNA 剪接复合物内的特定蛋白质-蛋白质相互作用。与该结构域进行 SRA 结合实验得到了阴性结果。我们通过 siRNA 敲低来检查 SRA/SRAP 的转录调节作用。在 MCF-7 细胞中，我们检查了对特定雌激素反应基因和 RNA-seq 鉴定为调节候选基因的基因的影响。仅能证实 SRA/SRAP 耗竭对一个基因的影响约为 20%。我们得出结论，必须重新评估当前的 SRAP 功能模型；我们认为，SRAP 可能在不同的环境中发挥作用，以稳定核内特定的分子间相互作用。
