Centro de Biología Molecular Severo Ochoa, CSIC-UAM , Madrid, Spain.
Laboratorio de Terapia Génica, Centro de Investigación y de Estudios Avanzados del I.P.N ., Ciudad de México, Mexico.
RNA Biol. 2020 Sep;17(9):1331-1341. doi: 10.1080/15476286.2020.1762054. Epub 2020 May 31.
Regulation of protein synthesis is an essential step of gene expression. This process is under the control of cis-acting RNA elements and trans-acting factors. Gemin5 is a multifunctional RNA-binding protein organized in distinct domains. The protein bears a non-canonical RNA-binding site, designated RBS1, at the C-terminal end. Among other cellular RNAs, the RBS1 region recognizes a sequence located within the coding region of Gemin5 mRNA, termed H12. Expression of RBS1 stimulates translation of RNA reporters carrying the H12 sequence, counteracting the negative effect of Gemin5 on global protein synthesis. A computational analysis of RBS1 protein and H12 RNA variability across the evolutionary scale predicts coevolving pairs of amino acids and nucleotides. RBS1 footprint and gel-shift assays indicated a positive correlation between the identified coevolving pairs and RNA-protein interaction. The coevolving residues of RBS1 contribute to the recognition of stem-loop SL1, an RNA structural element of H12 that contains the coevolving nucleotides. Indeed, RBS1 proteins carrying substitutions on the coevolving residues P or SS, drastically reduced SL1-binding. Unlike the wild type RBS1 protein, expression of these mutant proteins in cells failed to enhance translation stimulation of mRNA reporters carrying the H12 sequence. Therefore, the PXSS motif within the RBS1 domain of Gemin5 and the RNA structural motif SL1 of its mRNA appears to play a key role in fine-tuning the expression level of this essential protein.
蛋白质合成的调控是基因表达的一个重要步骤。这个过程受顺式作用 RNA 元件和反式作用因子的控制。Gemin5 是一种多功能 RNA 结合蛋白，由不同的结构域组成。该蛋白在 C 末端具有一个非典型的 RNA 结合位点，称为 RBS1。在其他细胞 RNA 中，RBS1 区域识别 Gemin5 mRNA 编码区内部的一段序列，称为 H12。RBS1 的表达刺激携带 H12 序列的 RNA 报告基因的翻译，抵消了 Gemin5 对全球蛋白质合成的负面影响。对跨进化尺度的 RBS1 蛋白和 H12 RNA 变异性的计算分析预测了氨基酸和核苷酸的共进化对。RBS1 足迹和凝胶迁移分析表明，鉴定出的共进化对与 RNA-蛋白相互作用之间存在正相关。RBS1 的共进化残基有助于识别茎环 SL1，这是 H12 的一种 RNA 结构元件，其中包含共进化核苷酸。事实上，携带共进化残基 P 或 SS 取代的 RBS1 蛋白，大大降低了 SL1 的结合。与野生型 RBS1 蛋白不同，这些突变蛋白在细胞中的表达未能增强携带 H12 序列的 mRNA 报告基因的翻译刺激。因此，Gemin5 的 RBS1 结构域内的 PXSS 基序及其 mRNA 的 RNA 结构基序 SL1 似乎在精细调节这种必需蛋白的表达水平方面起着关键作用。
