"mRNA Metabolism in Normal and Pathological Cells"; Centre de Génétique et de Physiologie Moléculaire et Cellulaire, CNRS UMR, Université Lyon 1, Villeurbanne, France.
PLoS One. 2013;8(3):e59137. doi: 10.1371/journal.pone.0059137. Epub 2013 Mar 11.
SR proteins exhibit diverse functions ranging from their role in constitutive and alternative splicing, to virtually all aspects of mRNA metabolism. These findings have attracted growing interest in deciphering the regulatory mechanisms that control the tissue-specific expression of these SR proteins. In this study, we show that SRSF5 protein decreases drastically during erythroid cell differentiation, contrasting with a concomitant upregulation of SRSF5 mRNA level. Proteasome chemical inhibition provided strong evidence that endogenous SRSF5 protein, as well as protein deriving from stably transfected SRSF5 cDNA, are both targeted to proteolysis as the cells undergo terminal differentiation. Consistently, functional experiments show that overexpression of SRSF5 enhances a specific endogenous pre-mRNA splicing event in proliferating cells, but not in differentiating cells, due to proteasome-mediated targeting of both endogenous and transfection-derived SRSF5. Further investigation of the relationship between SRSF5 structure and its post-translation regulation and function, suggested that the RNA recognition motifs of SRSF5 are sufficient to activate pre-mRNA splicing, whereas proteasome-mediated proteolysis of SRSF5 requires the presence of the C-terminal RS domain of the protein. Phosphorylation of SR proteins is a key post-translation regulation that promotes their activity and subcellular availability. We here show that inhibition of the CDC2-like kinase (CLK) family and mutation of the AKT phosphorylation site Ser86 on SRSF5, have no effect on SRSF5 stability. We reasoned that at least AKT and CLK signaling pathways are not involved in proteasome-induced turnover of SRSF5 during late erythroid development.
SR 蛋白具有多种功能，从组成型和选择性剪接，到 mRNA 代谢的几乎所有方面。这些发现引起了人们越来越大的兴趣，以揭示控制这些 SR 蛋白组织特异性表达的调节机制。在这项研究中，我们表明，SRSF5 蛋白在红细胞分化过程中急剧减少，与 SRSF5 mRNA 水平的同时上调形成对比。蛋白酶体化学抑制提供了强有力的证据，表明内源性 SRSF5 蛋白以及来自稳定转染的 SRSF5 cDNA 的蛋白都作为细胞经历终末分化而被靶向蛋白酶体降解。一致地，功能实验表明，SRSF5 的过表达增强了增殖细胞中特定的内源性前体 mRNA 剪接事件，但在分化细胞中则没有，这是由于内源性和转染源 SRSF5 都被蛋白酶体介导靶向。进一步研究 SRSF5 结构与其翻译后调节和功能之间的关系表明，SRSF5 的 RNA 识别基序足以激活前体 mRNA 剪接，而 SRSF5 的蛋白酶体介导的蛋白水解需要该蛋白的 C 末端 RS 结构域的存在。SR 蛋白的磷酸化是一种关键的翻译后调节，可促进其活性和亚细胞可用性。我们在这里表明，CDC2 样激酶 (CLK) 家族的抑制和 SRSF5 上 AKT 磷酸化位点 Ser86 的突变对 SRSF5 的稳定性没有影响。我们推断，至少 AKT 和 CLK 信号通路不参与晚期红细胞发育过程中蛋白酶体诱导的 SRSF5 周转。
