Molecular genetic analysis of the RNA-mediated signaling pathways

本研究旨在阐明RNA结合蛋白调控信号转导通路的调控机制。我们利用裂殖酵母模型系统和分子遗传学方法来鉴定调节包括MAP激酶通路在内的各种信号通路的RNA结合蛋白。我们鉴定了几种RNA结合蛋白，包括编码高度保守的KH型RNA结合蛋白的Rncl。Rncl过表达降低了PmklMAPK信号传导的活化，并且Rncl缺失增加了PmklMAPK信号传导的磷酸化水平，因此表明Rncl充当PmklMAPK信号传导的负调节剂。我们证明Rncl结合并稳定Pmpl mRNA，其编码用于Pmkl MAPK的MAPK磷酸酶。此外，Pmkl MAPK磷酸化Rncl，其调节Rncl的RNA结合活性以结合并稳定Pmpl mRNA。我们还鉴定了编码tRNA脱氨酶突变的tad 3 -1突变体。值得注意的是，我们证明tad 3 -1突变体在G1/S和G2/M转变时表现出细胞周期进展缺陷，从而表明摆动tRNA修饰对裂殖酵母的细胞周期至关重要。我们还确定了一个RRM型RNA结合蛋白，稳定的mRNA编码肌动蛋白结合蛋白。值得注意的是，MAPK Pmk 1直接磷酸化RRM型RNA结合蛋白，这种磷酸化负调节RNA结合蛋白结合靶mRNA的活性。MAPK通路长期以来被认为是通过磷酸化各种转录因子来调节细胞信号传导的转录调节因子。我们的数据表明，一种新的范式涉及MAPK介导的调节转录后RNA代谢。

This research aims to unravel the regulatory mechanisms of the signal transduction pathway regulated by RNA-binding proteins. We utilized the fission yeast model system and molecular genetic approach to identify RNA binding proteins that regulate various signaling pathways including MAP kinase pathways. We identified several RNA-binding proteins including Rncl that encodes a highly conserved KH-type RNA-binding protein. Rncl overexpression decreased the activation of the Pmkl MAPK signaling and Rncl deletion increased the phosphorylation level of the Pmk1 MAPK signaling thus indicating that Rncl acts as a negative regulator of the Pmkl MAPK signaling. We demonstrated that Rncl binds and stabilizes Pmpl mRNA that encodes a MAPK phosphatase for Pmkl MAPK Moreover, Pmk1 MAPK phosphorylates Rncl which regulates the RNA-binding activity of Rncl to bind and stabilize Pmpl mRNA. We also identified the tad3-1 mutant that encodes a mutation in the tRNA deaminase. Notably, we demonstrated that the tad3-1 mutants displayed defects in cell cycle progression at G1/S and G2/M transitions thus indicating that the wobble tRNA modification is essential to cell cycle in fission yeast. We also identified an RRM-type RNA binding protein that stabilizes the mRNA encoding an actin-binding protein. Notably, the MAPK Pmk1 directly phosphorylates the RRM-type RNA-binding protein and this phosphorylation negatively regulates the activity of the RNA-binding protein to bind target mRNAs. The MAPK pathways have long been known as a transcriptional regulator of cellular signaling by phosphorylating various transcription factors. Our data suggest a novel paradigm involving the MAPK-mediated regulation of the post-transcriptional RNAmetabolism.