Human FASTK preferentially binds single-stranded and G-rich RNA.

Daria M Dawidziak, Dawid A Dzadz, Mikołaj I Kuska, Madhuri Kanavalli, Maria M Klimecka, Matthew Merski, Katarzyna J Bandyra, Maria W Górna
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Abstract

Fas-activated serine/threonine kinase (FASTK) is the founding member of the FASTKD protein family, which was shown to regulate the fate of mRNA molecules on multiple levels. The mitochondrial variant of FASTK co-localizes with mitochondrial RNA granules and regulates the degradation of mitochondrial mRNAs, whereas the cytoplasmic and nuclear forms of FASTK are involved in the regulation of alternative splicing, cytoplasmic RNA granule formation, and mRNA translation. Despite these multiple roles of FASTK in mRNA biology, the exact rules of RNA recognition by this protein remained undetermined. Here, we demonstrate direct RNA binding by purified human FASTK and show its preference for single-stranded G-rich oligonucleotides, including those with a tendency to form RNA G-quadruplexes. Addition of FASTK alone was sufficient to achieve protection of mitochondrial mRNAs from degradation by the degradosome. Structural characterization by SAXS (Small-Angle X-ray Scattering) showed that FASTK in solution is a monomer with an extended conformation. Point mutagenesis studies supported the structural predictions of an exposed RNA-binding interface in the central helical region, preceded by a smaller, flexibly attached helical N-terminal domain. We provide the first such extensive in vitro characterization of the RNA binding properties for a representative of the FASTKD protein family and suggest how these intrinsic properties may underlie FASTK function in mRNA metabolism.

人类FASTK优先结合单链和富含g的RNA。
fas激活的丝氨酸/苏氨酸激酶(FASTK)是FASTKD蛋白家族的创始成员,它被证明在多个水平上调节mRNA分子的命运。FASTK的线粒体变体与线粒体RNA颗粒共定位并调节线粒体mRNA的降解,而细胞质和细胞核形式的FASTK则参与调节选择性剪接、细胞质RNA颗粒形成和mRNA翻译。尽管FASTK在mRNA生物学中有这些多重作用,但该蛋白识别RNA的确切规则仍未确定。在这里,我们证明了纯化的人类FASTK与RNA的直接结合,并显示了它对单链富含g的寡核苷酸的偏好,包括那些倾向于形成RNA g四联体的寡核苷酸。仅添加FASTK就足以保护线粒体mrna免受降解体的降解。小角x射线散射(SAXS)表征表明,溶液中的FASTK是一个具有扩展构象的单体。点诱变研究支持了在中心螺旋区暴露的rna结合界面的结构预测,在此之前是一个更小的、灵活附着的螺旋n端结构域。我们首次在体外对FASTKD蛋白家族的一个代表进行了广泛的RNA结合特性表征,并提出了这些内在特性可能是FASTK在mRNA代谢中功能的基础。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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