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Reconstitution of 3′ end processing of mammalian pre-mRNA reveals a central role of RBBP6
Genes & Development ( IF 10.5 ) Pub Date : 2022-02-01 , DOI: 10.1101/gad.349217.121
Moritz Schmidt 1 , Florian Kluge 1 , Felix Sandmeir 2 , Uwe Kühn 1 , Peter Schäfer 1 , Christian Tüting 1 , Christian Ihling 3 , Elena Conti 2 , Elmar Wahle 1
Affiliation  

The 3′ ends of almost all eukaryotic mRNAs are generated in an essential two-step processing reaction: endonucleolytic cleavage of an extended precursor followed by the addition of a poly(A) tail. By reconstituting the reaction from overproduced and purified proteins, we provide a minimal list of 14 polypeptides that are essential and two that are stimulatory for RNA processing. In a reaction depending on the polyadenylation signal AAUAAA, the reconstituted system cleaves pre-mRNA at a single preferred site corresponding to the one used in vivo. Among the proteins, cleavage factor I stimulates cleavage but is not essential, consistent with its prominent role in alternative polyadenylation. RBBP6 is required, with structural data showing it to contact and presumably activate the endonuclease CPSF73 through its DWNN domain. The C-terminal domain of RNA polymerase II is dispensable. ATP, but not its hydrolysis, supports RNA cleavage by binding to the hClp1 subunit of cleavage factor II with submicromolar affinity.

中文翻译:

哺乳动物前 mRNA 3' 端加工的重构揭示了 RBBP6 的核心作用

几乎所有真核生物 mRNA 的 3' 末端都是在基本的两步加工反应中产生的:延伸前体的核酸内切裂解,然后添加 poly(A) 尾。通过从过量产生和纯化的蛋白质中重构反应,我们提供了 14 种必不可少的多肽和两种对 RNA 加工有刺激作用的最小列表。在取决于多腺苷酸化信号 AAUAAA 的反应中,重组系统在与体内使用的位置相对应的单个优选位点处切割前体 mRNA。在这些蛋白质中,切割因子 I 刺激切割,但不是必需的,这与其在替代多聚腺苷酸化中的突出作用一致。需要 RBBP6,结构数据显示它通过其 DWNN 结构域接触并可能激活内切核酸酶 CPSF73。RNA 聚合酶 II 的 C 末端结构域是可有可无的。ATP(但不是其水解)通过以亚微摩尔亲和力与切割因子 II 的 hClp1 亚基结合来支持 RNA 切割。
更新日期:2022-02-01
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