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Structural and functional studies on protein factors involved in pre-mRNA 3' processing.

机译：对涉及前mRNA 3'加工的蛋白质因子的结构和功能研究。

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摘要

The 3' end formation of most eukaryotic messenger RNA precursors (pre-mRNAs) involves two reactions, an endonuclease cleavage followed by polyadenylation. A large number of protein factors have been identified as members of 3' processing machinery. Among them is cleavage stimulation factor (CstF), a heterotrimeric protein complex essential for both the efficiency and the specificity of the cleavage reaction. Its largest subunit, CstF-77, can mediates both the assembly of the CstF complex and the interactions with other protein factors in the 3' processing machinery. Here we determined the crystal structure of the HAT (h&barbelow;alf a&barbelow; T&barbelow;PR) domain of murine CstF-77, as well as its C-terminal sub-domain, the HAT-C domain. Our studies showed that the HAT domain of CstF-77 is formed of two distinct sub-domains, the HAT-N and the HAT-C sub-domains. Moreover, CstF-77 forms a homodimer mediated by the HAT-C sub-domain. This dimerization event has been further verified by various methods including static light-scattering studies, yeast two-hybrid assays, analytical ultracentrifugation measurements, and small angle X-ray scattering experiments. Our studies supported a dimeric association of the CstF complex in pre-mRNA 3' processing machinery.;Although a lot has been learned about pre-mRNA 3' end formation in the nucleus, a similar process in the mitochondria is much less understood. To this end, we conducted structural and functional studies on human mitochondria poly(A) polymerase, hPAPD1. This protein shares high structural similarity to that of the canonical PAPs for having similar palm domain (the catalytic domain) and fingers domain. However, hPAPD1 lacks the RNA binding domain which resides on the C-terminus of canonical PAPs, making it more similar to a cytoplasmic PAP, GLD-2. In the crystal structure of hPAPD1, we observed a unique N-terminal domain, which involves in the dimerization of this protein. Neither this domain nor the dimeric association has been reported for other PAPs before. In our following polyadenylation assays, we found that dimerization is critical for the activity of hPAPD1. Electrophoretic mobility shift assays (EMSA) also indicated this dimeric association may be involved in the recruitment of substrate RNA.
机译：大多数真核生物信使RNA前体（pre-mRNA）的3'端形成涉及两个反应，即核酸内切酶裂解，然后进行聚腺苷酸化。大量的蛋白质因子已经被确定为3'加工机制的成员。其中包括裂解刺激因子（CstF），这是一种对裂解反应的效率和特异性均必不可少的异三聚体蛋白复合物。它最大的亚基CstF-77可以介导CstF复合体的组装以及3'加工机械中与其他蛋白质因子的相互作用。在这里，我们确定了鼠CstF-77的HAT（h）的晶体结构，以及它的C末端亚结构域，即HAT-C域。我们的研究表明，CstF-77的HAT域由两个不同的子域形成，即HAT-N和HAT-C子域。此外，CstF-77形成由HAT-C子域介导的同型二聚体。此二聚化事件已通过多种方法进一步验证，包括静态光散射研究，酵母双杂交测定，超离心分析测量和小角度X射线散射实验。我们的研究支持前mRNA 3'加工机器中CstF复合物的二聚体缔合。尽管已经了解了许多有关前mRNA 3'端在细胞核中形成的知识，但线粒体中的类似过程却鲜为人知。为此，我们对人线粒体多聚（A）聚合酶hPAPD1进行了结构和功能研究。由于具有相似的棕榈结构域（催化结构域）和指状结构域，该蛋白质与规范PAP具有高度的结构相似性。但是，hPAPD1缺少位于经典PAP C末端的RNA结合结构域，使其与细胞质PAP GLD-2更相似。在hPAPD1的晶体结构中，我们观察到一个独特的N末端结构域，该结构涉及该蛋白的二聚化。之前尚未报道该域或二聚体关联用于其他PAP。在我们随后的聚腺苷酸化测定中，我们发现二聚化对于hPAPD1的活性至关重要。电泳迁移率变动分析（EMSA）也表明这种二聚体缔合可能与底物RNA的募集有关。

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  • 作者

    Bai, Yun.;

  • 作者单位

    Columbia University.;

  • 授予单位 Columbia University.;
  • 学科 Biophysics General.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 226 p.
  • 总页数 226
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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