Structural biology of pre-mRNA 3'-end processing

描述(申请人提供)：大多数真核信使RNA前体(前mRNAs)必须在细胞核内经过共转录处理后才能作为细胞质中的mRNAs发挥作用。加工事件包括5‘端封端、剪接和3’端多聚腺苷化。Pre-mRNAs的3‘端多聚腺苷化分两步进行--Pre-mRNA在其3’端附近的特定位置被核酸内切切割，然后添加Poly(A)尾。虽然切割反应看起来可能是简单的生化反应，但它的执行需要大量的蛋白质因子，包括切割和多腺苷化特异性因子(CPSF)复合体、切割刺激因子(CstF)复合体、切割因子I和II以及聚(A)聚合酶(PAP)。CPSF复合体包含CPSF-30、CPSF-73、CPSF-100和CPSF-160四个亚基，CstF复合体包含三个亚基：CstF-50、CstF-和CstF-77。 尽管对切割反应所需的大量蛋白质进行了表征，但实际催化水解酶的核酸内切酶的身份目前尚不清楚。最近的证据表明CPSF-73可能是切割反应的内切酶，尽管没有直接的实验证据证明这一活性。此外，目前关于这些重要蛋白质的结构信息很少。目前已知的只有PAP的结构和CstF-的核糖核酸结合域。为了填补这一知识空白，我们最近确定了人CPSF-73、酵母CPSF-100、小鼠CstF-77的晶体结构，并进行了初步的生化和突变研究，以评估来自结构的信息。这些初步结果为进一步对这些蛋白质进行生化、生物物理和结构研究奠定了基础，这些蛋白质在前mRNA3‘端加工中起着至关重要的作用。

DESCRIPTION (provided by applicant): Most eukaryotic messenger RNA precursors (pre-mRNAs) must undergo co-transcriptional processing in the nucleus before they can function as mRNAs in the cytoplasm. The processing events include 5' capping, splicing, and 3' polyadenylation. The 3' polyadenylation of pre-mRNAs occurs in two steps - endonucleolytic cleavage of the pre-mRNA at a specific site near its 3'-end and then the addition of the poly(A) tail. While the cleavage reaction may appear to be simple biochemically, it requires a large number of protein factors for its execution, including the cleavage and polyadenylation specificity factor (CPSF) complex, the cleavage stimulation factor (CstF) complex, cleavage factors I and II, and poly(A) polymerase (PAP). The CPSF complex contains four subunits, CPSF-30, CPSF-73, CPSF-100, and CPSF-160, and the CstF complex contains three subunits, CstF-50, CstF-64, and CstF-77. Despite the characterization of this large number of proteins that are required for the cleavage reaction, the identity of the endonuclease that actually catalyzes the hydrolysis is currently unknown. Recent evidence suggests CPSF-73 could be the endonuclease for the cleavage reaction, although no direct experimental evidence demonstrating this activity is available. Moreover, there is currently little structural information on these important proteins. Only the structures of PAP and the RNA binding domain of CstF-64 are known. To fill this gap in our knowledge, we have recently determined the crystal structures of human CPSF-73, yeast CPSF-100, murine CstF-77, and carried out preliminary biochemical and mutagenesis studies to assess the information from the structures. These initial results set the stage for further biochemical, biophysical, and structural studies on these proteins with crucial roles in pre-mRNA 3'-end processing.