Structural biology of pre-mRNA 3'-end processing

描述（由申请人提供）：大多数真核信使RNA前体（pre- mrna）必须在细胞核中经历共转录加工，才能在细胞质中发挥mrna的作用。加工过程包括5‘盖帽、剪接和3’聚腺苷酸化。前mrna的3‘聚腺苷化发生在两个步骤-前mrna在其3’端附近的特定位点的核内裂解，然后加入聚(a)尾部。虽然裂解反应在生物化学上看起来很简单，但它需要大量的蛋白质因子来完成，包括裂解和聚腺苷酸化特异性因子（CPSF）复合物、裂解刺激因子（CstF）复合物、裂解因子I和裂解因子II以及聚(a)聚合酶（PAP）。CPSF复合物包含CPSF-30、CPSF-73、CPSF-100和CPSF-160四个亚基，CstF复合物包含CstF-50、CstF-64和CstF-77三个亚基。

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.