3`-Processing of mRNA: Composition and dynamics of the processing complex and reconstitution of selected factors

真核mrna的3'端是通过转录后加工、核内溶解裂解和聚腺苷化产生的。这两种反应都是由一个大得惊人的蛋白质复合物催化的。人们对其组成和各组成部分的功能了解甚少。其中一个成分是裂解和聚腺苷化特异性因子（CPSF），它结合底物RNA中的聚腺苷化信号AAUAAA。CPSF的四个亚基是已知的，其中一个对于反应的第二阶段聚腺苷化可能是必不可少的。可能，至少存在一个额外的亚基。为了研究单个亚基的作用，我们想从重组亚基重构CPSF。本提案的第二个目标是通过亲和色谱法纯化加工复合物，并通过质谱法对其蛋白质组成进行完整分析。我们将使用生化分析来确认新的复杂成分在3'加工中的作用。亲和纯化和复合体组成分析的方法也将用于研究前mrna切割后加工复合体的变化。

3' ends of eukaryotic mRNAs are generated by post-transcriptional processing, endonucleolytic cleavage followed by polyadenylation. Both reactions are catalyzed by a surprisingly large protein complex. Its composition and the functions of its components are poorly understood. One component is cleavage and polyadenylation specificity factor (CPSF), which binds the polyadenylation signal AAUAAA in the substrate RNA. Four subunits of CPSF are known, one of which is possibly dispensable for the second phase of the reaction, polyadenylation. Probably, at least one additional subunit exists. We want to reconstitute CPSF from recombinant subunits in order to study the role of individual subunits. The second goal of this proposal is the purification of the processing complex by affinity chromatography and the complete analysis of its protein composition by means of mass spectrometry. We will use biochemical assays to confirm a role of novel complex components in 3' processing. The method of affinity purification foll owed by analysis of complex composition will also be used to investigate changes in the processing complex upon cleavage of the pre-mRNA.