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

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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.