An ordered pathway of assembly of components required for polyadenylation site recognition and processing

Abstract

Four HeLa cell nuclear factors that are required for specific pre-mRNA cleavage and polyadenylation have been extensively purified, thereby permitting an investigation of the role of each in the 3' processing reaction. Two factors, termed PF1 and PF2, are required for specific polyadenylation of the cleaved RNA. PF1 is a poly(A) polymerase, and PF2 is a factor that confers AAUAAA specificity to the reaction. Both of these factors, along with two additional factors termed CF1 and CF2, are required for the endonucleolytic cleavage of the pre-mRNA. The ability of each of these factors to form specific complexes with the pre-mRNA was assayed using native gel electrophoresis. Two distinct complexes were detected. PF2 forms an initial complex with the pre-RNA, dependent on the AAUAAA sequence element but independent of specific downstream sequences. Formation of the PF2-RNA complex permits the subsequent interaction of CF1 and the formation of a second, larger complex. CF1 binding requires the downstream sequence element in addition to PF2 binding. Whereas the PF2-RNA complex is unstable and dissociates rapidly, the ternary complex formed by CF1, PF2, and RNA is stable. Thus, the interaction of CF1, dependent on the downstream sequence element, can be viewed as a commitment of the poly(A) site for processing. On the addition of the poly(A) polymerase (PF1) and factor CF2, the pre-mRNA is specifically cleaved at the poly(A) site.