Participation of Proteins of the CPSF Complex in Polyadenylation of Transcripts Read by RNA Polymerase III from SINEs

Abstract

SINEs are mobile genetic elements of multicellular eukaryotes that arose during evolution from various tRNAs, as well as from 5S rRNA and 7SL RNA. Like the genes of these RNAs, SINEs are transcribed by RNA polymerase III. The transcripts of some mammalian SINEs have the capability of AAUAAA-dependent polyadenylation, which is unique for transcript generated by RNA polymerase III. Despite a certain similarity with canonical polyadenylation of mRNAs (transcripts of RNA polymerase II), these processes apparently differ significantly. The purpose of this work is to evaluate how important for polyadenylation of SINE transcripts are proteins of the CPSF complex formed by mPSF and mCF subcomplexes which direct mRNA polyadenylation. In HeLa cells, siRNA knockdowns of the CPSF components were carried out, after which the cells were transfected with plasmid constructs containing SINEs. A decrease in polyadenylation of the SINE transcripts as a result of the knockdown of the proteins was evaluated by Northern-hybridization. It turned out that the CPSF components, such as Wdr33 and CPSF30, contributed to the polyadenylation of SINE transcriptions, while the knockdown of CPSF100, CPSF73, and symplekin did not reduce the polyadenylation of these transcripts. Wdr33 and CPSF30, along with the CPSF160 and Fip1 previously studied, are components of the subcomplex mPSF responsible for mRNA polyadenylation. Thus, the available data suggest the importance of all mPSF proteins for polyadenylation of SINE transcripts. At the same time, CPSF100, CPSF73, and symplekin, forming the subcomplex mCF, are responsible for the cleavage of pre-mRNA; therefore, their non-participation in the polyadenylation of SINE transcriptions seems quite natural.