Cleavage and Polyadenylation of Pre-mRNA

During the course of transcription, the initial RNA product synthesized by RNA polymerase-II, called a primary transcript, undergoes several processing steps including capping, splicing and polyadenylation before a functional mRNA is produced. RNA polymerase-II initiates transcription at the first nucleotide of the first exon of a gene. Shortly after transcription begins, the 5' end of the nascent RNA is capped with 7-methyl-guanylate. Transcription by RNA polymerase-II terminates at any one of the multiple sites approximately 0.5-2kb downstream from the 3' end of the last exon in the transcript. The 3' end of a functional mRNA is generated by endonucleolytic cleavage at the poly-A site located at the 3' end of the final exon. A stretch of 100-250 adenine residues is added to the 3'-hydroxyl group left by the cleavage reaction. Finally, introns are removed by RNA splicing before the mature RNA is transported to the cytoplasm. All these processes are coupled reactions that influence each other. Capping and polyadenylation are important for the efficacy of translation, but not always absolutely required.Polyadenylation is the covalent linkage of a polyadenylyl moiety to an mRNA molecule. It is part of the route to producing mature mRNA for translation. Polyadenylation is initiated by cleavage of the mRNA which has been transcribed considerably beyond the adenylation start site. A 350 kDa protein complex called cleavage and polyadenylation specificity factor (CPSF), composed of 3 or 4 different polypeptides, first forms an unstable complex with the upstream AU-rich poly-A signal (AAUAAA sequence). Then at least 3 additional proteins - a 200kDa heterotrimer called cleavage stimulatory factor (CstF) and 2 as yet poorly characterized cleavage factors, CFI and CFII bind to the CPSF-RNA Complex. CstF binds to the GU or U rich sequence and this interaction stabilizes the multiprotein complex. Finally, a poly A-polymerase (PAP) binds to the complex before cleavage can occur. This requirement for PAP binding links cleavage and polyadenylation, so that the free 3' ends generated are rapidly polyadenylated. Assembly of this large, multiprotein cleavage-polyadenylation complex around the AU-rich poly-A signal in an RNA transcript is analogous in many ways to formation of the transcription-intiation complex at the AT-rich TATA box of a template DNA molecule.Following cleavage at the poly-A site, polyadenylation proceeds in two phases. Addition of the first approx. 12 adenine residues occurs slowly, followed by rapid addition of up to 200-250 more residues. The rapid phase requires the binding of multiple copies of a poly-A binding protein containing the RNP motif. This protein is designated PABII to distinguish it from the first poly-A binding protein characterized, which binds to the poly-A tail of cytoplasmic mRNAs. PABII binds to the short A tail initially added by PAP, stimulating polymerization of additional adenine residues by PAP. By an unknown mechanism, PABII slows polymerization greatly when the poly-A tail reaches a length of 200-250 residues. Poly-A tails in yeast have a length of 70-90 while bacterial (E.coli) tails are about 15-40. Mature mRNA moves to the nuclear surface and is exported through nuclear pores. This is an energy requiring, as well as likely a carrier-mediated process. In eukaryotes, polyadenylation protects the mRNA molecule from exonucleases and is important for transcription termination, export of the mRNA from the nucleus and translation.