At the 3′ end, most eukaryotic mRNAs have a string of 80-250 adenylate residues called the poly(A) tail. The poly(A) tail is added in a multistep process after the mRNA transcript has been synthesized. Two sequences are required for cleavage and polyadenylation (addition of ploy(A) tail) of the mRNA: 1) A highly conserved polyadenylation signal sequence AAUAAA, found 10 to 30 nucleotides upstream (on the 5′ side) of the cleavage; 2) A less well-defined sequence rich in G and U or U residues only, found 20 to 40 nucleotides downstream of the cleavage site. The cleavage generates the free 3′-OH group that defines the end of the mRNA, to which adenylate residues are added. Following proteins are required for cleavage and polyadenylation of pre-mRNAs.
1) Cleavage and polyadenylation specificity factor (CPSF): It is 360 kDa large complex made up of 4 polypeptides. It forms an unstable complex with the AAUAAA sequence.
2) Cleavage stimulatory factor (CStF1): 200 kDa heterotrimer.
3) Cleavage Factor I and II (CFI and CFII)
After CPSF has bound to the AAUAAA on the pre-mRNA, the CStF1, CFI, and CFII bind to the CPSF-mRNA complex. Interaction between CStF and GU or U-rich less well-defined sequence stabilizes the multiprotein complex. Finally, polyadenylate polymerase (poly(A) polymerase or PAP) binds to the complex before the cleavage can occur. The PAP binding links cleavage and polyadenylation, so that the free 3′ end generated after cleavage is rapidly polyadenylated. The assembly of this large multiprotein cleavage-polyadenylation complex around the AAUAAA signal in a pre-mRNA is analogous in many ways to the formation of the transcription-initiation complex at the TATA box.
Following cleavage at the poly(A) site, polyaenylation proceeds in two phases. Addition of the first 12 or so A residues occurs slowly, followed by a rapid addition of upto 200-250 A residues. The rapid phase requires the binding of multiple copies of poly(A) binding protein (PABII). PABII stimulates polymerization of additional A residues by PAP. PABII is also responsible for signaling PAP to terminate polymerization when te poly(A) tail reaches a length of 200-250 residues.