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PolyadenylationComments

Polyadenylation

Just as transcription initiation and capping are coupled, transcription termination and the 3’ cleavage and polyadenylation reactions are functionally linked. The Cleavage/Polyadenylation reaction is carried out the polyadenylation complex, a large multi-protein complex assembled from CPSF, CStF, CF1, and CF2. What signals (i.e. RNA sequence) in a pre-mRNA specify the site of polyadenylation? Some pre-mRNA's have multiple poly(a) signals, and cleavage and polyadenylation at these different signals can include or exclude various 3' terminal exons.

What mechanism can account for the fact that RNAs transcribed by RNA polymerase II are capped and usually polyadenylated, while these types of RNA processing do not occur on RNAs transcribed by RNA polymerase I or III? To ensure that only RNAP II transcripts are capped, the RNAP II CTD is phosphorylated by TFIIH, and the phosphorylated CTD recruits capping enzymes. After ~25-30 nucleotides have been transcribed and the nascent pre-mRNA is emerging from RNAP, the first transcribed nucleotide (the 5' end) is bound via a 5'-5' triphosphate linkage to a 7-methylguanosine. The 2’-hydroxyls of the first two transcribed nucleotides are also often methylated.

Upstream BindingUpstream AAUAAA bound by Cleavage and Polyadenylation Specificity Factor (CPSF; 4 subunits).
Downstream BindingDownstream G/U rich element is bound by the Cleavage Stimulatory Factor (CStF; 3 subunits).
Additional FactorsRNA cleavage requires Cleavage Factors (CF1 and 2); the endonuclease is the 73kd subunit of CPSF.
Binding of PAPThe PolyA polymerase (PAP) binds, activating cleavage of the 3' end.
Fragment DigestedThe free cleaved 3’ piece is rapidly degraded by 5’-3’ exonucleases.
Start of Poly(A) TailPoly(A) Polymerase slowly adds ~12 adenosine residues to the 3’ end of the 5’ product RNA.
Poly(A) Binding PrtnThe 12 adenosine residues are a binding site for PolyA Binding Protein (PABPII).
PAP StimulationBound PABPII stimulates PAP to quickly add ~250 adenosines, forming a poly(A) tail bound by many PABPII's.

Coupling 3' Cleavage & Transcription Termination

Antitermination ModelThe polyA sequence is recognized as it leaves the polymerase and causes loss of elongation factors and/or gain of termination factors.
Torpedo ModelThe free 5’ end after cleavage of the polyA site recruits exonucleases that rapidly degrade the RNA and induce release from the template when they reach the polymerase.

Lymphocytes

How is the site of polyadenylation related to transcription termination by RNA polymerase II? What is the mechanism that controls the change in the polyadenylation site of immunoglobulin heavy chain µ pre-mRNA as immature B lymphocytes develop into IgM secreting plasma cells? B lymphocytes carry IgM, and upon exposure to their complementary antigen become antibody-secreting plasma cells. This switch is accomplished by a change in the IgM mRNA poly(A) site. There are two poly(A) sites, with the upstream site requiring more CStF. In B cells, CstF64 levels are low; in plasma cells, CStF64 expression is unregulated.