Eukaryotes encode long precursor RNA transcripts, which are spliced, polyadenylated and capped to produce a messenger RNA that can be translated.
| Event | Where? | Overview |
|---|---|---|
| Capping | Nucleus | Guanosine is added to the 5′ end of the mRNA for stability, transport and translation initiation. |
| Polyadenylation | Nucleus | The 3’ end of the RNA is cleaved and a Poly-A tail is added by the polyadenylation complex. |
| Splicing | Cytoplasm | During splicing, introns are excised and exons are ligated together by the very large spliceosome. |
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. 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.
| Step | Overview |
|---|---|
| Upstream Binding | Upstream AAUAAA bound by Cleavage and Polyadenylation Specificity Factor (CPSF; 4 subunits). |
| Downstream Binding | Downstream G/U rich element is bound by the Cleavage Stimulatory Factor (CStF; 3 subunits). |
| Additional Factors | RNA cleavage requires Cleavage Factors (CF1 and 2); the endonuclease is the 73kd subunit of CPSF. |
| Binding of PAP | The PolyA polymerase (PAP) binds, activating cleavage of the 3′ end. |
| Fragment Digested | The free cleaved 3’ piece is rapidly degraded by 5’-3’ exonucleases. |
| Start of Poly(A) Tail | Poly(A) Polymerase slowly adds ~12 adenosine residues to the 3’ end of the 5’ product RNA. |
| Poly(A) Binding Prtn | The 12 adenosine residues are a binding site for PolyA Binding Protein (PABPII). |
| PAP Stimulation | Bound PABPII stimulates PAP to quickly add ~250 adenosines, forming a poly(A) tail bound by many PABPII’s. |
| Theory | Overview |
|---|---|
| Antitermination Model | The polyA sequence is recognized as it leaves the polymerase and causes loss of elongation factors and/or gain of termination factors. |
| Torpedo Model | The 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. |
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