As discussed previously, eukaryotic mRNAs are capped at their 5' end and polyadenylated at their 3' end. In vitro assays for these reactions have been developed, and several of the enzymatic activities have been identified. These will be reviewed in this section. Polyadenylation is not limited to eukaryotes. Several mRNAs in E. coli are polyadenylated as well. This is a fairly new area of study.
Fig. 3.3.5. mRNAs can be modified on the 5’ and 3’ ends.
Modification at the 5' end: Cap Structure
The "cap" is a methylated 5'‑GMP that is linked via its 5' phosphate to the b‑phosphoryl of the initiating nucleotide (usually A); see Fig. 3.3.6. Capping occurs shortly after transcription has begun. It occurs in a series of enzymatic steps (Fig. 3.3.7):
- Remove the g‑phosphoryl of the initiating nucleotide (RNA triphosphatase)
- Link a GMP to the b‑phosphoryl of the initiating nucleotide (mRNA guanylyl transferase). The GMP is derived from GTP, and is linked by its 5' phosphate to the 5' diphosphate of the initiating nucleotide. Pyrophosphate is released.
- The N‑7 of the cap GMP is methylated (methyl transferase), donor is S‑adenosyl methionine.
- Subsequent methylations occur on the 2' OH of the first two nucleotides of the mRNA.
Capping has been implicated in having a role in efficiency of translation and in mRNA stability.
Fig. 3.3.6. Structure of the 5’ cap on eukaryotic mRNAs.
Figure 3.3.7. Stepwise synthesis of the 5’ cap.
Several proteins are required for cleavage and polyadenylation at the 3' end:
- CPSF is a tetrameric specificity factor; it recognizes and binds to the AAUAAA polyadenylation signal.
- CFI and CFII are cleavage factors.
- PAP is the polyA polymerase.
- CFI, CFII and PAP form a complex that binds to the nascent RNA at the cleavage site, directed by the CPSF specificity factor.
- CstF is an additional protein implicated in this process in vitro, but its precise function is currently unknown.