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. |
Part of RNA processing in eukaryotes is addition of the 5′ mRNA Cap, which is essential for mRNA stability, transport, and translation (by guiding ribosomal assembly at the RNA’s 5′ end). To ensure that only RNAP II transcripts are capped, the RNAP II CTD is phosphorylated, 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.
Making the Cap occurs as follows:
|
The Cap binds the nucleic Cap Binding Complex which guides splicing, RNA stabilization and transport into the cytoplasm. Once in the cytoplasm, the Cap binds the eIF4 complex, which helps initiate translation via binding the mRNA to the assembling ribosome, and defining the AUG start codon. By defining a single start codon, the Cap ensures that the mRNA encodes the same polypeptide whenever translated. This is a key difference from prokaryotic mRNAs, which are often polycystronic.
The Cap Binding Complex (CBC) is a nuclear protein with two subunits: the 20kD (CBP20) subunit binds Cap; 80kD (CBP80) subunit engages cofactors. The Cap Binding Complex is needed for RNA splicing, stabilizing the RNA, and transport out of the nucleus.
| Subunit | Overview |
| CBP20 | CBP20 is very specific for the cap structure. To achieve this, it engages in numerous aromatic stacking, hydrogen bonding, and other interactions with the 7-methyl G and the triphosphate linkage. |
|---|---|
| CBP80 | CBP80 engages in protein-protein interactions. |
Once in the cytoplasm, the fully processed mRNA exchanges CBC for another Cap-binding protein: the E subunit of eukaryotic Initiation Factor 4 (eIF4E). The eIF4 complex delivers the mRNA to the small ribosomal subunit and helps identify the start codon during translation initiation.
| Next Steps | In In RNA processing: transcription initiation is coupled to capping; transcription termination is coupled to 3’ cleavage and polyadenylation. |
|---|
|
|
