Part of RNA processing in eukaryotes is addition of the 5' mRNA Cap, which is essential for mRNA splicing, 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 5' mRNA Cap occurs as follows:
- Cleave the 5’ triphosphate of the primary transcript;
- add a guanosine residue via a 5’-5’ linkage;
- methylate the cap guanosine;
- methylate the 2’ hydroxyls of the first several bases.
The Cap recruits proteins that guide splicing, transport and translation.
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 eIF4E, 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.
Cap Binding Complex
The Cap Binding Complex is needed for RNA splicing, RNA stability, and RNA transport out of the nucleus.
The Cap Binding Complex (CBC) is a nuclear protein with two subunits: the 20kD (CBP20) subunit binds Cap; 80kD (CBP80) subunit engages cofactors.
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 engages in protein-protein interactions.
Once in the cytoplasm, the fully processed mRNA exchanges the Cap Binding Complex (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.