Five prime cap
In molecular biology, the 5′ cap is a specially altered nucleotide on the 5′ end of precursor messenger RNA and some other primary transcripts as found in eukaryotes. The process of 5′ capping is vital to creating mature messenger RNA, which is then able to undergo translation. Capping ensures the messenger RNA's stability while it undergoes translation in the process of protein synthesis, and is a highly regulated process that occurs in the cell nucleus. Because this only occurs in the nucleus, mitochondrial and chloroplast mRNA are not capped.
5′ cap structure
The 5′ cap is found on the 5′ end of an mRNA molecule and consists of a guanine nucleotide connected to the mRNA via an unusual 5′ to 5′ triphosphate linkage. This guanosine is methylated on the 7 position directly after capping in vivo by a methyl transferase. It is referred to as a 7-methylguanylate cap, abbreviated m7G. Further modifications include the possible methylation of the 2′ hydroxy-groups of the first 2 ribose sugars of the 5′ end of the mRNA. The methylation of both 2′ hydroxy-groups is shown on the diagram. The 5′ cap looks like the 3′ end of an RNA molecule (the 5′ carbon of the cap ribose is bonded, and the 3′ unbonded). This provides significant resistance to 5′ exonucleases.
The starting point is the unaltered 5′ end of an RNA molecule. This features a final nucleotide followed by three phosphate groups attached to the 5′ carbon.
- One of the terminal phosphate groups is removed (by RNA terminal phosphatase), leaving two terminal phosphates i.e. 5'ppNp[Np]n
- GMP is added to the terminal phosphates (by a guanylyl transferase), losing two phosphate groups (from the GTP substrate) in the process. This results in the 5′–5′ triphosphate linkage. Producing 5'GpppNp[Np]n
- The 7-nitrogen of guanine is methylated (by a methyl transferase). Producing 5'm7GpppNp[Np]n
- Cap-Adjacent modifications can occur, normally to the first and second nucleotides (Producing up to 5'm7GpppNmpNmp[Np]n)
- If the immediately cap-adjacent nucleotide is 2'-O-Ribose methyl-Adenosine, (i.e the structure is 5'm7GpppAmp[Np]n) it can be further methylated at the N6 methyl position to form Cap1 m6Am (i.e. 5'm7Gpppm6Amp[Np]n)
5′ capping targeting
The Capping Enzyme Complex (CEC) required for capping is found bound to the RNA polymerase II before transcription starts. As soon as the 5′ end of the new transcript emerges the enzymes transfer to it and begin the capping process (this is a similar kind of mechanism to ensure capping as for polyadenylation).
5′ cap function
The 5′ cap has 4 main functions:
- Regulation of nuclear export.
- Prevention of degradation by exonucleases.
- Promotion of translation (see ribosome and translation).
- Promotion of 5′ proximal intron excision.
Nuclear export of RNA is regulated by the Cap binding complex (CBC), which binds exclusively to capped RNA. The CBC is then recognized by the nuclear pore complex and exported. Once in the cytoplasm after the pioneer round of translation, the CBC is replaced by the translation factors eIF-4E and eIF-4G. This complex is then recognized by other translation initiation machinery including the ribosome.
Cap prevents 5′ degradation in two ways. First, degradation of the mRNA by 5′ exonucleases is prevented (as mentioned above) by functionally looking like a 3′ end. Second, the CBC complex and the eIF-4E/eIF-4G block the access of decapping enzymes to the cap. This increases the half-life of the mRNA, essential in eukaryotes as the export process takes significant time.
Decapping of an mRNA is catalyzed by the decapping complex made up of at least Dcp1 and Dcp2, which must compete with eIF-4E to bind the cap. Thus the 5′ cap is a marker of an actively translating mRNA and is used by cells to regulate mRNA half-lives in response to new stimuli. Undesirable mRNAs are sent to P-bodies for temporary storage or decapping, the details of which are still being resolved.
The mechanism of 5′ proximal intron excision promotion is not well understood, but the 5′ cap appears to loop around and interact with the spliceosome in the splicing process, promoting intron excision.
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