Five-prime Cap

The five-prime cap, usually written 5′ cap, is a chemical modification at the beginning of most eukaryotic messenger RNAs (mRNAs). Its characteristic structure is a 7-methylguanosine residue joined to the first transcribed nucleotide by an unusual 5′–5′ triphosphate linkage. The cap helps distinguish an RNA as a substrate for processing and translation, provides a binding site for regulatory proteins, and protects the RNA against certain degradation pathways. It is normally added early during transcription, rather than after the entire RNA has been synthesized. (pubmed.ncbi.nlm.nih.gov)

Chemical structure and cap types

The designation 5′ refers to the numbering of carbon atoms in the ribose sugar of an RNA nucleotide. Ordinary RNA chains connect successive nucleotides through 3′–5′ phosphodiester bonds. The cap differs: the 5′ position of its guanosine is connected through three phosphate groups to the 5′ position of the first transcribed nucleotide. The cap guanosine is therefore oriented oppositely to the nucleotides in the RNA chain. Its guanine base is methylated at nitrogen position 7, producing m⁷G. (pubmed.ncbi.nlm.nih.gov)

The principal canonical cap structures differ in methylation of the nucleotides immediately following the cap:

Here, N₁ and N₂ denote the first and second transcribed nucleotides, and m denotes ribose 2′-O-methylation. The numbering of cap types does not count methyl groups on the cap guanosine. In humans, the enzymes CMTR1 and CMTR2 methylate the first and second transcribed nucleotide, respectively. (pmc.ncbi.nlm.nih.gov)

These modifications are chemically distinct. N7 methylation modifies the cap’s guanine base, whereas 2′-O-methylation modifies a sugar in the RNA itself. Their biological effects consequently need not be identical: the former is central to recognition by conventional cap-binding proteins, while the latter also influences recognition by antiviral proteins. (pmc.ncbi.nlm.nih.gov)

Formation during transcription

Canonical nuclear capping is coupled to transcription by RNA polymerase II. Capping enzymes associate with its phosphorylated carboxy-terminal domain, positioning them near the emerging RNA. The cap is added when the transcript is only about 20–30 nucleotides long, before most subsequent RNA-processing events. (pmc.ncbi.nlm.nih.gov)

Formation of cap 0 requires three sequential enzymatic activities:

1. RNA triphosphatase removes the outermost phosphate from the RNA’s original 5′ triphosphate, leaving a diphosphate end.
2. RNA guanylyltransferase transfers a guanosine monophosphate group from guanosine triphosphate to this end, creating the 5′–5′ triphosphate bridge.
3. Guanine-N7 methyltransferase methylates the added guanosine, using S-adenosylmethionine as the methyl donor.

The resulting cap 0 can undergo additional cap-adjacent methylation to produce cap 1 or cap 2. Different organisms package the catalytic activities into different combinations of proteins; the conserved feature is the reaction sequence, not a universally identical enzyme complex. (pmc.ncbi.nlm.nih.gov)

Recruitment to RNA polymerase II helps explain why capping is selective rather than an indiscriminate modification of all cellular RNA. It also makes capping part of the coordinated production of an RNA–protein particle, linking transcription to later processing and use of the transcript. (pmc.ncbi.nlm.nih.gov)

Biological functions

RNA processing and nuclear export

In the cell nucleus, the cap is recognized by the nuclear cap-binding complex (CBC), whose principal subunits are CBP20 and CBP80. Cap binding provides a platform for interactions with other proteins involved in RNA maturation. CBC promotes pre-mRNA splicing, including assembly of the spliceosome, and participates in 3′-end formation and export of RNA from the nucleus. Thus, a modification at one end of a transcript can affect processing elsewhere along it. (pubmed.ncbi.nlm.nih.gov)

The cap and the poly(A) tail are separate structures. The cap is attached to the 5′ end, whereas polyadenylation usually adds a chain of adenosine residues at the 3′ end. Their formation can be coordinated through RNA-processing proteins, but one modification is not a chemical extension of the other. (pmc.ncbi.nlm.nih.gov)

Translation initiation

During conventional cap-dependent translation, the cap is recognized by eukaryotic initiation factor 4E (eIF4E). This recognition contributes to recruitment of the machinery that initiates protein synthesis. Cap chemistry and orientation therefore influence how efficiently an mRNA is translated. (pubmed.ncbi.nlm.nih.gov)

The cap is not itself a coding sequence or a signal specifying an amino acid. It acts through molecular recognition: initiation factors bind the cap and help establish a functional translation-initiation complex. Experiments with synthetic capped RNAs demonstrate that changes improving resistance to degradation do not necessarily improve translation, because the same changes may weaken binding to eIF4E. (pmc.ncbi.nlm.nih.gov)

Protection against degradation

A capped 5′ end is resistant to exonucleases that require a 5′ monophosphate substrate. This protection is selective, not absolute: cellular enzymes can remove the cap, and RNA can also be degraded through other routes. Cap recognition, cap removal, and RNA degradation are therefore interconnected aspects of gene expression. (pmc.ncbi.nlm.nih.gov)

Recognition by antiviral proteins

Cap-adjacent 2′-O-methylation contributes to discrimination between cellular and foreign RNA by components of innate immunity. In experimental systems, viral RNAs lacking this modification are more susceptible to restriction by IFIT-family proteins, which can inhibit their translation or accumulation. Studies of West Nile virus, poxvirus, and coronavirus mutants established that this methylation can help viral RNA escape IFIT-mediated restriction. (pubmed.ncbi.nlm.nih.gov)

This is not a universal binary test of whether an RNA is “self.” In the West Nile virus experiments, loss of 2′-O-methylation altered sensitivity to interferon-induced antiviral effectors without increasing interferon induction in the infected fibroblasts studied. Cap structure influences particular recognition pathways in particular contexts. (pubmed.ncbi.nlm.nih.gov)

Decapping and cytoplasmic recapping

Messenger RNA decapping removes the protective cap and can direct an RNA into degradation. The enzyme DCP2 hydrolyzes capped RNA to release m⁷GDP, leaving an RNA with a 5′ monophosphate. This end is a substrate for the 5′–3′ exonuclease XRN1. DCP2 activity is regulated by interacting proteins, linking cap removal to the wider control of mRNA turnover. (pubmed.ncbi.nlm.nih.gov)

Capping is not confined to newly synthesized nuclear transcripts. A mammalian cytoplasmic complex has been identified that converts 5′-monophosphate RNA into a substrate for guanylylation and adds a cap. Experiments also identified capped, 5′-truncated RNA products. These findings establish that some RNA ends can be capped outside the original transcription process; they do not imply that every decapped RNA is routinely rescued. (pmc.ncbi.nlm.nih.gov)

Viral use of caps

Some viruses produce RNA with canonical-looking caps and cap-adjacent methylation. Such modifications allow viral transcripts to interact with cellular translation machinery and can reduce their susceptibility to antiviral restriction. (pubmed.ncbi.nlm.nih.gov)

Influenza virus acquires capped material from host RNA through cap-snatching. Its transcription machinery uses capped host-derived RNA fragments as primers for viral RNA synthesis. This differs from assembling a new cap through the ordinary cellular capping reaction sequence. Transfer of a host mRNA cap into influenza transcripts was demonstrated experimentally in 1979. (pubmed.ncbi.nlm.nih.gov)

Discovery and experimental applications

The cap’s unusual structure was established through studies of methylated viral RNA in the mid-1970s. Research on reovirus mRNA identified a blocked 5′ terminus containing N7-methylguanosine, a 5′–5′ triphosphate linkage, and a methylated first transcribed nucleotide. These experiments supplied direct chemical evidence for the structure now called the mRNA cap. (pubmed.ncbi.nlm.nih.gov)

Synthetic cap analogues are important tools for studying translation and RNA stability. They can be incorporated during in vitro transcription, but conventional dinucleotide analogues may enter the RNA in either the correct or reverse orientation. Anti-reverse cap analogues (ARCAs) modify the cap guanosine so that incorporation occurs in the intended orientation. Their development enabled more controlled comparisons of capped transcripts and improved translation in the experimental systems tested. (pubmed.ncbi.nlm.nih.gov)

Other synthetic analogues modify the phosphate bridge to alter susceptibility to decapping. Their performance illustrates a central constraint in cap engineering: an effective cap must combine chemical stability with appropriate recognition by cap-binding proteins. Greater resistance to one degradation enzyme alone does not guarantee greater protein production. (pmc.ncbi.nlm.nih.gov)

References

1. Reovirus messenger RNA contains a methylated, blocked 5'-terminal structure: m-7G(5')ppp(5')G-MpCppubmed.ncbi.nlm.nih.gov
2. 5'-Terminal m-7G(5')ppp(5')G-m-p in vivo: identification in reovirus genome RNApubmed.ncbi.nlm.nih.gov
3. mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domainpmc.ncbi.nlm.nih.gov
4. 5′-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase IIpmc.ncbi.nlm.nih.gov
5. Structural analysis of human 2′-O-ribose methyltransferases involved in mRNA cap structure formationpmc.ncbi.nlm.nih.gov
6. A nuclear cap binding protein complex involved in pre-mRNA splicingpubmed.ncbi.nlm.nih.gov
7. The nuclear cap-binding complex interacts with the U4/U6·U5 tri-snRNP and promotes spliceosome assembly in mammalian cellspmc.ncbi.nlm.nih.gov
8. Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl (3'-deoxy)GpppGpubmed.ncbi.nlm.nih.gov
9. Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cellspmc.ncbi.nlm.nih.gov
10. 2'-O methylation of the viral mRNA cap evades host restriction by IFIT family memberspubmed.ncbi.nlm.nih.gov
11. The hDcp2 protein is a mammalian mRNA decapping enzymepubmed.ncbi.nlm.nih.gov
12. The activation of the decapping enzyme DCP2 by DCP1 occurs on the EDC4 scaffold and involves a conserved loop in DCP1pmc.ncbi.nlm.nih.gov