5′ cap structure (cap-2).Ribose structure showing the positions of the 2′, 3′ and 5′ carbons.
Ineukaryotes, the 5′ cap (cap-0), found on the 5′ end of an mRNA molecule, consists of aguanine nucleotide connected to mRNA via an unusual 5′ to 5′triphosphate linkage. Thisguanosine ismethylated on the 7 position directly after cappingin vivo by amethyltransferase.[3][4][5][6] It is referred to as a7-methylguanylate cap, abbreviated m7G. The Cap-0 is the base cap structure, however, the first and second transcribed nucleotides can also be 2' O-methylated, leading to the Cap-1 and Cap-2 structures, respectively. This is more common in higher eukaryotes and thought to be part of the innate immune system to recognize mRNAs from other organisms.[7]
In multicellular eukaryotes and some viruses,[8] further modifications can be made, including the methylation of the 2′hydroxy-groups of the first tworibose sugars of the 5′ end of the mRNA. cap-1 has a methylated 2′-hydroxy group on the first ribose sugar, while cap-2 has methylated 2′-hydroxy groups on the first two ribose sugars, shown on the right. The 5′ cap is chemically similar to the3′ 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.[9]
Small nuclear RNAs contain unique 5′-caps. Sm-class snRNAs are found with 5′-trimethylguanosine caps, while Lsm-class snRNAs are found with 5′-monomethylphosphate caps.[10]
The starting point for capping with 7-methylguanylate is the unaltered 5′ end of an RNA molecule, which terminates at a triphosphate group. This features a final nucleotide followed by three phosphate groups attached to the 5′ carbon.[3] The capping process is initiated before the completion of transcription, as the nascent pre-mRNA is being synthesized.
One of the terminal phosphate groups is removed byRNA triphosphatase, leaving a bisphosphate group (i.e. 5′(ppN)[pN]n);
GTP is added to the terminal bisphosphate bymRNA guanylyltransferase, losing apyrophosphate from the GTP substrate in the process. This results in the 5′–5′ triphosphate linkage, producing 5′(Gp)(ppN)[pN]n;
Cap-adjacent modifications can occur, normally to the first and second nucleotides, producing up to 5′(m7Gp)(ppN*)(pN*)[pN]n (cap-1 and cap-2);[8]
If the nearest cap-adjacent nucleotide is2′-O-ribose methyl-adenosine (i.e. 5′(m7Gp)(ppAm)[pN]n), it can be further methylated at the N6 methyl position to formN6-methyladenosine, resulting in 5′(m7Gp)(ppm6Am)[pN]n.[3]
The mechanism of capping with NAD+, NADH, or 3′-dephospho-coenzyme A is different. Capping with NAD+, NADH, or 3′-dephospho-coenzyme A is accomplished through an "ab initio capping mechanism," in which NAD+, NADH, or 3′-desphospho-coenzyme A serves as a "non-canonical initiating nucleotide" (NCIN) fortranscription initiation byRNA polymerase and thereby directly is incorporated into the RNA product.[11] Both bacterial RNA polymerase and eukaryoticRNA polymerase II are able to carry out this "ab initio capping mechanism".[11]
For capping with 7-methylguanylate, thecapping enzyme complex (CEC) binds toRNA polymerase II before transcription starts. As soon as the 5′ end of the new transcript emerges from RNA polymerase II, the CEC carries out the capping process (this kind of mechanism ensures capping, as withpolyadenylation).[13][14][15][16] The enzymes for capping can only bind toRNA polymerase II, ensuring specificity to only these transcripts, which are almost entirely mRNA.[14][16]
Capping with NAD+, NADH, or 3′-dephospho-coenzyme A is targeted bypromoter sequence.[11] Capping with NAD+, NADH, or 3′-dephospho-coenzyme A occurs only at promoters that have certain sequences at and immediately upstream of the transcription start site and therefore occurs only for RNAs synthesized from certain promoters.[11]
Nuclear export of RNA is regulated by thecap binding complex (CBC), which binds exclusively to 7-methylguanylate-capped RNA. The CBC is then recognized by thenuclear pore complex and exported. Once in the cytoplasm after the pioneer round of translation, the CBC is replaced by the translation factorseIF4E andeIF4G of theeIF4F complex.[6] This complex is then recognized by other translation initiation machinery including the ribosome.[23]
Capping with 7-methylguanylate 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 and eIF4E/eIF4G block the access of decapping enzymes to the cap. This increases thehalf-life of the mRNA, essential in eukaryotes as the export and translation processes take significant time.
Decapping of a 7-methylguanylate-capped mRNA is catalyzed by the decapping complex made up of at least Dcp1 and Dcp2, which must compete with eIF4E to bind the cap. Thus the 7-methylguanylate 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 toP-bodies for temporary storage or decapping, the details of which are still being resolved.[24]
The mechanism of 5′ proximal intron excision promotion is not well understood, but the 7-methylguanylate cap appears to loop around and interact with thespliceosome in the splicing process, promoting intron excision.
^abSonenberg N, Gingras AC (April 1998). "The mRNA 5′ cap-binding protein eIF4E and control of cell growth".Current Opinion in Cell Biology.10 (2):268–275.doi:10.1016/S0955-0674(98)80150-6.PMID9561852.
^Matera AG, Terns RM, Terns MP (March 2007). "Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs".Nature Reviews. Molecular Cell Biology.8 (3):209–220.doi:10.1038/nrm2124.PMID17318225.S2CID30268055.
