The 5′ End of Eukaryotic mRNA Is Capped

KEY CONCEPTS
- A 5′ cap is formed by adding a G to the terminal base of the transcript via a 5′–5′ link.
- The capping process takes place during transcription and may be important for release from pausing of transcription.
- The 5′ cap of most mRNA is monomethylated, but some small noncoding RNAs are trimethylated.
- The cap structure is recognized by protein factors to influence mRNA stability, splicing, export, and translation.
Transcription starts with a nucleoside triphosphate (usually a purine, A or G). The first nucleotide retains its 5′-triphosphate group and makes the usual phosphodiester bond from its 3′ position to the 5′ position of the next nucleotide. The initial sequence of the transcript can be represented as:
5′ppp^A /_G pNpNpNp …
However, when the mature mRNA is treated in vitro with enzymes that should degrade it into individual nucleotides, the 5′ end does not give rise to the expected nucleoside triphosphate. Instead it contains two nucleotides that are connected by a 5′–5′ triphosphate linkage and also bear a methyl group. The terminal base is always a guanine that is added to the original RNA molecule after transcription.
Addition of the 5′ terminal G is catalyzed by a nuclear enzyme, guanylyl-transferase (GT). In mammals, GT has two enzymatic activities, one functioning as the triphosphatase to remove the two phosphates in GTP and the other as the guanylyl-transferase to fuse the guanine to the original 5′-triphosphate terminus of the RNA. In yeast, these two activities are carried out by two separate enzymes. The new G residue added to the end of the RNA is in the reverse orientation from all the other nucleotides:
5′Gppp + 5′pppApNpNp … → Gppp5′–5′ApNpNp … + pp + p
This structure is called a cap. It is a substrate for several methylation events. FIGURE 1 shows the full structure of a cap after all possible methyl groups have been added. The most important event is the addition of a single methyl group at the 7 position of the terminal guanine, which is carried out by guanine-7- methyltransferase (MT).

FIGURE 1. The cap blocks the 5′ end of mRNA and can be methylated at several positions.
Although the capping process can be accomplished in vitro using purified enzymes, the reaction normally takes place during transcription. Shortly after transcription initiation, Pol II is paused about 30 nucleotides downstream from the initiation site, waiting for the recruitment of the capping enzymes to add the cap to the 5′ end of nascent RNA. Without this protection, nascent RNA may be vulnerable to attack by 5′–3′ exonucleases, and such trimming may induce the Pol II complex to fall off of the DNA template. Thus, the process of capping is important for Pol II to enter the productive mode of elongation to transcribe the rest of the gene. In this regard, the pausing mechanism for 5′ capping represents a checkpoint for transcription reinitiation from the initial pausing site.

In a population of eukaryotic mRNAs, every molecule contains only one methyl group in the terminal guanine, generally referred to as a monomethylated cap. In contrast, some other small noncoding RNAs, such as those involved in RNA splicing in the spliceosome , are further methylated to contain three methyl groups in the terminal guanine. This structure is called a trimethylated cap.
The enzymes for these additional methyl transfers are present in the cytoplasm. This may ensure that only some specialized RNAs are further modified at their caps. One of the major functions for the formation of a cap is to protect the mRNA from degradation. In fact, enzymatic decapping represents one of the major mechanisms to regulate mRNA turnover in eukaryotic cells . In the nucleus, the cap is recognized and bound by the cap binding CBP20/80 heterodimer. This binding event stimulates splicing of the first intron and, via a direct interaction with the mRNA export machinery (TREX complex), facilitates mRNA export out of the nucleus. Once reaching the cytoplasm, a different set of proteins (eIF4F) binds the cap to initiate translation of the mRNA in the cytoplasm.