The 5 end of eukaryotic mRNA is capped

KEY TERMS:

• A cap is the structure at the 5 end of eukaryotic mRNA, introduced after transcription by linking the terminal phosphate of 5 GTP to the terminal base of the mRNA. The added G (and sometimes some other bases) are methylated, giving a structure of the form ⁷MeG5ppp5Np . . .
• A cap 0 at the 5 end of mRNA has only a methyl group on 7-guanine.
• A cap 1 at the 5 end of mRNA has methyl groups on the terminal 7-guanine and the 2-O position of the next base.
• A cap 2 has three methyl groups (7-guanine, 2-O position of next base, and N⁶ adenine) at the 5 end of mRNA.

KEY CONCEPTS:

• A 5 cap is formed by adding a G to the terminal base of the transcript via a 5–5 link. 1-3 methyl groups are added to the base or ribose of the new terminal guanosine. 

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:

5ppp^A/_GpNpNpNp...

But 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, connected by a 5–5 triphosphate linkage and also bearing methyl groups. 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. The reaction occurs so soon after transcription has started that it is not possible to detect more than trace amounts of the original 5 triphosphate end in the nuclear RNA. The overall reaction can be represented as a condensation between GTP and the original 5 triphosphate terminus of the RNA. Thus

5          5

Gppp + pppApNpNp...

5–– 5

GpppApNpNp... + pp + p

The new G residue added to the end of the RNA is in the reverse orientation from all the other nucleotides.

This structure is called a cap. It is a substrate for several methylation events. Figure 5.18 shows the full structure of a cap after all possible methyl groups have been added. Types of caps are distinguished by how many of these methylations have occurred:

• The first methylation occurs in all eukaryotes, and consists of the addition of a methyl group to the 7 position of the terminal guanine. A cap that possesses this single methyl group is known as a cap 0. This is as far as the reaction proceeds in unicellular eukaryotes. The enzyme responsible for this modification is called guanine-7-methyltransferase.
• The next step is to add another methyl group, to the 2–O position of the penultimate base (which was actually the original first base of the transcript before any modifications were made). This reaction is catalyzed by another enzyme (2–O-methyl-transferase). A cap with the two methyl groups is called cap 1. This is the predominant type of cap in all eukaryotes except unicellular organisms.
• In a small minority of cases in higher eukaryotes, another methyl group is added to the second base. This happens only when the position is occupied by adenine; the reaction involves addition of a methyl group at the N⁶ position. The enzyme responsible acts only on an adenosine substrate that already has the methyl group in the 2–O position.
• In some species, a methyl group is added to the third base of the capped mRNA. The substrate for this reaction is the cap 1 mRNA that already possesses two methyl groups. The third-base modification is always a 2–O ribose methylation. This creates the cap 2 type. This cap usually represents less than 10-15% of the total capped population.

In a population of eukaryotic mRNAs, every molecule is capped. The proportions of the different types of cap are characteristic for a particular organism. We do not know whether the structure of a particular mRNA is invariant or can have more than one type of cap.

In addition to the methylation involved in capping, a low frequency of internal methylation occurs in the mRNA only of higher eukaryotes. This is accomplished by the generation of N⁶ methyladenine residues at a frequency of about one modification per 1000 bases. There are 1-2 methyladenines in a typical higher eukaryotic mRNA, although their presence is not obligatory, since some mRNAs do not have any (for review see Bannerjee, 1980).