The Chemistry Of Rna Splicing

Sequences within the RNA Determine Where Splicing Occurs

We now consider the molecular mechanisms of the splicing reaction. How are the introns and exons distinguished from each other? How are introns removed? How are exons joined with high precision? The borders between introns and exons are marked by specific nucleotide sequences within the pre-mRNAs. These sequences delineate where splicing will occur. Thus, as shown in Figure 13-2, the exon-intron boundary—that is, the boundary at the 5' end of the intron—is marked by a sequence called the 5f splice site. The intron-exon boundary at the 3' end of the intron is marked by the 3' splice site. (The 5' and 3' splice situs were sometimes referred to as the donor and acceptor sites, respectively, but this nomenclature is rarely used today.)

The figure shows a third sequence necessary for splicing. This is called the branch point site for branch point sequence). It is found entirely within the intron, usually close to its 3' end, and is followed by a poiypyrimidine tract [Py tract), as shown.

The consensus sequence for each of these elements is shown in Figure 13-2, The most highly conserved sequences are the GU in the 5' splice site, the AG in the 3' splice site, and the A at the branch site. These highly conserved nucleotides are all found within the intron itself—perhaps not surprisingly, as the sequence of the exons, in contrast to the introns. is constrained by the need to encode the specific amino acids of the protein product.

The Intron Is Removed in a Form Called a Lariat as the Flanking Exons Are Joined

Let us begin by considering the chemistry of splicing, which is achieved by two successive transesterification reactions in which phosphodiester linkages within the pre-mRNA are broken and new ones are formed (Figure 13-3). The first reaction is triggered by the 2' OH of the conserved A at the branch site. This group acts as a nucleophile to attack the phosphoryi group of the conserved G in the 5' splice site. (This is an SN2 reaction that proceeds through a pen-tavalent phosphorous intermediate.) As a consequence, the phosphodiester bond between the sugar and the phosphate at the junction between the intron and the exon is cleaved and the freed 5' end of the intron is joined to the A within the branch site. Thus, in addition to (he 5' and 3' backbone linkages, a third phosphodiester extends from the 2'OH of that A to create a three-way junction (hence its description as a branch point). The structure of the three-way junction is shown in Figure 13-4.

Notice thai the 5' exon is a leaving group in the first transesterifi-cation reaction. In the second reaction, the 5' exon (more precisely, the newly liberated 3'OH of the 5' exon) reverses its role and becomes a nucleophile that attacks the phosphoryi group at the 3' splice site (Figure 13-3). This second reaction has two consequences. First, and most importantly, it joins the 5' and 3' exons;

5" exon Intron 31 exon r ~~~~~~ " " T

s'JJQ^IT^agu jtT^^^w^-iuiAijEBfla'

i ' / i i 5' spltce site branch site 3' splice site

Figure 13-2 Sequences at the intron-exon boundary. Shown in the figure are the consensus sequences for both the 5' and 1' splice sites, and also the conserved A at the branch site As in other cases of consensus sequences, where two alternative bases are similarly favored, those bases are both indicated at that posftion- In this figure, the consensus sequences shown are for humans. This is true for all other figures, unless otherwise stated.

FIGURE 13-3 The splicing reaction.

Shown are the two steps of the splicing reaction described in the text. In the first step, the RNA forms a loop structure, which is shown in detail tn the next figure

5' exon inlron 3f exon

5' exon inlron 3f exon

rntron lariat

rntron lariat

spliced exons thus, this is the step in which the two coding sequences are actually "spliced" together. Second, this same reaction liberates the intron, which serves as a leaving group. Because the 5' end of the intron had been joined to the branch point A in the first transesterification reaction, the newly liberated intron has the shape of a lariat.

In the two reaction steps, there is no net gain in the number of chemical bonds—two phosphodiester bonds are broken, and two new ones made. As it is just a question of shuffling bonds, no energy input is demanded by the chemistry of this process. But, as we shall see

FIGURE 13-4 The structure of the three-way junction formed during the splicing reaction.

M 5-end

W fO

0 OH

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    How are xons joined with high precision?
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