Splicing reaction

The mechanisms whereby introns are removed from the primary transcript in the nucleus, exons are ligated to form the mRNA molecule, and the mRNA molecule is transported to the cytoplasm are being elucidated. Four different splicing reaction mechanisms have been described. The one most frequently used in eukaryotic cells is described below. Although the sequences of nu-... 

Residues involved in the protein splicing reaction are shown by a single character code of amino acids. N- and C-termini are indicated by NH2 and COOH respectively. 

Fig. 12 The spliceosome splicing reaction. In the first step, the 2 -OH of an adenosine residue that is conserved in the intron attacks the phosphorus at the 5 splice site and generates an intron-3 -exon 2 intermediate and a free 5 exon 1. In the second step, the free 3 -OH of the 5 exon attacks the phosphorus at the 3 splice site to produce ligated exons and an excised intron...

Of the five snRNAs, U2 and U6 interact with the reaction site (the 5 splice site and the branch point) in the first chemical step. These two snRNAs are known to anneal together to form a stable-based paired structure in the absence of proteins and in the presence of ions as shown in Fig. 13, with U2 acting as an inducer molecule that displaces the U4 (that is an antisense molecule that regulates the catalytic function of U6 RNA) from the initially formed U4-U6 duplex. The secondary (or higher ordered) structure of the U2-U6 complex consists of the active site of the spliceosome. Recent data suggests that these two snRNAs function as the catalytic domain of the spliceosome that catalyzes the first step of the splicing reaction [145]. 

FIGURE 18. Schematic representation of the self-splicing reaction catalyzed by group 1 introns. G is the guanosine nucleotide cofactor, P a phosphate linker... 

The recombination process is not as precise as the site-specific recombination described earlier, so additional variation occurs in the sequence at the V-J junction. This increases the overall variation by a factor of at least 2.5, thus the cells can generate about 2.5 X 1,200 = 3,000 different V-J combinations. The final joining of the V-J combination to the C region is accomplished by an RNA-splicing reaction after transcription, a process described in Chapter 26. 

Some components of the splicing apparatus appear to be tethered to the CTD of RNA polymerase II, suggesting an interesting model for the splicing reaction. As the first splice junction is synthesized, it is bound by... 

The fourth class of introns, found in certain tRNAs, is distinguished from the group I and II introns in that the splicing reaction requires ATP and an endonuclease. The splicing endonuclease cleaves the phosphodiester bonds at both ends of the intron, and the two exons are joined by a mechanism similar to the DNA ligase reaction (see Fig. 25-16). 

Because the intron itself is chemically altered during the splicing reaction—its ends are cleaved—it may appear to lack one key enzymatic property the ability to catalyze multiple reactions. Closer inspection has shown that after excision, the 414 nucleotide intron from Tetrahymena rRNA can, in vitro, act as a true enzyme (but in vivo it is quickly degraded). A series of... 

The known catalytic repertoire of ribozymes continues to expand. Some virusoids, small RNAs associated with plant RNA viruses, include a structure that promotes a self-cleavage reaction the hammerhead ribozyme illustrated in Figure 26-25 is in this class, catalyzing the hydrolysis of an internal phosphodiester bond. The splicing reaction that occurs in a spliceosome seems to rely on a catalytic center formed by the U2, U5, and U6 snRNAs (Fig. 26-16). And perhaps most important, an RNA component of ribosomes catalyzes the synthesis of proteins (Chapter 27). 

Pre-tRNAs. In the removal of type I introns the formation of specific stem and loop structures directs the splicing reactions (Fig. 28-18).47 597 Stems and loop structures already exist in tRNA precursors. Cleavage sites are usually located just to the 3 side of the anti-... 

Why do cells ever splice proteins It isn t clear. However, a curious fact is that many inteins are homing endonucleases.h k The genes for these nucleases are often present in introns in mRNA, and the homing endonuclease often cuts DNA in such a way as to initiate movement of its own gene (Chapter 27). The endonuclease itself is found in the center of the intein between the two end domains, which contain the catalytic centers for the splicing reaction. 

The mechanism of self-splicing in this case is somewhat different from that observed in the spliceosome reaction (fig. 28.21). First the 3 hydroxyl group of the guanosine cofactor attacks the phosphodiester bond at the 5 splice site. This is followed by another transesterification reaction in which the 3 hydroxyl group of the upstream RNA attacks the phosphodiester bond at the 3 splice site, thereby completing the splicing reaction. The final reaction products include the spliced rRNA and the excised oligonucleotide. 

Since its initial discovery, self-splicing has been found to occur for RNAs from a wide variety of organisms. Certain precursor RNAs that exhibit self-splicing produce lariats, just like those seen in the commonly observed splicing reactions that are catalyzed by spliceosomes (see fig. 28.19). These findings suggest that at one time all splicing reactions were RNA-catalyzed. 

Ribozyme activities are not confined to splicing reactions. Among the large number of RNases, the most sophisticated enzymes are probably those involved in processing because they must attack preRNAs at specific sites. In his studies on... 

There are six ribozymes that have been successfully modified and/or adapted for use in therapeutic and functional genomic applications. These are the group I introns, RNAse P, the hammerhead and hairpin motifs, the hepatitis delta ribozyme and the reverse splicing reaction of group II introns. Each of these ribozymes requires a divalent metal cation for activity (usually Mg++), which may participate in the chemistry of the cleavage/ligation reaction and/or may be important for maintaining the structure of the ribozyme. 

Certain pre-mRNAs contain more than one set of signal sequences for 3 end cleavage and polyadenylation. In some cases, the location of the alternative polyadenylation sites is such that, depending on the site chosen, particular exons may be lost or retained in the subsequent splicing reactions (Fig. 7). Here the effect is to change the coding capacity of the final mRNA so that different proteins are produced depending on the polyadenylation site used. In other... 

The small nuclear ribonucleoprotein particles (snRNPs or snurps ) that carry out the splicing reaction use RNA-RNA basepairing to select the splice sites. Almost all intron-exon junctions contain the sequence AG-GU with the GU beginning the intron sequence. Furthermore, the consensus sequence for the beginning of the intron has a longer sequence complementary to the U1 RNA. Thus, assembly of the splicing complex, called the spliceosome, starts when the RNA component of the U1 snRNP base pairs with the junction between the 3 end of the exon and the 5 end of the intron. See Figure 12-13. 

---