Splicing steps

After the U1 snRNP binds to the pre-mRNA (step a, Fig. 28-22)614 the U2 snRNP binds to another almost invariant sequence CURACU found 20 to 55 nucleotides upstream of the 3 junction.608,615-617 The A in this sequence becomes a branch point. It is brought close to the 5 splice site with the aid of a preassembled complex of snRNPs U4, U6, and U5. In this complex U4 and U6 are tightly paired, additional proteins are also present,618 21 and enhancers may be located in adjacent exons.617 Upon binding of U6 to the 5 splice site, the U1 and U4 snRNPs are released (step b, Fig. 28-22) and the 2 -OH of the branch point adenosine attacks the backbone phosphorus atom (step c) at the 5 splice junction forming a lariat intermediate. The 3 end created at the 5 junction must now be held and brought close to the 3 splice junction, which is located with the aid of U5 snRNP.622 The 3 splice junction, utilized in the second splicing step (step d, Fig. 28-22) has the consensus sequence (T/C)N(C/T)AG G. 

The first splicing step is dependent upon a divalent metal ion, but the second is not.623 Both steps appear to be in-line nucleophilic displacement reactions.624 Additional splicing factors are needed for formation of the U4, U6, U5 complex and its function in the second splicing step, which also appears to require ATP.621 622... 

Yeast protein L30, which is not homologous to any bacterial protein, controls its own synthesis by a feedback inhibition at the mRNA splicing step. L30 binds to its own pre-mRNA near the 5 splice site, blocking completion of the spliceosome assembly (Chapter 28).159... 

Splicing is a processing step of the pre-mRNA to become a mature transcript. This involves the excision of intervening noncoding sequences (introns) from coding sequences (exons) by a multiple protein complex, the spliceosome. After splicing the mRNA molecule is ready for translation, since it contains a continuous sequence that encode an entire protein. 

Chong, S., Montello, G.E., Zhang, A., Cantor, E.J., Liao, W., Xu, M.-Q., and Benner, J. (1998) Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucl. Acids Res. 26, 5109-5115. 

The removal of introns from pre-messenger RNAs in eukaryotes is catalyzed by the spliceosome, which is a large ribonucleoprotein consisting of at least 70 proteins and five small nuclear RNAs (snRNA) [144]. This splicing pathway involves two phosphotransfer reactions. In the first step, the 5 splice site is attacked by a 2 hydroxy group of an adenosine nucleotide within the intron [indicated by A in Fig. 12] that corresponds to the branch point in the lariat intermediate (Fig. 12,middle). In the second step, the 3 -OH group of the free 5 exon attacks the phosphodiester bond between the intron and... 

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]. 

Fig. 16A,B Proposed mechanism of the pre-mRNA splicesomal splicing. A catalytic metal ion is essential for both steps A the first step B the second step. A catalytic magnesium ion is inferred to directly coordinate the 3 oxyanion leaving group in the transition state. Catalysts which deprotonate from the nucleophiles have still remained unclear [152]...

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