Splicing branch points

Figure 28.30. Splicing Branch Point. The structure of the branch point in the lariat intermediate in which the adenylate residue is joined to three nucleotides hy phosphodiester bonds. The new 2 -to-5 linkage is shown in red, and the usual 3 -to-5 linkages are shown in blue.

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... 

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 28-22 Assembly and action of the spliceosomal complex. Four special sequence elements control the process the 5 and 3 splice sites, the branch point (adenosine A), and a polypyrimidine tract. The snRNP particle U1 locates the 5 splice site and U2 the branch point. The tri-snRNP U4 U6 U5 then binds, U6 recognizing the 5 splice site, and U1 and U4 are released. The 2 -OH of the branch point adenosine attacks the phos-phodiester linkage to form a lariet intermediate, which releases the intron in a lariet form in the final step. After Valcarcel and Green.612...

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 selection of appropriate introns is important for efficient RNA processing and to eliminate the utilization of the cryptic splice site in the coding region. Introns with splice sites and branch points that closely match the established consensus sequences are spliced more efficiently and accurately than ones that do not. Several introns have been used widely in mammalian expression vectors, including the second intron of the rabbit, 0-globin gene (O Hare et al., 1981), and intron A of CMV (Hartikka et al., 1996). Some expression vectors include a hybrid intron, e.g., the 5 portion of an adenovirus sequence and the 3 portion of an immunoglobulin sequence (Choi et al., 1991). 

Brown. J. W. S. 1986. A catalogue of splice junction and putative branch-point sequences from plant introns. Nucl. Acid Res. 14, 9549-9559. 

Fig. 3 Human consensus BPS. (a) Pictogram and (b) WebLogo presentations of BPS. Position 0 represents the branch point, (c) Representative sequences and positions of splicing cu-elements...

The 3 end of an intron and the 5 end of an exon carry a consensus sequence of CAG G, where the vertical line represents the intron/exon boundary. The AG dinucleotide is scanned from the branch point and the first AG is recognized as the 3 end of the intron (Chen et al 2000). In a patient with congenital myasthenic syndrome, we identified duplication of a 16-nt segment comprised of 8 intronic and 8 exonic nucleotides at the intron 10/exon 10 boundary of CHRNE encoding the acetylcholine receptor epsilon subunit (Ohno et al 2005). We found that the upstream AG of the duplicated segment is exclusively used for splicing and that one or two mutations in the upstream BPS had no effect whereas complete deletion of the upstream BPS partially activated the downstream AG. Similar exclusive activation of the upstream AG is reported in HEXB (Dlott et al., 1990) and SLC4A1 (Bianchi et al, 1997). Creation of a cryptic AG dinucleotide close to the 3 end of an intron should be carefully scrutinized in mutation analysis. 

Figure 2 Details of two successive trans-esterification reactions. In the first step, the 2 -OH group of the branch point adenosine nucleophilically attacks the phosphate at the junction of the S exon and intron (S splice site), resulting in the formation of a new S -2 phosphodiester bond between the first nucleotide of the intron and the branch point adenosine (lariat structure formation) and breakage of an old 3 -S phosphodiester bond between the last nucleotide of the S exon and the first nucleotide of the intron (cut-off S exon formation). In the second step, the 3 -OH group of the cut-off S exon nucleophilically attacks the phosphate at the junction of the intron and 3 exon, ligating the two exons (mRNA formation) and releasing the lariat intron. The phosphates at the S splice site (red) and at the 3 splice site (green) and the branch point adenosine and its 2 -OH group are pictured. The lines represent the intron and boxes depict exons (El and E2).

D6 is a hairpin stmcture that harbors the branch point adenosine, whose 2 -OH group acts as the nucleophile for the first step of splicing in the branching pathway. In addition, D6 contains an contact that forms the ri-ri contact with D2. This element is a GNRA tetraloop-receptor interaction that is believed to mediate a conformational switch between the two steps of splicing. 

In the first step of splicing, the 2 -OH group of the branch point adenosine attacks the scissile phosphate at the designated 5 -splice site. It is not established how the nucleophile is activated however, it is likely that the 2 -OH is coordinated to a metal ion in the core that could facilitate deprotonation (17). 

Figure 28.31. Spliceosome Assembly. U1 (blue) binds the 5 splice site and U2 (red) to the branch point. A preformed U4-U5-U6 complex then joins the assembly to form the complete spliceosome.

Figure 29.32 Spliceosome assembly and action. U1 binds the 5 splice site and U2 binds to the branch point. A preformed U4-US-U6 complex then joins the assembly to form the complete spliceosome. The U6 snRNA re-folds and binds the 5 splice site, displacing Ul, Extensive Interactions between U6 and U2 displace U4. Then, in the first transesterification step, the branch-site adenosine attacks the S splice site, making a lariat Intermediate. US holds the two exons in close proximity, and the second transesterification takes place, with the S splice-site hydroxyl group attacking the 3 splice site. These reactions result in the mature spliced mRNA and a lariat form of the intron bound by U2. US. and U6. After T. Villa, j. A. Pletss. and C, Guthrie. Cell 109(2002) H9-1S2.]...

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