Splicing consensus sequences

Figure 37-12. Consensus sequences at splice junctions. The 5 (donor or left) and 3 (acceptor or right) sequences are shown. Also shown is the yeast consensus sequence (UACUAAQ for the branch site. In mammalian cells, this consensus sequence is PyNPyPy-PuAPy, where Py is a pyrimidine, Pu is a purine, and N is any nucleotide. The branch site is located 20-40 nucleotides upstream from the 3 site.

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). 

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. 

Figure 11.5. Mutation of intron splice sites involved in formation of a mutant allele of CYP2D6. (A) Normal intron consensus sequences of mammalian gene. (B) Mutation of the CYP2D6 splice site that moves the splice site downstream to the next 3 consensus sequence, altering the reading frame of the protein and resulting in a premature stop codon (CYP2D6 4) and a truncated, inactive protein.

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. 

In the late 1970s and early 1980s, the sequences of many eukaryotic genes became available for study, thus making it possible to conduct a comparative analysis to identify important sequence elements in pre-mRNA. Careful inspection of higher eukaryotic genes revealed several consensus sequences in introns at or near the 5 and 3 splice sites (12, 13). The 5 splice site consensus sequence is G/GURAGU (/ represents the 5 exon-intron junction R depicts a purine the underlined dinucleotide GU is invariant). The 3 splice site is YAG/G (here, / represents the 3 intron-exon junction Y is a pyrimidine the... 

The main mechanistic differences between the two spliceo-somes occur at the stage of intron recognition, rather than catalysis. Indeed, recognition of the 5 splice site and the branch site occurs simultaneously by the U11-U12 di-snRNP (141). Furthermore, there is a requirement among U12-dependent introns for 5 exon sequences to form U6atac-5 splice site interactions (142). Lastly, more constrained consensus sequences, as... 

Splicing is a facile complex operation that is carried out by spliceosomes, which are assemblies of proteins and small RNA molecules (Section 28.3.4). This enzymatic machinery recognizes signals in the nascent RNA that specify the splice sites. Introns nearly always begin with GU and end with an AG that is preceded by a pyrimidine-rich tract (Figure 5.35). This consensus sequence is part of the signal for splicing. 

Figure 5.35. Consensus Sequence for the Splicing of mRNA Precursors.

S, and 28S ribosomal RNA (Section 29.3.1). The other ribosomal RNA molecule (5S rRNA, Section 29.3.1) and all the transfer RNA molecules (Section 29.1.2) are synthesized by RNA polymerase III, which is located in the nucleoplasm rather than in nucleoli. RNA polymerase II, which also is located in the nucleoplasm, synthesizes the precursors of messenger RNA as well as several small RNA molecules, such as those of the splicing apparatus (Section 28.3.5). Although all eukaryotic RNA polymerases are homologous to one another and to prokaryotic RNA polymerase, RNA polymerase II contains a unique carboxyl-terminal domain on the 220-kd subunit this domain is unusual because it contains multiple repeats of a YSPTSPS consensus sequence. The activities of RNA polymerase II are regulated by phosphorylation on the serine and threonine residues of the carboxyl-terminal domain. Another major distinction among the polymerases lies in their responses to the toxin a -amanitin, a cyclic octapeptide that contains several modified... 

Figure 28.27. Splice Sites. Consensus sequences for the 5 splice site and the 3 splice site are shown. Py stands for pyrimidine.

Figure 4.35 Consensus sequence for the splicing of mRNA precursors.

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