Splice junction

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.

Rl. Raben, N., Sherman, J., Miller, E, Mena, H., and Plotz, P., A5 splice junction mutation leading to exon deletion in an Ashkenazic Jewish family with phosphofructokinase deficiency (Tarui disease). J. Biol. Chem. 268,4963-4967 (1993). 

T24. Tsujino, S., Tonin, P Shanske, S., Nohria, V., Boustany, R.-M., Lewis, D Chen, Y.-T., and DiMauro, S., A splice junction mutation in a new myopathic variant of phosphoglycerate kinase deficiency (PGK North Carolina). Ann. Neurol. 35,349-353 (1994). 

Ibeanu GC, Blaisdell ], Ferguson R], Ghanayem Bl, Brosen K, Benhamou S et al. A novel transversion in the intron 5 donor splice junction of CYP2C19 and a sequence polymorphism in exon 3 contribute to the poor metabolizer phenotype for the anticonvulsant drug S-me-phenytoin. J Pharmacol Exp Ther 1999 290[2] 635—640. 

Otterness DM, Szumlanski CL, Wood TC et al. Human thiopurine methyltransferase pharmacogenetics. Kindred with a terminal exon splice junction mutation that results in loss of activity. J Clin Invest 1998 101 1036-1044. 

Fusion vectors are available that combine a recombinant protein with a mutant mini intein segment (not containing an endonuclease domain) and followed by a chitin binding domain (CBD Zhang et al., 2001). These mutants typically also have an alanine substitution that replaces the cysteine or serine/threonine usually found on the C-extein splice junction. Alanine... 

To make mRNA, the primary transcript must be spliced to bring the protein-coding sequences (exons) together and to remove the intervening sequences (introns). The splice signals consist of a 5 and a 3 set of sequences that are always found at splice junctions. However, this is generally believed to provide too little information to recognize a splice site specifically and correctly. Some sequences in the intron are also important. 

C. Splicing errors alter the critical sequence around an intron-exon splice junction. 

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 RNA world hypothesis requires a nucleotide polymer to reproduce itself. Can a ribozyme bring about its own synthesis in a template-directed manner The self-splicing rRNA intron of Tetrahymena (Fig. 26-26) catalyzes the reversible attack of a guanosine residue on the 5 splice junction (Fig. 26-37). If the 5 splice site and the internal guide sequence are removed from the intron, the rest of the intron can bind RNA strands paired with short oligonucleotides. Part of the remaining intact intron effectively acts as a template for the... 

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. 

Conventionally, the variants are characterized by coamplification with wild-type sequences using reverse transcription polymerase chain reaction (RT-PCR). However, this approach focuses on small regions of the known wild-type mRNA. Because of this threshold detection, spliced transcripts expressed at low levels may fall below the threshold of detection. To avoid this and other limitations of the conventional RT-PCR technique, the targeted amplification method can be used (Poola et al., 2000). This method involves the targeted amplification of the alternatively spliced molecules as separate gene populations using specific primers designed for the alternative splice junctions, without coamplification of wild-type molecules. 

Figure 4.8 The intein system as used for fusion proteins (Xu, 2000). The fusion proteins undergo self-cleavage at the upstream splice junction. The amino acids that participate directly are shown, and the remainder of the intein and target protein are drawn as boxes. Usually, the intein is conjugated to a chitin-binding domain, so purification of the fusion protein can occur by using a chitin-conjugated column. Self-cleavage can occur overnight.

The term splicing refers to the process by which introns are removed and the mRNA put back together to form a continuous coding sequence in the 5 -3 direction. Remembering how accurate this process must be is important. If only a single nucleotide of an intron were left in the processed mRNA, the protein made from that mRNA would be non-functional, because the ribosome would read the wrong codons. The cellular machinery that splices pre-mRNAs uses information at the splice junctions to determine where to cut and where to rejoin the mRNA. Removal of introns from transcripts containing more than one intron usually occurs in a preferred but not exclusive order. Several pathways are used. 

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

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