Splice canonical

The UniProt entry for human NTE lists three variants attributed to alternative splicing (UNIPROT, 2008). Although UniProt has adopted isoform-1 as the canonical sequence, this chapter defines human NTE as isoform-2, which is the sequence initially reported by Lush et al. (1998), consisting of 1,327 amino acids and having a molecular weight without posttranslational modifications of 146 kDa. The protein has a transmembrane domain near the N-terminus (residues 9-31) and three putative cyclic nucleotide binding domains (residues 163-262, 480-573, and 597-689), in addition to the patatin domain (residues 933-1,099), which contains the active site (Wijeyesakere et al., 2007). 

The modified Mth RIRl, Mxe GyrA, and Ssp DnaB mini-inteins have been recently applied to the isolation of proteins with an N-terminal cysteine residues (29,30). These inteins undergo temperature- and pH-dependent C-termi-nal cleavage when the N-terminal cysteine residue of the intein is substituted with alanine (Table 2). The target protein is recombinantly expressed as a fusion protein with the C-terminal intein tag (31) (Fig. 3B). After intein splicing the protein that possesses N-terminal cysteine is generated. Moreover, such a protein can be obtained by total chemical synthesis and different chemical labels or non-canonical amino acids can be site-specifically incorporated into the sequence. 

Since the discovery of split genes it was observed that practically all introns contain two very conservative dinucleotides. The donor site has GT exactly at the intron s 5 -boundary and the acceptor site has AG exactly at its 3 -boundary [6, 7]. We call splice sites of this type canonical. Introns flanked by the standard GT-AG pairs excised from pre-mRNA by the spliceosome including Ul, U2, U4/U6 and U5 snRNPs [7]. Recently, a few examples of a new type of splice pair, a AT-AC, has been discovered. It is processed by a related, but different splicing machinery [8, 9]. AT-AC introns are excised by a novel type of spliceosome composed of snRNPs Ull, U12, U4atac/U6atac, and U5 [10, 11, 12]. Several other cases of non-canonical splice sites with... 

Of 43 337 pairs of donor and acceptor splice sites (splice pairs) 22 489 were supported by EST sequences. 98.71% of those contain canonical dinucleotides GT and AG for donor and acceptor sites. 0.56% hold non-canonical GC-AG splice site pairs. The reminder 0.73% occurs in a lot of small groups (with maximum size of 0.05%). 53.6% of canonical and just 27.3% of non-canonical splice pairs were supported by ESTs. Based on these figures it was supposed that at least half of annotated non-canonical sites presents annotation errors, as was shown in some previous works [8, 17]. 

The occurrence of canonical dinudeotides upstream or downstream in each EST-supported non-canonical splice site group was carefully analyzed. For example, the telethonin gene has only one intron annotated in positions 639-885. This junction is completely supported by ESTs and the annotated splice sites are GG-CA. Analyzing the telethonin sequence uncovered an occurrence of a canonical splice pair GT-AG just one position downstream from the annotated site (Figure 3.4). Taking into account that the non-canonical splice junctions occur very rarely, we can suspect that the canon-... 

Possible errors in EST-supported splice pairs. Example of an annotated non-canonical junction from the H. sapiens telethonin gene, intron 1 (Genbank accession AJ010063). This junction is... 

It was concluded that 99.24% of the splice site pairs should be GT-AG, 0.69% GC-AG, 0.05% AT-AC and finally only 0.02% could consist of other types of non-canonical splice sites (Table 3.3). Therefore, gene finding approaches using just standard GT-AG splice sites can potentially predict accurately 97% genes (assuming 4-5 exons per gene on average). If the... 

Errors found by comparing GenBank and human high throughput sequences for several annotated non-canonical splice sites. 

Frequencies of canonical and non-canonical splice sites in human genes. 

Burset M., Seledtsov I., Solovyev V. (2000) Analysis of canonical and non-canonical splice sites in mammalian genomes. Nucleic Acids Res., 28, 4364-4375. 

In early experiments, seqnence analysis of mutant clones indicated a single base substitntion in the gene with a G to A transition at the canonical 5 donor splice site of intron 12 and expression of a truncated unstable protein product. This protein lacked the C-terminal region, a structural change that yielded less than 1% of the normal activity of PAH. 

Thus far we have considered only protein-coding variants generated by alternative splicing from one TU. The FANTOM3 annotation revealed 82 non-canonical in-frame and nine frame-shifted protein fusion candidates produced from two TUs. Probably, the fusions are the result of transcriptional readthrough of two closely positioned head-to-tail oriented TUs followed by r/f-splicing of the hybrid pre-mRNA. Originally, the phenomenon was noted in a study of UEVl-KUA and predicted to be more widespread than expected (Thompson et al 2000). 

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