Cryptic Splice Site Usage Research Articles

Identification of Amino Acid Residues Contributing to Desensitization of the P2X2 Receptor Channel

The P2X2 receptor (P2X2R) is a member of the ATP-gated ion channels that mediate Ca2+ entry in several tissues, including the brain, adrenal medulla, and pituitary. Alternative usage of cryptic splice sites in the primary P2X2R transcript accounts for the existence of several transcript types, one of which (P2X2-2R) encodes a functional channel. P2X2-2R lacks a stretch of cytoplasmic C-terminal amino acids (Val370-Gln438) and exhibits rapid and complete desensitization, whereas P2X2R desensitizes slowly and incompletely. The role of the C terminus in P2X2R desensitization was studied by generating several channel mutants and monitoring intracellular free Ca2+ changes in transfected single GT1-7 neurons. Deletion studies indicated that the Arg371-Ile391 segment of the P2X2R is required for sustained Ca2+ influx. To identify the important residues within this segment, three contiguous amino acids were sequentially changed to alanine. Only two of these replacement mutants, at Arg371-Thr372-Pro373 and Lys374-His375-Pro376, had an enhanced rate of desensitization. Single amino acid deletions in the P2X2R C terminus and a series of insertions of wild-type sequences into the corresponding spliced site identified four residues, Pro373-Lys374-His375-Pro376, required for sustained Ca2+ influx through agonist-occupied wild-type channels. Thus, it is likely that the Pro373-Pro376 sequence of P2X2R represents a functional motif that is critical for the development of the slow desensitization profile observed in these channels. Consequently, deletion of this motif by alternative splicing provides an effective mechanism for generating a channel with controlled Ca2+ influx.

The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: Causes and consequences

A total of 101 different examples of point mutations, which lie in the vicinity of mRNA splice junctions, and which have been held to be responsible for a human genetic disease by altering the accuracy of efficiency of mRNA splicing, have been collated. These data comprise 62 mutations at 5' splice sites, 26 at 3' splice sites and 13 that result in the creation of novel splice sites. It is estimated that up to 15% of all point mutations causing human genetic disease result in an mRNA splicing defect. Of the 5' splice site mutations, 60% involved the invariant GT dinucleotide; mutations were found to be non-randomly distributed with an excess over expectation at positions +1 and +2, and apparent deficiencies at positions -1 and -2. Of the 3' splice site mutations, 87% involved the invariant AG dinucleotide; an excess of mutations over expectation was noted at position -2. This non-randomness of mutation reflects the evolutionary conservation apparent in splice site consensus sequences drawn up previously from primate genes, and is most probably attributable to detection bias resulting from the differing phenotypic severity of specific lesions. The spectrum of point mutations was also drastically skewed: purines were significantly over-represented as substituting nucleotides, perhaps because of steric hindrance (e.g. in U1 snRNA binding at 5' splice sites). Furthermore, splice sites affected by point mutations resulting in human genetic disease were markedly different from the splice site consensus sequences. When similarity was quantified by a 'consensus value', both extremely low and extremely high values were notably absent from the wild-type sequences of the mutated splice sites. Splice sites of intermediate similarity to the consensus sequence may thus be more prone to the deleterious effects of mutation. Regarding the phenotypic effects of mutations on mRNA splicing, exon skipping occurred more frequently than cryptic splice site usage. Evidence is presented that indicates that, at least for 5' splice site mutations, cryptic splice site usage is favoured under conditions where (1) a number of such sites are present in the immediate vicinity and (2) these sites exhibit sufficient homology to the splice site consensus sequence for them to be able to compete successfully with the mutated splice site. The novel concept of a "potential for cryptic splice site usage" value was introduced in order to quantify these characteristics, and to predict the relative proportion of exon skipping vs cryptic splice site utilization consequent to the introduction of a mutation at a normal splice site.

Mutation of 3' splice sites in two different class I genes results in different usage of cryptic splice sites.

To determine the pattern of alternative splicing at the 5' end of class I genes, the 3' splice sites bordering exon 2 of the H-2Dd and H-2Kd genes were mutated from AG to GG (H-2Dd) or CG (H-2Kd). The mutant genes were transfected into L cells, and RNA from clones expressing these Ag was used for analysis by RNase and S1 nuclease mapping techniques. The first intervening sequence of both class I genes contains several potential 3' splice acceptor sites. However, a clear preference for only one site was detected in each of the H-2Dd and H-2Kd mRNA. Examination of the endogenous H-Dd and H-2Kd class I transcripts in normal murine tissues and in tumors demonstrated that the alternatively spliced mRNAs were produced, but at a low frequency. Infection of transfected L cells or tumor lines with vesicular stomatitis virus altered the level of differentially spliced message in these cells.