Why CRYP-SKIP?

Removal of introns from precursor messenger RNA (pre-mRNA) by splicing is a critical step in eukaryotic gene expression. This process is mediated by a large RNA-protein complex termed spliceosome, which consists of several small nuclear ribonucleoproteins (snRNPs) and a large number of non-snRNP splicing factors. The spliceosome is assembled on the pre-mRNA in a step-wise process. Analysis of mutations that were identified in genes deficient in patients with hereditary disorders revealed that up to 50% may alter pre-mRNA splicing. Although spliceosome assembly, intron lariat formation, or exon ligation may be impaired by DNA alterations anywhere in introns and exons, most human splicing mutations have been found in splice-site consensus sequences, particularly in GT and AG dinucleotides that define 5' and 3' intron ends, respectively (1).

The vast majority of splice-site mutations leads to

exon skipping1

exon skipping

or

aberrant splice-site activation,

aberrant splice-site activation



1Legend: introns are shown as lines, exons are shown as cylinders. Mutations are schematically denoted by stars. Wild-type and mutated splicing pattern is shown above and below the primary transcript, respectively.

both in the germline and in somatic cells. The two outcomes may have profoundly different phenotypic consequences that are often impossible to predict, because mutation screening of most disease genes does not routinely involve analysis of RNA samples from affected individuals and their family members.

Activation of aberrant splice-sites has been shown to reflect the availability of splice-site consensus sequences and the density of several types of auxiliary splicing sequences, particularly in the sequence between authentic and aberrant splice sites (2). The auxiliary signals, which are thought to act through altering secondary structure and/or binding sites of splicing regulatory proteins are often necessary for accurate splice-site selection (2-8), but their relative importance is poorly understood.

What CRYP-SKIP does?

CRYP-SKIP users submit sequences of mutated alleles containing one exon (strictly in UPPER case) and at least 4 bp of flanking intronic sequence (in lower case). The server analyzes significant predictor variables of cryptic splice-site activation (CR-E) and exon skipping (EXSK) using a regularly updated logistic regression model (2). This model employs a large number of human sequences that were shown to sustain disease-causing mutations leading to the two aberrant splicing outcomes (2). These sequences are available from public databases of aberrant 3' and 5' splice sites (9,10). CRYP-SKIP determines numerical values for predictor variables that were used in the regression model and calculates the overall probability of cryptic splice-site activation in exon for each submitted sequence.

The server output is a single page containing exonic sequence with intronic flanks. The sequence contains predicted cryptic 5' splice sites (red marks) and 3' splice sites (blue marks); their size reflects their relative intrinsic strength. The sum of 5' splice sites is equal to the PCR-E value. The size of 3' splice-site marks is at present equal to their NNSplice score; their PCR-E values are under development). Canonical or natural splice sites of the exon are not marked; they are determined by the submitter as boundaries between upper and lower case sequences. Exonic sequence is highlighted in light blue. The page also contains a summary table with a list of the most important predictor variables, their calculated values and the overall probability of cryptic splice site activation as opposed to exon skipping, termed PCR-E. PCR-E (shown graphically as a red pointer on a dial scale on the right) takes values between zero and one, with higher values speaking in favour of cryptic splice-site activation and lower values in favour of exon skipping. Densities of putative exonic splicing silencers or PESSs (4,5), NN splice-site scores (11), splicing silencers, as determined by a fluorescence activated screen (7), and scores for the SR protein SF2/ASF (12) together with exon length have been incorporated in the updated regression model. Comparisons of each predictor variable between sequences that underwent splice-site activation or exon skipping and average human introns or exons were published previously (2 and references therein).

How to cite CRYP-SKIP?

Divina, P., Kvitkovicova, A., Buratti, E., Vorechovsky, I. (2009) Ab initio prediction of mutation-induced cryptic splice-site activation and exon skipping. Eur J Hum Genet., 17, 759-765. [Pubmed] [Fulltext]

References

  1. Cooper, D.N. and Krawczak, M. (1993) Human Gene Mutation. BIOS Scientific Publishers, Oxford.
  2. Kralovicova, J. and Vorechovsky, I. (2007) Global control of aberrant splice site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition. Nucleic Acids Res., 35, 6399-6413. [PubMed] [Fulltext]
  3. Cartegni, L., Chew, S.L. and Krainer, A.R. (2002) Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat. Rev. Genet., 3, 285-298. [PubMed]
  4. Zhang, X.H. and Chasin, L.A. (2004) Computational definition of sequence motifs governing constitutive exon splicing. Genes Dev., 18, 1241-1250. [PubMed] [Fulltext]
  5. Zhang, X.H., Kangsamaksin, T., Chao, M.S., Banerjee, J.K. and Chasin, L.A. (2005) Exon inclusion is dependent on predictable exonic splicing enhancers. Mol. Cell. Biol., 25, 7323-7332. [PubMed] [Fulltext]
  6. Fairbrother, W.G., Yeh, R.F., Sharp, P.A. and Burge, C.B. (2002) Predictive identification of exonic splicing enhancers in human genes. Science, 297, 1007-1013. [PubMed]
  7. Wang, Z., Rolish, M.E., Yeo, G., Tung, V., Mawson, M. and Burge, C.B. (2004) Systematic identification and analysis of exonic splicing silencers. Cell, 119, 831-845. [PubMed]
  8. Wang, Z., Xiao, X., Van Nostrand, E. and Burge, C.B. (2006) General and specific functions of exonic splicing silencers in splicing control. Mol. Cell, 23, 61-70. [PubMed]
  9. Vorechovsky, I. (2006) Aberrant 3' splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization. Nucleic Acids Res., 34, 4630-4641. [PubMed] [Fulltext]
  10. Buratti, E., Chivers, M.C., Kralovicova, J., Romano, M., Baralle, M., Krainer, A.R. and Vorechovsky, I. (2007) Aberrant 5' splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization. Nucleic Acids Res., 35, 4250-4263. [PubMed] [Fulltext]
  11. Reese, M.G., Eeckman, F.H., Kulp, D. and Haussler, D. (1997) Improved splice site detection in Genie. J. Comput. Biol., 4, 311-323. [PubMed]
  12. Smith, P.J., Zhang, C., Wang, J., Chew, S.L., Zhang, M.Q. and Krainer, A.R. (2006) An increased specificity score matrix for the prediction of SF2/ASF-specific exonic splicing enhancers. Hum. Mol. Genet., 15, 2490-2508. [PubMed] [Fulltext]

Terms of use

CRYP-SKIP may be used by non-profit organisations and by others for non-commercial purposes without further express permission. Use for any commercial purpose will require written permission to use the database and accompanying scripts.

No part of the web site http://cryp-skip.img.cas.cz/ (the 'Site') and all data therein may be used for commercial approaches without the written consent.

Commercial users of CRYP-SKIP should contact:

Dr I Vorechovsky

School of Medicine

Southampton University Hospital

MP808

Tremona Road

Southampton SO16 6YD

United Kingdom


Tel: +44 2380 796425

Fax: +44 2380 794264

E-mail: igvo@soton.ac.uk

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