If a mutation is in the splice site (donor), how can we bioinformatically predict its function in silico? For example if the site is broken or not, is there any exon skipping, if no then how to find the next splice site?
Many mutations of the splice site have been published, for
example, IVS1þ1G>A, IVS3þ5_6GA>AC, and IVS5-1G>C
in EVC, and IVS4þ2T>C in EVC2. Tompson et al. [2007]
also reported mutation in EVC (IVS13þ5G>T) produced three alternative splicing in affected individuals.
How can we defend this mutation without expression studies, which is very difficult in Pakistan.
I am sorry to say that without experimental assay, you cannot tell anything ! You can reveal/hide a donor/acceptor splice site, reveal/hide a enhancer/silencer binding site, and there is competition between splicing factors as well...
Not doable without experiment, except if you do not care to conclude whatever result...
Expression assay is your final prove for the mutation at splice site. Otherwise, you can just predict that a protein is going to be truncated.
you can use these links and attached fils. I hope it can help you
http://www.ensembl.org/index.html
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0018599
http://www.nature.com/gim/journal/v16/n7/full/gim2013176a.html
I think you can first to evaluate it if it's a real mutation or a SNP, it's better population based (some population has relative higher mutations in certain spots). If it's confirmed a mutation, then you can calculate the influence of it on splice site strength, that will be one major force to cause alternative splicing. It can be further go to see if it's influence the annealing with U1, U2, U4,5,6 annealing with the site. Then, search it to see if this mutation gain or loss of ISS, ISE. Berge lab has some tools to identify ISS/ISE. Furthermore, if you're interested,check if this single mutation will the editing of RNA, etc. If certain intron has other regulation functions, then the story will be more complicated.
Splice acceptor and donor consensus sequences are highly conserved, and mutations in the invariant 'ag' acceptor or 'gt' donor sites are unlikely to be common SNVs. In vertebrates, the consensus sequences necessary for intron splicing during the processing of pre-mRNA are cag|G (3' or "acceptor" splice site) and MAG|gtragt (5' or ”donor" splice site)(intron-exon boundaries are indicated by '|'). Mutation of a donor splice site usually results in the following: 1. exon skipping (most common), which can lead to frameshift and a premature termination codon, leading nonsense mediated decay if it occurs before the last exon, or an in-frame deletion, which may lead to protein truncation; or 2. It may activate cryptic splice sites. You can validate your bioinformatics prediction by sequencing cDNA. Also, MutationTaster (http://www.mutationtaster.org/) incorporates a splice site prediction program (NNSplice), but, in my experience, it is not very conservative, so I would not rely too heavily on that prediction.
I am sorry to say that without experimental assay, you cannot tell anything ! You can reveal/hide a donor/acceptor splice site, reveal/hide a enhancer/silencer binding site, and there is competition between splicing factors as well...
Not doable without experiment, except if you do not care to conclude whatever result...
Hi,
I agree with Florent. Recent publications conclude that one may use the in silico predictions as a first selection of mutations that possibly affect splicing. But they are still inaccurate even in determinig whether a variant will affect splicing or not - and they are not capable of predicting the splicing outcome (exon skipping, cryptic splice site utilization, intron retention). So the experimental validation is necessary.
If you want to try anyway, I prefer Sroogle engine (covering several programs for both the splice sites and the splicing regulatory elements predictions). In addition, there is a nice publication evaluating the outcome of splice site predictors: Houdayer et al., 2012. You might be interested.
All the best,
Lucie
http://sroogle.tau.ac.il/
http://www.ncbi.nlm.nih.gov/pubmed/22505045
You may use Human Splice Finder http://www.umd.be/HSF3/
It will not only check for your variant itself but also explore a nearby target that has a potential splicing effect once the original splicing site is abolished.
Best of luck
Musharraf
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