Hey Everyone,
I recently identified a mutation in a gene that is causing disease. There are 30 exons in this gene. Exon 29 is protein coding, except for the last 11 bp, which is where the 3' UTR starts. Basically, my mutation is the last base pair in exon 29, which is a G to an A (c.*11G>A) so it's really early on in the 3'UTR. RT-PCR show's that this activates a cryptic donor splice site, that is 153bp downstream of the natural splice site. The last exon (exon 30) is entirely 3' UTR.
I attached a schematic to illustrate what I'm seeing. Just wondering how I can determine the effect of this 153 bp on the 3'UTR of this gene?
Any advice would be greatly appreciated,
Justin
Hi Justin,
do you have RNA for this sample? best would be to pcr the fragment out to confirm the alternative splicing.
Concerning the effect;
did you consider checking whether there are any potential miRNA binding sites in the section that is inserted into the UTR?
This could coincide with transcriptional downregulation; again, if you have RNA, and some other samples that don't have this mutation, you could see whether there is any obvious change in mRNA levels in this sample.
Arnoud
Thanks, for you're reply. I did RT-PCR, TA-cloning and sequenced my alternatively spliced fragment. I experimentally confirmed that this does in fact activate a cryptic donor splice site 153bp downstream of the natural splice site. What I'm wondering now is how this is affecting the 3' UTR.
perhaps you can explore whether there are any miRNA binding sites created using this tool perhaps?
http://www.mirdb.org/custom.html
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