Effect of premature transcription termination on gene transcript/protein

Question

[Nicolas Rosewick]

Hi,

I did some mutation screening in order to find some mutation that introduce novel polyA sites within gene introns. In summary I've a table like that

chr    position   orientation
chr1     155656             +
chr2     454545             -
chr10   1212121             +

Where "orientation" defined the orientation of the polyA ("+" means that only sense transcript may stops at this polyA).

Question : How can I test the effects of these novel polyA on the gene harboring this mutation (in term of trucated transcript/protein and protein domains). And how can I check if this mutation / and truncated protein/transcript is present in some cancer types ?

Thanks

[WouterDeCoster]

I assume you mean wet lab validation?

[Devon Ryan]

You might annotate the distance to the nearest upstream AAUAAA, since that'll signal transcriptional termination more than a a polyA stretch. Are you able to do GROseq or RiboSeq or something like that? Those tend to make it a bit easier to define transcript bounds, since you get pausing there (I have no clue if the library prep is possible in this sort of situation).

[Nicolas Rosewick]

I did some modified RACE to catch poly-A tailed transcript, and then HTS on a miSeq.

[Devon Ryan]

That's a good idea. I imagine you weren't lucky enough to be able to sequence through the polyA on the 3' end...

Anyway, I wonder if Kallisto or Salmon might help here. You could create new entries with the novel termination sites and see if those have much higher TPMs in any of the appropriate samples compared to the inappropriate samples. I'm not sure how exact the quantification will end up being, but that's worth a shot.

[Nicolas Rosewick]

Thanks Devon. Good idea but I don't think my RACE experiments is quantitative in term of gene expression. What I was thinking is to extract the protein domains informations for each gene (available in biomart ENSEMBL) and to check across all genes involved in these premature transcriptional termination if there is an enrichment for specific protein domains that are truncated and removed from the truncated protein. Any other ideas are welcome :)