alternative splicing. - (Sep/12/2008 )
I am working in a project that concern a gene that was proved to be associated with an autoimmune disease, last year, we discovered that the RNA of this gene has two alternatively spliced isoforms, the full-length and An isoform lacking exon two (delta 2 )
The delta 2 isoform is constitutively expressed in B cells as for the full- length, but they are expressed in different levels. In our model we think that delta 2 is a rescue isoform that has a role in minimizing the B-cells activation, so more like reducing the probability to produce autoantibodies.
My main goal now is to understand how the delta two isoform appears, and how the splicing machinery produces this isoform,
My question is which approach should i take to have more understanding for this case?
In the last months i have constructed minigenes that contains the first three exons, with different mutations in the branch site and in some noncoding SNPs and each one of the exons is surrounded by 500 bp upstream and down stream and this 5oo bp represents a part of the intronic sequences for these exons, then, i transfected this minigenes to HEK293 cells.
I got a relatively good results, but not so charming, so i was thinking about checking what proteins in the splicosome complex is missing or participating in enhancing or silincing the exon skipping in this Transcript.
So i need some tips about what can i do and how should i complete.
do any body know about a softwase or website that can help in finding the splicinf factors that can bind to specific sequences? like those for the transcription factors.
Please, any suggestions