I was wondering if anybody has good advice how to analyse the possible presence of an IRES element in a certain mRNA. I recently ran into a novel starting Exon which does not contain any coding information but has a size of appr. 900bp and is alternatively spliced instead of the Exon usually found in the databases. The coding information for the protein isn't altered by this alternative Exon anyhow.
I thought about using online tools for analysis of the sequence of my mRNA of interest, but could not find any suitable tool yet. Alternatively I would also consider experimental approaches via inhibition of 5'.Cap-dependent translation to investigate if a certain mRNA exhibits an IRES element for translation initiation, but I am not aware of any suitable antibiotics that function on CAP-dependent initiation of translation.
Try transcribingand translating the mRNA in a rabbit reticulocyte lysate, with a reporter (Like firefly luciferase) at the end of the mRNA. You could try inserting the reporter at different positions in the mRNA, working inwards from the end. You could assay for the firefly luciferase to know if you have an IRES in your mRNA. Based on where you get your IRES, you could further define the positions.
Initially, you could try analysing your RNA molecule in silico, to see if there are "IRES-like" structures in your mRNA
I'd look into the the hypoxia expression research. To the best of my knowledge the only mammalian IRES elements discovered to date have been in those genes that remained expressed during hypoxic stress. It seems that these have evolved to support survival during such a high stress state as low oxygen. These IRES elements have likely been sequenced and so that might give you an indication as to whether your EXON has any sequence similarity to those. Also, the means of discovering these will likely be described in the reports.
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