|M.Sc Student||Nochi Ayelet|
|Subject||Identification of Genes whose Translation is Modulated by|
IRES Element in Saccharomyces cerevisiae
|Department||Department of Medicine||Supervisor||Professor Mordechai Choder|
Initiation of protein synthesis in eukaryotes requires recruitment of the small (40S) ribosomal subunit to the mRNA, by binding of eIF4E to m7GpppN (cap). However, some mRNAs contain internal ribosomal entry site (IRES), an RNA element that can direct the 40S ribosome to internal mRNA site(s).
In order to identify yeast IRESes, members of the lab constructed a bicstronic vector which contains a library of 5'-UTR of yeast genes in between the two cistrons, and found potential elements that function as IRESes. We focused on a subgroup of these potential IRESes present in the following genes: TDH1,2,3 ,GPM1, FBA1, TAF25 and JAC1, and decided to determine if these IRESes are recognized also in their natural mRNA context.
The genes were tagged with Myc9. Expression of the tagged genes detected the full length protein, and also a shorter product in the case of TDH1,2,3, FBA1 and GPM1. These N-terminally truncated proteins and their lengths were consistent with the presence of the IRESes within their ORFs.
We examined this possibility by creating different point mutations. We found that the mutations that disrupted the translation of the full length protein also disrupted the synthesis of the truncated ones.
We could find no conditions that permit differential synthesis of the truncated proteins and the full length ones.
It is possible that the IRESes found in the bicistronic context are not recognized in the natural mRNA context. Alternatively, these IRESes may promote translation of the full length ORF, or translation of an ORF which is not in frame with the major ORF.