Assembling an orthogonal translation compartment in S. cerevisiae

Spliced leader trans-splicing occurs in many eukaryotes, including nematodes such as Caenorhabditis elegans and Ascaris suum. It involves the transfer of a short RNA sequence called the spliced leader (SL), with a 5' terminal trimethyl guanosine cap, onto the 5' end of messenger RNAs (mRNAs). The spliced leader is donated by SL RNA and replaces the 5'untranslated region of the target pre-mRNA and provides the 5' end cap structure necessary for mRNA translation. I exploited this process in attempt to assemble an orthogonal translation system in Saccharomyces cerevisiae. SL trans-splicing is absent from fungi, and like most eukaryotes, yeast endogenous mRNAs are monomethyl guanosine capped. A key part of the orthogonal system is the ability to selectively cap specific RNAs with trimethylguanosine via spliced leader trans-splicing. These RNAs could then be recognised by nematode eIF4E proteins such as Ascaris eIF4E-3, that can bind trimethylguanosine capped mRNA, leading to "compartmentalised" translation. I expressed nematode SL RNAs and synthetic pre-mRNA targets in the yeast Saccharomyces cerevisiae and found that this is sufficient to confer trans-splicing activity, albeit at a low level. The analysis of trans-spliced transcripts revealed that there are preferred splice acceptor sites. The obtained results provide the first conformation of trans-splicing activity in yeast cells. Moreover, I employed the nematode translation initiation factor Ascaris eIF4E-3 as an exogenous translation initiation factor in S. cerevisiae. This investigation provides the first demonstration that Ascaris eIF4E-3 is functional in yeast translation. Finally, I used a luciferase reporter system to assay yeast trans-splicing at the protein level. In cells expressing AscariseIF4E-3 and SL RNA, the expression the reporter gene is enhanced by spliced leader trans-splicing.