|M.Sc Student||Yahav Yosefzon|
|Subject||Role of 3'UTR Motifs in mRNA Localization and Protein|
|Department||Department of Biology||Supervisor||Professor Arava Yoav|
|Full Thesis text|
Mature mRNA in eukaryotes has a tripartite structure consisting of 5'UTR, ORF and 3'UTR. While the ORF encodes the functional entity - the protein, the UTRs are known in their involvement in post-transcriptional regulation of gene expression. This regulation is achieved by sequence and structural elements which are located in the UTRs and serve as recognition sites for RNA-binding proteins (RBPs).
In this work we aimed to explore possible involvement of 3'UTR elements and RBPs that bind them, in post-transcriptional regulation. Our initial model transcript was the S.cerevisiae PMP1 gene, which encodes a membrane protein and its mRNA was found to associate with membranes. We recently showed that the 3'UTR of PMP1 mRNA is responsible for its membrane association. PMP1 3'UTR contain four sequence elements comprising of UG dinucleotide repeats, which were found to be important for its membrane association. The involvement of UG repeats in membrane association may be exerted through RBPs that bind them and target them to membranes.
Puf proteins are evolutionary conserved RBPs that bind 3'UTRs through a PUM domain. A previous study revealed that Puf2p binds mRNAs that encode membrane proteins, including PMP1. By affinity-purification we found that mutations in the UG repeats significantly decreased Puf2p binding. However, electro mobility shift assay (EMSA) with recombinant purified Puf2-PUM domain and fragments of the UG repeats, revealed that the interaction of Puf2p with the UG repeats is probably indirect. Through bioinformatics analysis we identified sequence element (UAAUNNNUAAU "UA motif") that is highly enriched in the 3'UTRs of Puf2p targets. EMSA revealed that Puf2p indeed binds this consensus motif. Importantly, Puf2p binds similar sequence derived from PMP1 3'UTR. This suggests that Puf2p interacts with PMP1 3'UTR through the UA motif. To explore Puf2p function in post-transcriptional regulation, we tested its effect on PMP1 membrane association by velocity sedimentation analysis and by isolation of membrane bound polysomes and we found that apparently Puf2p is not involved in PMP1 membrane association.
Our results suggest that PMP1 3'UTR contains two motifs: UG repeats which important for PMP1 membrane association and affect Puf2p binding, and a novel UA motif that serves as Puf2p binding site. The factors which are necessary for PMP1 membrane association and the functional role of Puf2p are yet to be explored. This may provide better understanding of the molecular interaction between the UG repeats and the UA motif and for the interplay between multiple, different elements in 3'UTRs.