|Ph.D Thesis||Department of Biology|
|Supervisor:||Assoc. Prof. Arava Yoav|
|Full Thesis text|
Eukaryotic mRNAs can be divided to three functional regions: the ORF that encodes for the polypeptide chain, the 5'UTR that mainly regulates the initiation stage of translation, and the 3'UTR that participates in several aspects of mRNA metabolism including translation and stability. An mRNA closed loop structure in which the cap, the PolyA tail and the stop codon region were shown to interact with each other through their associated proteins was proposed to explain how 3'UTR can mediate translation control. Yet, whether these interactions are stable in-vivo and within endogenous mRNAs, and whether additional intra-molecular interactions exist is unknown.
To explore this, we cleaved several yeast polysomal mRNAs at their stop codon region and separated the resulting fragments in sucrose gradient followed by Northern analysis. Sedimentation of 3'UTR fragments in heavy fractions might point to its association with heavy complexes.
Several endogenous mRNAs were tested, and a bi-modal sedimentation pattern was observed for the 3’UTR of all of them: some sediment as free of ribosomal subunits and some sediment in heavy fractions. The heavy 3'UTR appeared to sediment similarly to the ORF-containing fragment, yet the extent of co-sedimentation differed between mRNAs. Similar observations were obtained by an alternative assay, which was based on immunoprecipitation. Various treatments that are expected to interfere with the cap to polyA interactions had no effect on the co-sedimentation pattern. Moreover, the 3'UTR appeared to co-sediment with regions from all along the ORF. We found that the interaction depends on features from both the ORF and the 3'UTR. Taken together, these results indicate extensive associations between 3'UTRs and their ORFs, which vary between genes, and imply that in-vivo, polyribosomal mRNAs exist in a compact structure that contains multiple contacts between the 3'UTR and the ORF.
Analysis of the 3'UTR of PMA1 revealed that it has additional sedimentation position; it sediments in polysomal fractions that represent association with complexes the size of ribosomes, which are different from the sedimentation position of its ORF. Co-immunoprecipitation of ribosomes and their associated fragments revealed significant Co-Immunoprecipitation for the 3'UTR of PMA1 but not of control 3'UTR. This suggests association of ribosomes with PMA1 3'UTR.
Taken together, we suggest the existence of extensive intra-molecular association between translated and untranslated regions. These associations may be through translating ribosomes, or by RNA-RNA base-pairing and probably allow general and specific regulation by the 3'UTR. The nature of this regulation is yet to be determined.