טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentSamra Nitzan
SubjectDeciphering the Translation Independent Localization
Mechanism of mRNAs to the ER Membrane
DepartmentDepartment of Biology
Supervisor Professor Yoav Arava
Full Thesis textFull thesis text - English Version


Abstract

In eukaryotic cells proteins are sorted to each organelle to maintain their proper function. Proteins destined for secretion or the plasma membrane, are targeted to the endoplasmic reticulum (ER) by either of two distinct mechanisms: co and post translation localization. In post-translation pathway, the protein is targeted after its complete synthesis, with the help of many chaperones. In the co-translational pathway the targeting occurs while the protein is translated. An N-terminal polypeptide is recognized by the signal recognition particle (SRP), and the mRNA-ribosome-polypeptide complex is transported to the vicinity of the ER and recognized by SRP receptor. Recently, growing evidence indicates that many mRNAs encoding ER-targeted proteins associate with the ER membrane in translation independent manner. This suggests a novel mode of targeting.

In this study, our goal was to characterize the translation independent localization mechanism by finding proteins that are involved in this process. Our model system was the Saccharomyces cerevisiae plasma membrane protein 1 (PMP1) gene, which encodes small subunit of the plasma membrane H-ATPase. Previous studies in our lab showed that PMP1 mRNA is localized to the ER in translation independent manner through elements in its 3' untranslated region (UTR). We hypothesized that RBPs mediate this localization through binding to the elements in PMP1 3' UTR. To identify such proteins we used the RNA-binding protein purification and identification (RaPID) method that allows isolation of a specific RNA with its associated proteins.

Novel proteins were identified as associated with PMP1 mRNA; many of them are RNA binding proteins (RBPs). In order to test which candidate proteins influence PMP1 mRNA localization, its sedimentation in sucrose gradient was probed in deletion strain of these proteins. This analysis did not reveal a change in the ER localization of PMP1 mRNA in any of the tested proteins. 

One of the identified proteins, the translation elongation factor YEF3, was further analyzed. Co-immunoprecipitation (co-IP) with TAP-tagged Yef3p confirmed its association with PMP1 mRNA. Furthermore, deletion of the 3' UTR region reduced Yef3p association. This suggests a possible role for Yef3p in PMP1 mRNA localization.

In summary, our study found new proteins associated with PMP1 mRNA and tested their role in its localization to the ER. Further tests are needed to determine the post-transcriptional role of these proteins. Furthermore, the regulatory role of Yef3 protein and its interaction with PMP1 3' UTR are yet to be determined and should be further explored.