טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentLoya Adi
SubjectA Possible Role for the 3'UTR of PMP1 in Association with
Lipid Rafts
DepartmentDepartment of Biology
Supervisor Professor Yoav Arava
Full Thesis textFull thesis text - English Version


Abstract

            All mRNAs contain 3 functional domains: the 5'UTR, the ORF and the 3'UTR. In this work we aimed to explore possible involvement of a 3'UTR in translational control. A database was composed for the lengths of ORFs and UTRs of Saccharomyces cerevisiae and the PMP1 transcript was identified from it. PMP1 has a 123nts ORF, and a 700nts 3'UTR. It encodes a small membrane-protein that is a part of the plasma-membrane H+-ATPase, and is likely to be located to lipid-rafts. It appeared from polysomal analysis that PMP1 mRNA sediments as associated with 10 or more ribosomes - a surprisingly high number for such a short ORF. Further analyses indicated that the heavy sedimentation is related to translation.

To explore whether ribosomes are associated also with the 3'UTR, a Ribosome Density Mapping (RDM) experiment was performed, in which ribosomal association of different mRNA regions is determined. The results revealed that the ORF region sediments as associated with 2-4 ribosomes, while the 3'UTR sediment as associated with 10 ribosomes or more and suggested that the 3'UTR is associated with a heavy complex that drags the PMP1 ORF to the heavy end of the gradient.

We tested the sedimentation of various organelles by western analysis. All the organelle markers but the lipid-raft marker appeared in the light fractions, indicating that under our experimental conditions these organelles disintegrate. The lipid-raft marker Gas1p appeared to sediment in the heavy fractions, suggesting that lipid-rafts remain intact and sediment in these fractions. The sedimentation of PMP1 was also tested in a strain depleted of lipid-rafts and revealed a strong shift in its sedimentation, supporting the notion that its heavy sedimentation is due to association with lipid-rafts.

The sequence of the 3'UTR contains a short putative ORF (YCR024C-b). To test its role we generated a plasmid with the PMP1 transcript deleted of this region. Polysomal separation revealed that the deleted transcript sediments as associated with 2-4 ribosomes, thus confirming the RDM results. Moreover, it pinpoints the importance of YCR024C-b in the heavy sedimentation of the entire transcript. Western and IP analyses of Flag-Tagged YCR024C-b did not reveal any band at the expected size, suggesting that either the ORF is not translated or that degrades rapidly.

Taken together, our results suggest that the PMP1 transcript is associated with lipid-rafts during its translation. The 3'UTR is involved in this association, probably through YCR024C-b.