|M.Sc Student||Vaisberg Dmitri|
|Subject||The Impact of the Promoter and Sfp1 on the mRNA Fate in the|
Cytoplasm in Saccharomyces cervisiae
|Department||Department of Medicine||Supervisor||PROF. Mordechai Choder|
|Full Thesis text - in Hebrew|
Our group previously discovered that the type and sequence of the promoter's UAS can regulate mRNA degradation in the cytoplasm. We used a system which included two constructs that are identical in their ORF and TATA region and produce an identical mRNA, but different in their UAS region. While one contains the ACT1 UAS and was referred to as "Construct A", the other contains the RPL30 UAS and was referred to as "Construct B".
In this work I was interested in finding whether the promoter is able to affect the entry of the mRNA into p-bodies (PB), cytoplasmatic granules in which mRNA degradation and processing take place. I also wanted to examine whether Sfp1, a specific transcription factor for PBFs (protein biosynthesis factors), is involved in the ability of the promoter to couple mRNA degradation and entry into PB with transcription.
In order to achieve these goals, the system of constructs A and B was used together with the MS2 mRNA live imaging system. I used NaN3 as stress inducer for PB formation and shot the cells under fluorescent light. The results of the research revealed that the promoter affects mRNA entry into the PB, and that promoter B causes more mRNA to enter the PB than promoter A.
The effect of Sfp1 was examined by a very similar protocol, using Sfp1 labeled with GFP. The results of these experiments indicated that mRNA B is much more colocalized with Sfp1 than mRNA A, and again this is determined by the promoter.
When the degradation of both constructs was examined using northern-blot, it was discovered that mRNA B underwent degradation at a faster rate in the absence of Sfp1p, in contrary to construct A. These results indicate that the Sfp1 is able to affect the mRNA degradation rate depending on the promoter which the mRNA is transcripted from. It was also viewed that incubation with NaN3 causes a severe mRNA degradation inhibition of most of the cellular mRNAs.
All of the results obtained lead to a development of a model in which the promoter couples different gene expression stages to the transcription; one of them is mRNA entry to the PB. The Sfp1 is also affected by the promoter and acts as gene expression mediator only for genes with PBF promoter. The work also found a possible new interaction between the PB accumulation and the degradation inhibition under stress conditions.