טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentAdawi Israa
SubjectEarly assembly of cytoplasmic mRNA decay factors to
mRNAs in the nucleus
DepartmentDepartment of Medicine
Supervisor Professor Mordechai Choder
Full Thesis textFull thesis text - English Version


Abstract

Gene Expression is traditionally divided into several stages, including mRNA synthesis and processing, export (in eukaryotes), translation and decay. Yet, gene expression can be viewed as a circular system in which all stages are mechanistically coupled and coordinated by master regulators. In my work, I investigated the possible coupling between these stages: translation, degradation and transcription .

Saccharomyces cerevisiae (baker’s yeast) is my research model. The fact that many cellular processes are conserved among different eukaryotic species-combined with the powerful genetic and molecular tools that are available- has made this yeast important experimental organism for a variety of basic problems in eukaryotic molecular biology.

In yeast, the transcription of RNA is done by RNA Polymerase II; which is composed of 12 subunits (Rpb1p-Rpb12p): A ten-subunit core and two-subunit complex comprising Rpb4p and Rpb7p (Rpb4/7). Rpb4p and Rpb7p normally associate with their cognate polymerase as a heterodimer, but it was found that Rpb4p and Rpb7p disassociate from the RNA polymerase together with the mRNA and they play roles in the translation and the degradation of the mRNA in the cytoplasm .

The major pathway of mRNA degradation in yeast initiates with deadenylation of the mRNA poly(A) tail whose full length in yeast is usually 60-80 bases. When the length of the poly(A) tail reaches 10-12 bases or less, the mRNA 5 cap [m(7)GpppN] is removed, followed by an exonucleolytic digestion of the unprotected RNA. Deadenylation is carried out by Pan2/Pan3  and the Ccr4/Caf1 poly(A) nuclease complexes, while decapping by Dcp1/Dcp2. Decapping is probably a point of no return in the degradation process, as it exposes the 5' end of the mRNA to the major 5'-to-3' exonuclease,Xrn1p, and facilitates its activity. Pat1p is considered to be the first decay factor recruited to mRNA, at a time when the mRNA is still associated with translation factors. It is still unclear how Pat1p is recruited to the mRNA and what controls this recruitment .

Rpb4/7p and Pat1p share similar properties: both are shuttling proteins; they shuttle between the cytoplasm and the nucleus, both bind mRNAs and both participate in the translation and the degradation of the mRNA. In addition, they interact with each other. While it is known that Rpb4/7p binds the mRNA in the nucleus during the transcription stage, it is still unknown how Pat1p is recruited to the mRNA. Based on these facts, the raised questions are: Does Pat1p like Rpb4/7p; bind the mRNA during the transcription stage in the nucleus? Is this possible interaction mediated by Rpb4/7p ?

Here I showed that Pat1p interacts with RNA Polymerase II and binds the mRNA in the nucleus. I found that blocking the import of Pat1p to the nucleus reduced the interaction between Pat1p and the mRNA. I showed also that Rpb4/7p mediates the interaction between Pat1p and the translated mRNA. I suggest that Pat1p is recruited to the mRNA co-transcriptionally by Rpb4/7p. Both Pat1p and the Rpb4/7p accompany the mRNA throughout its life cycle from the synthesis until the degradation, the free Pat1p and the free Rpb4/7p return back from the cytoplasm to the nucleus.