טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentShlomit Yehudai-Resheff
SubjectThe Molecular Mechanism of mRNA Polyadenylation and
Degradation in the Chloroplast
DepartmentDepartment of Biology
Supervisor Full Professor Schuster Gadi


Abstract

While trying to purify the chloroplast Poly(A) Polymerase, the polyadenylation activity was co purified with PNPase and indeed could not be separated from it. Purified PNPase was able to polyadenylate RNA molecules. The activity of PNPase was directed to RNA degradation or polymerization by manipulating physiologically relevant concentrations of Pi and ADP. Pi enhanced degradation, whereas ADP inhibited degradation and enhanced polymerization. These results suggest that polyadenylation and degradation of RNA in chloroplasts are performed by one enzyme, PNPase.

The second goal of this work was to elucidate the molecular mechanism of polyadenylation and degradation in the chloroplast.

An analysis of the amino acid sequence of PNPase shows that the protein is composed of two core domains related to RNase PH, two RNA-binding domains (KH and S1) and an alpha-helical domain. In order to define the molecular mechanism controlling the two opposite activities of this protein in the chloroplast, the ribonuclease, polymerase and RNA-binding properties of each domain were analyzed. The first core domain, which was predicted to be inactive in the bacterial enzyme, was active exclusively in the RNA degradation and not in the polymerization. Surprisingly, the second core domain was found to be active in degradation of only polyadenylated RNA, non-polyadenylated molecules were degraded only if first polymerized. The high affinity poly(A) binding site was localized to the S1 domain.

Phylogenetic and sequence alignment analyses of the two core domains shows that the two separate domains divided very early in development, resulting in the evolution of the bacterial and organelle PNPase and the exosome protein in Eukaryotes and some archea. Therefore, the combination of these domains in the PNPase and the exosomes can provide insight into an evolutionary conserved RNA degradation machine.