טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentHalpert Michal
SubjectExploring the Exo-and Endonucleolytic Activities
of RNase J as a Key Player in Chloroplast RNA
Degradation
DepartmentDepartment of Biology
Supervisor Professor Schuster Gadi
Full Thesis textFull thesis text - English Version


Abstract

Chloroplast gene expression is regulated mainly by posttranscriptional processes which are important for functionally activating an mRNA, as well as for ultimately leading to its degradation. RNA processing and decay in the chloroplast is catalyzed by nucleus-encoded ribonucleases and includes 5’, 3’ and intercistronic maturation of the polycistronic transcripts. These is carried out by exoribonucleases which remove nucleotides starting at either the 5’ or 3’ end, and endoribonucleases, which cleave the RNA internally.

The rate-limiting step of RNA decay in chloroplasts is believed to be an initial endonucleolytic cleavage. This step generates products that are accessible to exonucleases by removing protective features like strong secondary structures and RNA binding proteins present at the RNA ends. RNA binding proteins like the pentatricopeptide repeat (PPR) proteins are predicted to define RNA ends in chloroplasts by blocking 5’ to 3’ and 3’ to 5’ exonucleases after the polycistronic transcripts are stochastically being cleaved. RNase J, a well-defined endonuclease and 5' to 3' exonuclease in B. subtilis, is hypothesized to have similar properties in Arabidopsis chloroplasts and therefore responsible for initiating these unspecific intercistronic cleavages as well as for 5' end maturation of the RNA.

The primary aim of this work was to characterize the mode of activity and properties of the Arabidopsis RNase J. We expressed and purified the recombinant protein and then performed activity assays with different labeled RNA substrates. The Arabidopsis RNase J was found, as predicted, to be active as both an endonuclease and a 5’ to 3’ exonuclease. The endonucleolytic activity was found to be more robust as compared to the B. subtilis RNase J and was shown to be directed by the binding of PPR10 protein to the RNA substrate. Both Arabidopsis RNase J endonucleolytic and 5’ to 3’ exonucleolytic activities were found to be performed by a single active site and showed sensitivity to the presence of guanosines in the RNA substrate. Based on the crystal structure of the B. subtilis RNase J the Arabidopsis RNase J is likely to harbor an RNA binding pocket, probably located near the active site, which serves as a sensor for the 5' end of the RNA. While further studies are required in order to understand the importance of the unique GT1 domain found in the plant proteins, the C terminal part of the protein was shown to have an important function in maintaining the oligomeric structure of the protein.