|Ph.D Student||Schein Aleks|
|Subject||Analyzing Key Players in the Chloroplast RNA Degradation|
|Department||Department of Biology||Supervisor||Professor Gadi Schuster|
|Full Thesis text|
Chloroplast gene expression is regulated at the transcriptional, post-transcriptional and translational as well as post-translational levels. Since the rate of individual transcript's accumulation is dependent on the turnover rate and RNA degradation in the chloroplast is recognized as a primary mechanism controlling organelle's gene expression, unraveling the degradation mechanism is of great importance. The chloroplast RNA degradation mechanism is believed to be similar to the prokaryotic one. In E. coli, RNA degradation is triggered by the removal of the 5’ end phosphates followed by endonucleolytic cleavage by RNase E, an essential endoribonuclease, prominently involved in the bacterial RNA metabolism. Plant homologs of bacterial RNase E, encoded in the nuclear genome, have been proposed to function in the chloroplasts in a similar way. In this work we show that RNase E of Arabidopsis is located in the soluble fraction of the chloroplast as a high molecular weight complex. In order to characterize its endonucleolytic activity, Arabidopsis RNase E was expressed in bacteria and analyzed. Similar to its E. coli counterpart, the endonucleolytic activity of the Arabidopsis enzyme depends on the number of phosphates at the 5’ end, is inhibited by structured RNA, and preferentially cleaves A/U-rich sequences. The enzyme forms an oligomeric complex of ~680 kDa. The chloroplast localization and the similarity to the E. coli enzyme suggest that plant RNase E participates in the initial endonucleolytic cleavage of the polyadenylation-stimulated RNA degradation process in the chloroplast, perhaps in collaboration with the two other chloroplast endonucleases, RNase J and CSP41.
On the next level of RNA degradation in E. coli, RNA fragments, generated by RNase E cleavage, are polyadenylated by bacterial-type poly(A) polymerase (PAPI), a member of ribo-nucleotidyltransferase (rNTr) family; and polynucleotide phosphorylase (PNPase). Homologs of PAPI were also found in plants, but none was ever proved to have a role in chloroplast RNA degradation mechanism. This work presents biochemical analysis of several putative chloroplast rNTrs of Arabidopsis. We show that rNTr NP_174130, encoded by At1g28090 genetic locus, possesses weak poly(A) polymerase activity in vitro. Another putative chloroplast rNTr proved to be a tRNA CCA transfer enzyme.
Together, in this work the first analysis of an eukaryotic chloroplast endoribonuclease of the RNase E family and the initial characterization of a putative poly(A) polymerase located in the chloroplast were undertaken, showing that the polyadenylation-assisted RNA degradation is remarkably conserved in chloroplasts and E. coli, despite about 1.5 billion years of evolutionary separation.