Polynucleotide phosphorylase functions as both an exonuclease and a poly(A) polymerase in spinach chloroplasts
S Yehudai-Resheff1, M Hirsh, G Schuster
1Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Abstract:
The molecular mechanism of mRNA degradation in the chloroplast consists of sequential events including endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products, and exonucleolytic degradation by polynucleotide phosphorylase (PNPase). In Escherichia coli, polyadenylation is performed mainly by poly(A)-polymerase (PAP) I or by PNPase in its absence. While trying to purify the chloroplast PAP by following in vitro polyadenylation activity, it was found to copurify with PNPase and indeed could not be separated from it. Purified PNPase was able to polyadenylate RNA molecules with an activity similar to that of lysed chloroplasts. Both activities use ADP much more effectively than ATP and are inhibited by stem-loop structures. The activity of PNPase was directed to RNA degradation or polymerization by manipulating physiologically relevant concentrations of P(i) and ADP. As expected of a phosphorylase, P(i) enhanced degradation, whereas ADP inhibited degradation and enhanced polymerization. In addition, searching the complete Arabidopsis genome revealed several putative PAPs, none of which were preceded by a typical chloroplast transit peptide. These results suggest that there is no enzyme similar to E. coli PAP I in spinach chloroplasts and that polyadenylation and exonucleolytic degradation of RNA in spinach chloroplasts are performed by one enzyme, PNPase.
Insights
Spinach chloroplasts use polynucleotide phosphorylase (PNPase) for both RNA degradation and polyadenylation, unlike E. coli. This single enzyme, PNPase, manages these crucial mRNA processing steps.
Area of Science:
- Chloroplast molecular biology
- RNA metabolism
- Enzymology
Background:
- mRNA degradation in chloroplasts involves endonucleolytic cleavage, polyadenylation, and exonucleolytic degradation by polynucleotide phosphorylase (PNPase).
- In Escherichia coli, polyadenylation is primarily mediated by poly(A)-polymerase (PAP) I or PNPase.
- The presence and function of PAP in chloroplasts remain incompletely understood.
Purpose of the Study:
- To investigate the enzyme responsible for polyadenylation in spinach chloroplasts.
- To elucidate the molecular mechanism of mRNA processing and degradation in chloroplasts.
- To determine if a distinct PAP enzyme exists in chloroplasts or if PNPase performs this function.
Main Methods:
- Attempted purification of chloroplast poly(A)-polymerase (PAP) based on in vitro polyadenylation activity.
- Co-purification analysis of polyadenylation activity with polynucleotide phosphorylase (PNPase).
- Biochemical assays to characterize the enzymatic activities of purified PNPase, including RNA polymerization and degradation under varying substrate concentrations (ADP, P(i)).
- Bioinformatic search of the Arabidopsis genome for putative PAPs with chloroplast transit peptides.
Main Results:
- Chloroplast PAP activity co-purified with PNPase and could not be separated.
- Purified PNPase exhibited significant in vitro RNA polyadenylation activity, comparable to lysed chloroplasts.
- PNPase activity was modulated by physiological concentrations of inorganic phosphate (P(i)) and ADP, directing it towards degradation or polymerization.
- No putative PAPs with typical chloroplast transit peptides were identified in the Arabidopsis genome.
Conclusions:
- Spinach chloroplasts likely lack an enzyme analogous to E. coli PAP I.
- Polynucleotide phosphorylase (PNPase) in spinach chloroplasts is responsible for both polyadenylation and exonucleolytic degradation of RNA.
- A single enzyme, PNPase, performs multiple key roles in chloroplast mRNA metabolism.
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