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Free Content Mutational analysis of Arabidopsis chloroplast polynucleotide phosphorylase reveals roles for both RNase PH core domains in polyadenylation, RNA 3′‐end maturation and intron degradation

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Summary

Polynucleotide phosphorylase (PNPase) catalyzes RNA polymerization and 3′→5′ phosphorolysis in vitro, but its roles in plant organelles are poorly understood. Here, we have used in vivo and in vitro mutagenesis to study Arabidopsis chloroplast PNPase (cpPNPase). In mutants lacking cpPNPase activity, unusual RNA patterns were broadly observed, implicating cpPNPase in rRNA and mRNA 3′‐end maturation, and RNA degradation. Intron‐containing fragments also accumulated in mutants, and cpPNPase appears to be required for a degradation step following endonucleolytic cleavage of the excised lariat. Analysis of poly(A) tails, which destabilize chloroplast RNAs, indicated that PNPase and a poly(A) polymerase share the polymerization role in wild‐type plants. We also studied two lines carrying mutations in the first PNPase core domain, which does not harbor the catalytic site. These mutants had gene‐dependent and intermediate RNA phenotypes, suggesting that reduced enzyme activity differentially affects chloroplast transcripts. The interpretations of in vivo results were confirmed by in vitro analysis of recombinant enzymes, and showed that the first core domain affects overall catalytic activity. In summary, cpPNPase has a major role in maturing mRNA and rRNA 3′‐ends, but also participates in RNA degradation through exonucleolytic digestion and polyadenylation. These functions depend absolutely on the catalytic site within the second duplicated RNase PH domain, and appear to be modulated by the first RNase PH domain.
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Document Type: Research Article

Affiliations: 1: Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, NY 14853, USA 2: Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel

Publication date: August 1, 2011

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