3' To 5' Exoribonucleases In Arabidopsis Thaliana Plastids
Chloroplast genes and metabolic activities are regulated by the products of nuclear genes, acting mostly at the post-transcriptional level. Polynucleotide phosphorylase (PNP) and Ribonuclease R (RNR1) are the two known nucleus-encoded exoribonucleases and are necessary for the correct maturation and degradation of RNAs. PNP catalyzes both the processive 3' to 5' phosphorolysis of chloroplast RNA and its processive polymerization depending on the ratio between inorganic phosphate (Pi) and nucleotide diphosphates (NDPs). RNR1 is a hydrolytically processive 3' to 5' exoribonuclease that releases 5' monophosphate nucleotides. This study used both null mutants and single amino acid substitutions in the two core catalytic domains of PNP to investigate its role in many aspects of chloroplast RNA metabolism. The phenotypic characterization of null mutants (pnp1-1 to 1-3) showed a chlorotic phenotype in young leaves that became less severe as leaves matured, and molecular analysis demonstrated the involvement of PNP in the 3' maturation, stabilization and/or degradation of many chloroplast RNAs, as well as its importance in the excised intron lariat degradation pathway. Two mutations in the first core domain demonstrated a role for this region in PNP activity, however the residual activity of the PNP mutants permitted the construction of otherwise lethal pnp/rnr double mutants. The resulting rnr1 mutant plants with reduced PNP activity are chlorotic and display a global reduction in RNA abundance. Such a counterintuitive outcome following the loss of RNA degradation activity suggests a major importance of RNA maturation as a determinant of RNA stability. The detailed analysis of the double mutant transcriptome revealed that RNR1 completes the maturation of mRNAs 3' termini created by PNPase in a two-step maturation process. In contrast to the double mutant, the rnr1 single mutant, known to have a substantial decline in rRNA levels, over-accumulated most of the mRNA species examined when compared to the wild-type. Combined with the reduced number of ribosomes, it was not unexpected to find most of the excess mRNAs species present in non-polysomal fractions. Half-life measurements demonstrated a substantial increase in the stability of most mRNA species tested, supporting the hypothesis that RNR1 plays important roles in the maintenance of both chloroplast rRNA and mRNA homeostasis. Because of the dependence of PNPase activity on the Pi:NDP ratio in vitro, we investigated a potential link between PNP and phosphate (P) metabolism of the plant. We found that P-deprived pnp mutants develop aborted clusters of lateral roots while a global analysis of metabolites and transcripts supported the hypothesis that the activity of PNP is involved in plant acclimation to P availability. The most recent results, involving a double mutant lacking both PNP and the endonuclease RNase E, indicate that not only exonucleolytic processing but also endonucleolytic processing is involved in stabilizing chloroplast RNAs. Taken together, these studies deepen our knowledge about the role of RNases in the plastid RNA processing and decay pathways. Indeed, such double mutants reveal the importance of RNA maturation, perhaps as a quality control mechanism.
Chloroplast; rna; Exonuclease
Pleiss, Jeffrey A.; Hanson, Maureen R
Ph.D. of Plant Biology
Doctor of Philosophy
dissertation or thesis