Abstract
Transfer RNA (tRNA) precursors undergo endoribonucleolytic processing of their 5’ and 3’ ends. 5’ cleavage of the precursor transcript is performed by ribonuclease P (RNase P). While in most organisms RNase P is a ribonucleoprotein that harbors a catalytically active RNA component, human mitochondria and the chloroplasts (plastids) and mitochondria of seed plants possess protein-only RNase P enzymes (PRORPs). The plant organellar PRORP (PRORP1) has been characterized to some extent in vitro and by transient gene silencing, but the molecular, phenotypic and physiological consequences of its down-regulation in stable transgenic plants have not been assessed. Here we have addressed the function of the dually targeted organellar PRORP enzyme in vivo by generating stably transformed Arabidopsis plants in which expression of the PRORP1 gene was suppressed by RNA interference (RNAi). PRORP1 knock-down lines show defects in photosynthesis, while mitochondrial respiration is not appreciably affected. In both plastids and mitochondria, the effects of PRORP1 knock-down on the processing of individual tRNA species are highly variable. The drastic reduction in the levels of mature plastid tRNA-Phe(GAA) and tRNA-Arg(ACG) suggests that these two tRNA species limit plastid gene expression in the PRORP1 mutants and, hence, are causally responsible for the mutant phenotype.
Highlights
In all organisms, transfer RNAs are synthesized as precursor transcripts that undergo extensive post-transcriptional processing, before they can be aminoacylated and serve as amino acid donors in protein biosynthesis. Transfer RNA (tRNA) maturation involves processing of the 5’ and 3’ ends as well as extensive chemical modification of individual nucleosides [1,2,3,4]
Transformation of Arabidopsis thaliana plants with a hairpin-type RNA interference (RNAi) construct [24] targeted against PRORP1 yielded several independent transgenic lines, which were subsequently assayed for their phenotypes under standard greenhouse conditions
Our reverse genetic analysis of the dually targeted proteinaceous ribonuclease P (RNase P) revealed that the effects of knocked-down PRORP1 on the processing of individual tRNA species in chloroplasts and mitochondria are highly variable
Summary
Transfer RNAs (tRNAs) are synthesized as precursor transcripts that undergo extensive post-transcriptional processing, before they can be aminoacylated and serve as amino acid donors in protein biosynthesis. tRNA maturation involves processing of the 5’ and 3’ ends as well as extensive chemical modification of individual nucleosides [1,2,3,4]. Plant cells need tRNA-processing and tRNA-modifying enzymes in three distinct cellular compartments (nucleocytosolic compartment, mitochondria and plastids). Plant Organellar RNA Processing by RNase P involved in tRNA processing and tRNA modification have been identified for all three compartments Evidence for the existence of a different type of RNase P that lacks an RNA component was first provided for spinach chloroplasts [16]. Based on sequence similarity to MRPP3, RNA-free RNase P enzymes were identified from the model plant Arabidopsis thaliana [7, 8] and from the moss Physcomitrella patens [22]. PRORP1 can rescue an RNase Pdeficient Escherichia coli strain [7], providing further evidence for the protein being sufficient to faithfully perform tRNA 5’ maturation
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