Abstract
RNase P is a ubiquitous endonuclease that removes the 5′ leader sequence from pre-tRNAs in all organisms. In Arabidopsis thaliana, RNA-free proteinaceous RNase Ps (PRORPs) seem to be enzyme(s) for pre-tRNA 5′-end processing in organelles and the nucleus and are thought to have replaced the ribonucleoprotein RNase P variant. However, the evolution and function of plant PRORPs are not fully understood. Here, we identified and characterized three PRORP-like proteins, PpPPR_63, 67, and 104, in the basal land plant, the moss Physcomitrella patens. PpPPR_63 localizes to the nucleus, while PpPPR_67 and PpPPR_104 are found in both the mitochondria and chloroplasts. The three proteins displayed pre-tRNA 5′-end processing activity in vitro. Mutants with knockout (KO) of the PpPPR_63 gene displayed growth retardation of protonemal colonies, indicating that, unlike Arabidopsis nuclear RPORPs, the moss nuclear PpPPR_63 is not essential for viability. In the KO mutant, nuclear-encoded tRNAAsp (GUC) levels were slightly decreased, whereas most nuclear-encoded tRNA levels were not altered. This indicated that most of the cytosolic mature tRNAs were produced normally without proteinaceous RNase P-like PpPPR_63. Single PpPPR_67 or 104 gene KO mutants displayed different phenotypes of protonemal growth and chloroplast tRNAArg (ACG) accumulation. However, the levels of all other tRNAs were not altered in the KO mutants. In addition, in vitro RNase P assays showed that PpPPR_67 and PpPPR_104 efficiently cleaved chloroplast pre-tRNAArg (CCG) and pre-tRNAArg (UCU) but they cleaved pre-tRNAArg (ACG) with different efficiency. This suggests that the two proteins have overlapping function but their substrate specificity is not identical.
Highlights
RNase P is an endonuclease that removes the 59 leader sequence from precursor tRNAs
Arabidopsis thaliana, three MRPP3 homologs have been identified as RNase P enzymes; they are termed proteinaceous RNase P 1 (PRORP1), PRORP2, and PRORP3 [6]
Algae seem to possess both a ribonuceoprotein RNase P and a proteinaceous RNase Ps (PRORPs). The latter is not known to localize in either the organelles or the nucleus. From these differences in the status of PRORP enzymes in algae and plants, the following questions have arisen: When were plant PRORP genes duplicated during plant evolution, which PRORP protein was targeted to the organelles and which to the nucleus, and was ribonucleoprotein RNase P enzyme replaced by PRORP in plants? To answer these questions, we focused on PRORPs of the basal land plant bryophyte Physcomitrella patens
Summary
RNase P is an endonuclease that removes the 59 leader sequence from precursor tRNAs (pre-tRNAs) This endonucleolytic cleavage is an essential step in the production of mature tRNAs in all organisms, as well as in mitochondria and chloroplasts [1,2]. Human mitochondrial RNase P was identified as an RNA-free enzyme composed of three proteins called mitochondrial RNase P protein 1 (MRPP1), MRPP2, and MRPP3 [4]. Single PRORP2 or PRORP3 knockout mutant lines show wild-type phenotypes, whereas the homozygous double mutation in prorp prorp results in embryonic lethality [7]. This indicates that PRORP2 and 3 have redundant functions and are essential for embryogenesis and plant growth
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