A single Arabidopsis organellar protein has RNase P activity

Journal name:
Nature Structural & Molecular Biology
Volume:
17,
Pages:
740–744
Year published:
DOI:
doi:10.1038/nsmb.1812
Received
Accepted
Published online

Abstract

The ubiquitous endonuclease RNase P is responsible for the 5′ maturation of tRNA precursors. Until the discovery of human mitochondrial RNase P, these enzymes had typically been found to be ribonucleoproteins, the catalytic activity of which is associated with the RNA component. Here we show that, in Arabidopsis thaliana mitochondria and plastids, a single protein called 'proteinaceous RNase P' (PRORP1) can perform the endonucleolytic maturation of tRNA precursors that defines RNase P activity. In addition, PRORP1 is able to cleave tRNA-like structures involved in the maturation of plant mitochondrial mRNAs. Finally, we show that Arabidopsis PRORP1 can replace the bacterial ribonucleoprotein RNase P in Escherichia coli cells. PRORP2 and PRORP3, two paralogs of PRORP1, are both localized in the nucleus.

At a glance

Figures

  1. PRORP proteins define a novel family of putative nucleases present in a wide array of eukaryote lineages.
    Figure 1: PRORP proteins define a novel family of putative nucleases present in a wide array of eukaryote lineages.

    (a) PRORP1 contains an organellar targeting signal (TS) and 3 PPR motifs that could be involved in RNA binding. Land plant PRORPs also always contain a Lys/Arg–rich box in their N-terminal part. The C-terminal domain contains a metallonuclease domain corresponding to the predicted catalytic site of the protein. This domain is well conserved among species. The comparison of 181 PRORP orthologs in plants, animals and protists shows that three aspartates and one histidine are 100% conserved in all the sequences analyzed. The logo representing residue frequency at each position in a portion of the aligned sequences was obtained with http://weblogo.berkeley.edu. Asterisks show the two aspartates that were mutated to alanines to create a PRORP1 catalytic mutant. (b) Unrooted neighbor-joining phylogenic tree showing the occurrence of PRORP in eukaryote lineages. A sample of representative PRORP protein sequences from plants, animals and other evolutionarily distant eukaryotes were used for the phylogenetic analysis. Monocot and dicot stand for monocotyledon and dicotyledon plants. Supplementary Figure 2 shows a tree constructed with 113 PRORP sequences; bootstrap values >50 are indicated along branches.

  2. PRORP1 is mitochondrial and chloroplastic, whereas PRORP2 and PRORP3 are nuclear.
    Figure 2: PRORP1 is mitochondrial and chloroplastic, whereas PRORP2 and PRORP3 are nuclear.

    (a) Arabidopsis protoplasts were transformed with constructs expressing fusions of EYFP with PRORP proteins. EYFP shows the fluorescence of fusion proteins. C shows the autofluorescence of chlorophyll in panel 2, mitotracker fluorescence in panel 5 and DAPI fluorescence in panels 8 and 11. T shows the transmitted light image for the respective protoplasts. PRORP1-EYFP colocalizes with both chlorophyll (panels 1 and 2) and mitotracker signals (panels 4 and 5). PRORP2-EYFP and PRORP3-EYFP both colocalize with DAPI staining. White arrows, chloroplasts; orange arrows, mitochondria; red arrows, nuclei. Scale bars, 5 μm. (b) Three PRORP-specific antibodies were used to immunodetect PRORP proteins in purified fractions of Arabidopsis mitochondria (Mit), chloroplasts (Chl) and nuclei (N). Antibodies directed against the mitochondrial Nad9, chloroplast ribulose bisphosphate carboxylase/oxidase (RuBisCo) and nuclear histone 2B (H2B) were used to control the purity of the respective cell fractions. Molecular weights, left of panel, are given in kDa.

  3. PRORP1 is essential.
    Figure 3: PRORP1 is essential.

    (a) PRORP1 has seven exons; the coding sequence is shown in light gray and UTRs in dark gray. Similar to most PPR genes, the 5′ region of PRORP1 that encodes PPR motifs does not contain any intron. Two prorp1 mutant lines were identified. For both lines, the T-DNA insertion was mapped in the second exon of PRORP1. Numbers give the positions of the inserts compared to the gene initiation codon. LB and RB stand for left and right borders of the T-DNA insertion. For both mutant lines, no plant homozygous for the T-DNA insertion could be obtained. (b) Siliques of heterozygous PRORP1/prorp1 mutant plants are compared to a wild-type silique. Mutant siliques contain green and white seeds. Green seeds contain wild-type or PRORP1/prorp1 embryos (green arrows), whereas white seeds contain prorp1/prorp1 embryos (white arrows). (c) Green and white seeds were bleached with Hoyer solution and observed at 20× magnification. This revealed that white seeds contain embryos with development stopped at the globular stage (circled in gray), thus showing that prorp1 mutations result in embryo lethality.

  4. PRORP1 has RNase P activity.
    Figure 4: PRORP1 has RNase P activity.

    (a) The activity was assayed using 5′-end-labeled in vitro transcripts of the precursors of mitochondrial tRNACys, chloroplastic tRNAPhe and tRNA-like structures found in the mitochondrial transcripts orf138 and nad6 (Supplementary Fig. 6). Precursor transcripts are 144, 146, 162 and 195 nucleotides long respectively. RNase P endonucleolytic cleavage results in the release of 5′ leader sequences of 51, 51, 66 and 52 nucleotides, respectively. Lanes C, P and mP, precursor transcripts incubated alone (C), with PRORP1 (P) or with mutant PRORP1 (mP). After the reactions, RNA molecules were separated on 8% denaturing polyacrylamide gels and autoradiographed. The positions of RNA length markers (in nucleotides) are given at the right margin. (b) The precise cleavage sites of the tRNACys precursor and the tRNA-like structure found in orf138 by PRORP1 were mapped by comparing the alkaline (OH), RNase T1 and RNase P1 digestion profiles of precursor transcripts with 5′ leader sequences released by PRORP1 activity. RNase T1 cleaves RNA after G and generates 3′-phosphate ends, whereas RNase P1 cleaves preferentially after G as well but, like RNase P, generates 3′-OH ends. The observed cleavage sites correspond to the predicted cleavage sites of RNase P (gray arrows). RNA molecules were separated on a high-resolution 8% polyacrylamide gel and autoradiographed. (c) The cleavage product of tRNACys by PRORP1 was subjected to complete alkaline hydrolysis, and nucleoside phosphates were separated by two-dimensional thin-layer chromatography (2D TLC). Cleavage products were either subjected to alkaline phosphatase treatment (+) or left untreated (−) before hydrolysis. This showed that PRORP1 cleavage generates tRNAs with a 5′-terminal phosphate as expected for cleavage by RNase P.

  5. Arabidopsis PRORP1 can functionally replace E. coli RNase P in vivo.
    Figure 5: Arabidopsis PRORP1 can functionally replace E. coli RNase P in vivo.

    (a) LB agar plates of E. coli BW cells transformed with the empty vector pDG148 (S/X) (top), with the derivative plasmid encoding wild-type PRORP1 (middle) or with the plasmid coding for catalytically inactive PRORP1 D474A D475A (mPRORP, bottom); cells were grown under permissive (arabinose) or nonpermissive (glucose) conditions. (b) Western blot of total protein extracts from E. coli BW cells grown in the presence of arabinose and transformed with the same plasmids as in a. Immunodetection was performed with PRORP1-specific antibodies. T, total protein extract; IS, insoluble protein pellet; S, soluble protein fraction. The position of protein size markers (in kDa) is indicated on the left, and the arrow on the right marks the position of the PRORP1 signal. Proteins were separated by SDS-PAGE.

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Author information

  1. These authors contributed equally to this work.

    • Bernard Gutmann,
    • Andreas Taschner &
    • Markus Gößringer
  2. These authors contributed equally to this work.

    • Walter Rossmanith &
    • Philippe Giegé

Affiliations

  1. Institut de Biologie Moléculaire des Plantes du Centre National de la Recherche Scientifique, Strasbourg, France.

    • Anthony Gobert,
    • Bernard Gutmann &
    • Philippe Giegé
  2. Center for Anatomy & Cell Biology, Medical University of Vienna, Vienna, Austria.

    • Andreas Taschner,
    • Johann Holzmann &
    • Walter Rossmanith
  3. Institute for Pharmaceutical Chemistry, Philipps-Universität Marburg, Marburg, Germany.

    • Markus Gößringer &
    • Roland K Hartmann

Contributions

A.G., R.K.H., J.H., W.R. and P.G. conceived and designed the experiments; A.G., B.G., A.T., M.G. and P.G. performed the experiments; A.G., B.G., A.T., M.G., R.K.H., W.R. and P.G. analyzed the data; A.G., R.K.H., W.R. and P.G. wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

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