Figure 1.
PRORP proteins are widely distributed among eukaryotes.
(A) Schematic diagram of plant organellar PRORPs with a transit peptide (TP), RNA binding PPR motifs (P), and NYN metallonuclease domain (NYN). (B) Neighbor-joining phylogenetic tree of PRORPs and PRORP-like proteins. Representative PRORP protein sequences from evolutionarily distant plants were used for the phylogenic analysis. Experimentally determined localization of PRORPs is presented as chloroplast (chl), mitochondria (mit), or nucleus (nuc). Bootstrap values>50 are indicated along branches. Protein ID or accession numbers of PRORPs and PRORP-like proteins are listed in Table S1.
Figure 2.
Localization of PpPPR_63, 67, and 104.
(A) Observations of GFP fluorescence in various cells and tissues of the stable transgenic moss KI-10 line expressing PpPPR_63-GFP fusion protein. Schematic diagram of a moss plant and observed parts are in circles (a). Images are differential interference contrast (DIC) (b, d, f, h, j) and GFP (green) and chlorophyll (magenta) fluorescence (c, e, g, i, k). Single protonemal cell (b, c), protonemal cells (d, e), a bud (f, g), a young leaf (h, i), and mature leaf cells (j, k). Scale bars = 5 µm (b, c) or 20 µm (d–k). (B) Subcellular localization of N-terminal PpPPR_67-GFP fusion protein (N67-GFP, upper panels) or N-terminal PpPPR_104-GFP fusion protein (N104-GFP, bottom panels). For mitochondrial localization control, the γ subunit of mitochondrial ATPase and RFP fusion protein (Mt-RFP) was used. Fluorescence of GFP and RFP, the overlay of the two fluorescence images (Merged), and the corresponding Nomarski images (DIC) are shown.
Figure 3.
Pre-tRNA cleavage assay of P. patens PRORP-like proteins.
(A) Schematic diagrams of various recombinant proteins. Recombinant proteins r67, r104, r63, and rM63 indicate, respectively, PpPPR_67, PpPPR_104, PpPPR_63, and the mutant PpPPR_63, the two catalytic aspartates (DD) of which were substituted with alanines (AA). rC63 consists of the tag sequences and an NYN domain only of PpPPR_63. (B) Coomassie brilliant blue (CBB)-stained recombinant proteins (1 µg each). (C) RNase P activity was assayed by using 2 µg of in vitro-transcribed chloroplast (chl) pre-tRNAPhe, mitochondrial (mit) pre-tRNACys, or nuclear (nuc) pre-tRNAAsp and 100 ng of recombinant proteins. The reaction products were separated by using 8% denaturing PAGE and stained with ethidium bromide. (D) The 5′-ends of processed pre-tRNAs in (C) were determined by primer extension analysis. Processed pre-tRNAs without proteins (lanes 1 and 5), with r67 (lanes 2 and 6), with r104 (lanes 3 and 7), or with r63 (lanes 4 and 8) were reverse transcribed from the 5′-end-labeled primers underlined in (E). (E) The nucleotide sequences of chloroplast (chl) pre-tRNAPhe and mitochondrial (mit) pre-tRNACys are shown with small letters (5′ leader and 3′ trailer sequences) and capital letters (predicted mature tRNA). Arrowheads indicate the 5′-end positions determined in (D). Underlined sequences indicate the position of primers used for primer extension.
Figure 4.
Molecular and morphological phenotypes of the PpPPR_63 KO and complementation mosses.
(A) Schematic diagram of PpPPR_63 and construction of the KO locus. The nptII cassette was inserted into the 4th intron of the PpPPR_63 gene. (B) Detection of PpPPR_63 transcripts by RT-PCR. (C) Two-week-old protonemal colonies of the PpPPR_63 KO line (Δ63) and the complementation lines (F63 and M63). Bars = 5 mm. (D) One-week-old filamentous protonemata regenerated from cut leaves. (E) Steady-state levels of nuclear-encoded tRNAs.
Figure 5.
PpPPR_67 and PpPPR_104 KO mosses.
(A) Schematic structure of PpPPR_67 and 104 KO loci. The nptII cassette was inserted into the 5th exon of PpPPR_67 and the 4th exon of PpPPR_104. (B) Detection of PpPPR_67 and 104 transcripts by RT-PCR. (C) Two-week-old protonemata colonies of the PpPPR_67 KO line (Δ67) and the PpPPR_104 KO line (Δ104). Scale bars = 5 mm. (D) Steady-state levels of chloroplast (chl) tRNAArg isoacceptors with different anticodons, chl tRNAPhe (GAA) and mitochondrial (mit) tRNACys (GCA).
Figure 6.
Pre-tRNA cleavage assay of the recombinant proteins r67 and r104.
Reactions containing 500 nM of chloroplast 5′-32P-pre-tRNAArg (ACG) (upper), tRNAArg (CCG) (middle), or tRNAArg (UCU) (lower) and 500 nM of recombinant protein were quenched at 1, 5, 15, and 30 min points, resolved on 8% denaturing PAGE, and analyzed with a phosphoimager.
Figure 7.
Schematic summarizing the localization of three PRORP homologs,
PpPPR_63, 67, and 104 in P. patens. This study suggested that unidentified RNase P enzyme(s) including RNP-type RNase P may be present at least in the nucleus.