RNR1, a 3'-5' exoribonuclease belonging to the RNR superfamily, catalyzes 3' maturation of chloroplast ribosomal RNAs in Arabidopsis thaliana

Abstract

Arabidopsis thaliana chloroplasts contain at least two 3' to 5' exoribonucleases, polynucleotide phosphorylase (PNPase) and an RNase R homolog (RNR1). PNPase has been implicated in both mRNA and 23S rRNA 3' processing. However, the observed maturation defects do not affect chloroplast translation, suggesting that the overall role of PNPase in maturation of chloroplast rRNA is not essential. Here, we show that this role can be largely ascribed to RNR1, for which homozygous mutants germinate only on sucrose-containing media, and have white cotyledons and pale green rosette leaves. Accumulation of chloroplast-encoded mRNAs and tRNAs is unaffected in such mutants, suggesting that RNR1 activity is either unnecessary or redundant for their processing and turnover. However, accumulation of several chloroplast rRNA species is severely affected. High-resolution RNA gel blot analysis, and mapping of 5' and 3' ends, revealed that RNR1 is involved in the maturation of 23S, 16S and 5S rRNAs. The 3' extensions of the accumulating 5S rRNA precursors can be efficiently removed in vitro by purified RNR1, consistent with this view. Our data suggest that decreased accumulation of mature chloroplast ribosomal RNAs leads to a reduction in the number of translating ribosomes, ultimately compromising chloroplast protein abundance and thus plant growth and development.

Figure 1

RNR1 T-DNA insertion mutants. (A) Diagram of the RNR1 gene showing the positions of three T-DNA insertion alleles used in this work. The positions of T-DNA insertions were confirmed by PCR. (B) WT and homozygous rnr1 mutants were germinated on sucrose-containing media for 15 days (top panels), or grown on sucrose-containing media for 40 days and transferred to soil for an additional 9 days (bottom panels). (C) RT–PCR showing expression of the RNR1 gene in different tissues of WT Arabidopsis. Primers used in this analysis (RNR5′ and RNR3′) are indicated in (A).

Figure 2

Loss of RNR1 expression affects chloroplast biogenesis. (A) Transverse sections of cotyledons from 3-week-old WT and rnr1 plants stained with toluidine blue. Chloroplasts (cp) are visible in WT cotyledons as globular shapes. rnr1 cotyledons exhibit a severely disorganized structure and contain no visible chloroplasts. The magnification bar corresponds to 100 µm. (B) Transmission electron micrographs from cotyledons of 3-week-old WT and rnr1 plants. Mitochondria and plastids are indicated by m and cp, respectively. The magnification bars correspond to 1 µm.

Figure 3

mRNA and protein accumulation in rnr1 and WT plants. (A) One microgram of total RNA from WT or from rnr1-3 was separated in a 1.2% agarose–formaldehyde gel and analyzed with the probes indicated to the right of each blot. 28S rRNA accumulation is shown in the ethidium bromide-stained membrane below each experiment to confirm equal loading. (B) Immunoblot analysis compares accumulation of the proteins shown at right in rnr1 as compared with a dilution series of WT proteins. The stained filter is shown at the bottom as an estimate of gel loading.

Figure 4

Analysis of rRNA expression and processing. (A) Diagram of the rRNA operon and location of probes (A–F) used for RNA gel blot analyses, and sizes of transcripts (in kb) observed by the gel blot analysis shown in (B). (B) RNA gel blot analysis. One microgram of total RNA from WT or rnr1-3 was separated in 1.2% agarose–formaldehyde gels, and blots were probed as indicated above each blot, with transcripts identified shown to the right. 28S rRNA is shown in the ethidium bromide-stained membrane below each experiment to confirm equal loading. The transcripts marked by asterisks for probe D are mature 23S rRNA processed at hidden breaks (see text).

Figure 5

Mapping of 16S rRNA 5′ and 3′ ends by cRT–PCR. The 5′ and 3′ ends are shown by solid arrowheads for WT and by open arrowheads for rnr1, with numbers of corresponding clones obtained shown at each position. The 5′ and 3′ ends of the mature 16S rRNA sequence are underlined.

Figure 6

Analysis of 23S 3′ end processing by RNase protection. (A) The probe is indicated below the diagram of the 4.5S–5S region of the rrn operon. Sizes of protected RNAs found in (B) are indicated above the schematic. (B) Protected RNA from WT or from rnr1-3, as indicated above each lane, was separated in a 6% denaturing polyacrylamide gel. The positions of protected bands corresponding to mature and pre-23S RNAs are indicated at right, and size standards are at left.

Figure 7

Analysis of 4.5S and 3′ end processing. (A) An aliquot of 1.5 µg of total RNA from WT, rnr1-1, rnr1-2 and rnr1-3 was separated on a 12% acrylamide gel and stained with ethidium bromide (left panel) or analyzed with a 4.5S rRNA-specific probe (right panel). Migration and size (nt) of molecular weight markers are indicated. (B) The probe used in this analysis is indicated by the heavy arrow below the schematic of the 4.5S–5S–tRNA^Arg region of the rrn operon. Sizes of protected RNAs seen in (C) are indicated above the schematic. (C) Protected RNA from WT or from rnr1-3, as indicated above each lane, was separated in a 6% denaturing polyacrylamide gel. The positions of protected bands corresponding to mature and pre-4.5S rRNAs are indicated at right, and size standards are at left.

Figure 8

Ordered processing of the 23S–4.5S rRNA. (A) Analysis of 4.5S precursor rRNA accumulation. One microgram of total RNA from either WT or from rnr1-3 was separated in a 1.2% agarose–formaldehyde gel, and probed with the oligonucleotides indicated in the diagram above. 28S rRNA accumulation on the ethidium bromide-stained membrane is shown below each blot to confirm equal loading. (B) Mapping 23S–4.5S precursor 5′ ends by cRT–PCR. The 5′ ends are shown by solid arrowheads for WT and by open arrowheads for rnr1, with numbers of corresponding clones obtained shown at each position. The 5′ end of the mature 23S rRNA is underlined. (C) Mapping of 23S–4.5S precursor 3′ ends by cRT–PCR, labeled as in (B). The mature 4.5S and 5S rRNA 3′ ends are underlined.

Figure 9

Analysis of 5S rRNA 3′ end processing. (A) The probe is indicated by the heavy arrow below the diagram of the 4.5S–5S–tRNA^Arg region. Sizes of protected RNAs seen in (C) are indicated above the diagram. (B) Protected RNA from WT or from rnr1-3, as indicated above each lane, was separated in a 6% denaturing polyacrylamide gel. The positions of protected bands corresponding to mature and pre-5S rRNAs are indicated at the right, and size standards are at the left. (C) Mapping of 5S rRNA 5′ and 3′ ends by cRT–PCR. The genomic sequence is shown at the top, with the mature 3′ and 5′ ends underlined. 5S rRNA 3′ and 5′ ends obtained for WT and rnr1 plants are indicated with the number on the right representing the number of clones for each sequence. Nucleotides that could be present either at 3′ or at 5′ ends are indicated in the middle column. Adenosines that are added post-transcriptionally are in lowercase.

Figure 10

RNR1 accurately processes 5S rRNA 3′ ends in vitro. Affinity-purified RNR1-tag and a fraction from a mock purification were incubated with substrates corresponding to the mature 5S rRNA (5S) or to the same RNA with a 20 nt 3′ extension (5S + 20). Incubation times are indicated in minutes above each lane and the positions and putative secondary structures of substrate and product are indicated at the right and left, respectively.

Figure 11

Sucrose density gradient fractions from WT and rnr1 were analyzed by RNA gel blot with the probes indicated to the right of each panel. The 12 lanes from left to right indicate the gradient fractions, with 1 representing the top of the gradient and 12 representing the bottom of the gradient. An ethidium bromide-stained membrane for each sample is included; equal proportions of each fraction were loaded. The positions of ribosomal (<80S) and polysomal fractions were determined by running puromycin-treated samples in parallel to experimental samples, as indicated at the bottom.