Overaccumulation of the chloroplast antisense RNA AS5 is correlated with decreased abundance of 5S rRNA in vivo and inefficient 5S rRNA maturation in vitro

Abstract

Post-transcriptional regulation in the chloroplast is exerted by nucleus-encoded ribonucleases and RNA-binding proteins. One of these ribonucleases is RNR1, a 3'-to-5' exoribonuclease of the RNase II family. We have previously shown that Arabidopsis rnr1-null mutants exhibit specific abnormalities in the expression of the rRNA operon, including the accumulation of precursor 23S, 16S, and 4.5S species and a concomitant decrease in the mature species. 5S rRNA transcripts, however, accumulate to a very low level in both precursor and mature forms, suggesting that they are unstable in the rnr1 background. Here we demonstrate that rnr1 plants overaccumulate an antisense RNA, AS5, that is complementary to the 5S rRNA, its intergenic spacer, and the downstream trnR gene, which encodes tRNA(Arg), raising the possibility that AS5 destabilizes 5S rRNA or its precursor and/or blocks rRNA maturation. To investigate this, we used an in vitro system that supports 5S rRNA and trnR processing. We show that AS5 inhibits 5S rRNA maturation from a 5S-trnR precursor, and shorter versions of AS5 demonstrate that inhibition requires intergenic sequences. To test whether the sense and antisense RNAs form double-stranded regions in vitro, treatment with the single-strand-specific mung bean nuclease was used. These results suggest that 5S-AS5 duplexes interfere with a sense-strand secondary structure near the endonucleolytic cleavage site downstream from the 5S rRNA coding region. We hypothesize that these duplexes are degraded by a dsRNA-specific ribonuclease in vivo, contributing to the 5S rRNA deficiency observed in rnr1.

FIGURE 1.

Accumulation and processing of 5S rRNA and tRNA^Arg in rnr1-3. (A) RNA gel blot of 0.5 μg total RNA extracted from wild-type (WT) and rnr1-3 rosette leaves, probed for 5S rRNA (upper panel) and tRNA^Arg (lower panel). RNA loading is represented by 7SL, an snRNA (upper panel) and ethidium bromide staining of the 28S rRNA (lower panel). Transcript sizes are indicated at left, and RNA species are designated at right. (B) Quantitative RT-PCR determination of 5S rRNA and tRNA^Arg abundance. Expression levels are an average of three biological and at least two technical replicates for each sample, with error bars representing the standard deviation. The WT expression level was set to 1, and samples were normalized to actin mRNA.

FIGURE 2.

Identification and accumulation of asRNAs complementary to 5S-trnR in wild-type (WT) and rnr1-3 plants. (A) Representative ESTs corresponding to the antisense strand of the 5S-trnR region. The major AS5 species identified in rnr1-3 from sequencing of cRT-PCR products are indicated by dotted lines, with the 5′ and 3′ ends numbered according to the Arabidopsis chloroplast genome. PCR primer annealing positions are indicated by arrowheads, and the strand-specific probes used in C are shown above (Pb 1 and Pb 2). (B) AS5 accumulation was determined by qRT-PCR (left) as described in the legend to Figure 1. cRT-PCR (right) was used to amplify cDNAs for 5′ and 3′ end determination. Bands 1 and 2 refer to the sequences shown in panel A. (C) Agarose gel blot analysis of AS5 using 15 μg of total RNA. The probes used are indicated at the top of each gel, with the gene location shown in A. The ethidium bromide–stained gel (left) is shown to reflect loading. Sizes in kilobases correspond to known rRNA species. (D) Amplification of AS5 by RT-PCR from WT tissues, and rnr1-3 (last lane on right). Ubiquitin was used as a control.

FIGURE 3.

Inhibition of 5S rRNA maturation by AS5. (A) Representation of the synthetic 5S-trnR transcript (top) and the complementary AS5-1 and AS5-2 transcripts aligned to the corresponding genome positions. (+1) First nucleotide of intergenic region; (P) RNase P cleavage site. (B, left two lanes) Labeled transcripts serving as markers for the migration of mature 5S rRNA (m5S) and the 5S-trnR precursor cleaved at the RNase P site (5S + I), respectively. (Third lane) 5S-trnR precursor; (fourth lane) precursor incubated for 30 min in buffer without chloroplast proteins. (Last 12 lanes) Uniformly labeled 5S-trnR transcript incubated with the chloroplast protein extract and increasing amounts of trace-labeled AS5 inhibitors. Products were separated in a 5% denaturing polyacrylamide gel. Icons at the left indicate the sense substrates, and arrows on the right indicate the antisense substrates.

FIGURE 4.

AS5 sequence determinants for inhibition of 5S rRNA maturation. (A) Representation of sense and antisense substrates, as described in the legend to Figure 3. (B) Processing of labeled 5S-trnR in the absence or presence of antisense inhibitors, as described in the legend to Figure 3.

FIGURE 5.

Quantitative measurement of inhibition of 5S rRNA (A) and tRNA^Arg (B) maturation by AS5 substrates. The signal intensity of the mature transcripts (y-axis) was plotted against log₁₀ (fold excess of inhibitor used) (x-axis). Linear regressions were fitted to each data set to determine inhibition efficiency. 100% processing efficiency was defined as 5S rRNA or tRNA^Arg yield without inhibitor.

FIGURE 6.

Effect of AS5 inhibitors on the stability of mature 5S rRNA or various processing intermediates. The labeled intermediates were mature 5S rRNA (5S), a 33-nt extended precursor (5S + 33), a 232-nt extended precursor (5S + I), and the complete 5S-trnR substrate. Increasing amounts of AS5-1 (A), AS5-4 (B), and AS5-7 (C) were used with electrophoresis as described in the legend to Figure 3.

FIGURE 7.

Mapping intermolecular base-pairing between 5S-trnR and AS5 by treatment with mung bean nuclease. (A) Representation of the substrates used for the experiment in panels B and C. (+10) An endonuclease cleavage site; (P) RNAse P cleavage site. (B) Labeled 5S-trnR was incubated with the chloroplast protein extract in duplicate with or without a 500-fold excess of each AS5 inhibitor. Reaction products were treated (+) or not (−) with mung bean nuclease following RNA purification, and analyzed by 5% denaturing polyacrylamide gel electrophoresis. (A–I) Transcripts referred to in the text and in panel D, with F representing the putative full-length sense-antisense duplex for each AS5 inhibitor. Where clusters of bands occur, a vertical line marks the bands assigned to that cluster. (C) 5S-trnR was 5′-end-labeled and incubated with chloroplast protein extract with or without a 500-fold excess of the two inhibitors shown. RNA was extracted prior to treatment with MBN. (D) Interpretation of bands seen in panels B and C, as discussed in the text. (+40–60) The region of discontinuity susceptible to MBN digestion.

FIGURE 8.

S1 nuclease protection of tRNA^Arg precursors generated in vivo. (A) The 5S-trnR region of the chloroplast genome and the positions of protected fragments (dashed lines) observed in panel B. The 5′-labeled probe is shown below the diagram. (B) S1 nuclease protection. (Lane M) markers; (lane P) probe alone; (lanes WT and rnr1-3) protection using total RNA; (lane tRNA) negative control using yeast tRNA. Electrophoresis was in a 6% polyacrylamide gel, which is shown in two exposures. Open arrowheads indicate the positions of 5′ ends that are present both in the WT and rnr1-3.

FIGURE 9.

Analysis of 5S rRNA (A) or tRNA^Arg (B) processing in chloroplast RNase mutants. (A,B) Gel blot analysis of total RNA extracted from WT, rne, rnj, pnp, and rnr1-3 rosette leaves, separated in agarose (0.5 μg; left panels) or polyacrylamide (1.0 μg; right panels) gels. Relative loading of WT RNA is indicated above the blots. The right lane of the polyacrylamide gel in panel B is an in vitro transcript to serve as a size marker for the 5S-trnR dicistronic transcript. (Bands A–F) Transcripts discussed in the text. (C) Interpretation of bands observed in A and B, as discussed in the text.

FIGURE 10.

Model for AS5-mediated regulation of 5S maturation. (Top) AS5 abundance is modulated by RNR1. (Left side) 5S-trnR maturation is initiated by endonuclease cleavage either in the intergenic region by RNase E and/or J, or by RNase P. 5S-containing intermediates are trimmed by RNR1 and PNPase, whereas trnR-containing intermediates are trimmed 5′-to-3′ by RNase J, or are subject to RNase P cleavage. The tRNA^Arg 3′ end is created by RNase Z. (Right side) AS5 potentially regulates this maturation through either complete or partial duplexing with 5S rRNA precursors. This base-pairing would inhibit endonucleolytic cleavage downstream from the mature 5S rRNA 3′ end, and create a target for RNase III–like enzymes that would trigger degradation of both sense and antisense strands.