Overexpression of a natural chloroplast-encoded antisense RNA in tobacco destabilizes 5S rRNA and retards plant growth

Hotto, Amber M.; Huston, Zoe E; Stern, David B.

doi:10.1186/1471-2229-10-213

Cited by 37 publications

(35 citation statements)

References 66 publications

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“…We also note that the in vitro experiments used molar ratios of precursor and asRNA that may not represent the ratios of the bioactive molecules in vivo. On the other hand, our recent demonstration that overexpression of AS5 in tobacco chloroplasts destabilizes the 5S rRNA (Hotto et al 2010) supports the viewpoint that the in vitro system is a useful platform for dissecting the mechanism of AS5 action.…”

Section: In Vitro Dissection Of As5 Inhibitor Sequencesmentioning

confidence: 88%

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

Sharwood¹,

Hotto²,

Bollenbach³

et al. 2010

RNA

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Post-transcriptional regulation in the chloroplast is exerted by nucleus-encoded ribonucleases and RNA-binding proteins. One of these ribonucleases is RNR1, a 39-to-59 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.

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Section: In Vitro Dissection Of As5 Inhibitor Sequencesmentioning

confidence: 88%

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

Sharwood¹,

Hotto²,

Bollenbach³

et al. 2010

RNA

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“…Some functional RNAs are defined by 59-end RNA-binding proteins that stall 59 / 39 exonucleolytic activity (Drager et al 1998;Nickelsen et al 1999;Loiselay et al 2008;Johnson et al 2010;Prikryl et al 2010), but other protective mechanisms may also exist. Examples of RNAs probably not protected by PPR-type proteins include natural (i.e., accumulating in the WT) asRNAs (Georg et al 2010;Hotto et al 2010;Sharwood et al 2011), small RNAs involved in splicing or other functions (Goldschmidt-Clermont et al 1991;Vera and Sugiura 1994), as well as rRNAs, tRNAs, and any mRNAs that do not bind protective 59-end proteins. Some of these species may escape RNase J degradation by virtue of associating with proteins, such as ribosomal proteins, splicing factors, or translation factors.…”

Section: Discussionmentioning

confidence: 99%

“…We reasoned that several recent reports, including our own, had described the accumulation of chloroplast antisense or non-coding RNAs (Lung et al 2006;Georg et al 2010;Hotto et al 2010), and that these might be substrates for RNase J. If this were the case, they could overaccumulate in mutant material.…”

Section: Diffuse Transcripts and Novel Bands Are Mainly Antisense Rnasmentioning

confidence: 99%

Chloroplast RNase J compensates for inefficient transcription termination by removal of antisense RNA

Sharwood¹,

Halpert²,

Luro³

et al. 2011

RNA

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Ribonuclease J is an essential enzyme, and the Bacillus subtilis ortholog possesses both endoribonuclease and 59 / 39 exoribonuclease activities. Chloroplasts also contain RNase J, which has been postulated to participate, as both an exo-and endonuclease, in the maturation of polycistronic mRNAs. Here we have examined recombinant Arabidopsis RNase J and found both 59 / 39 exoribonuclease and endonucleolytic activities. Virus-induced gene silencing was used to reduce RNase J expression in Arabidopsis and Nicotiana benthamiana, leading to chlorosis but surprisingly few disruptions in the cleavage of polycistronic rRNA and mRNA precursors. In contrast, antisense RNAs accumulated massively, suggesting that the failure of chloroplast RNA polymerase to terminate effectively leads to extensive symmetric transcription products that are normally eliminated by RNase J. Mung bean nuclease digestion and polysome analysis revealed that this antisense RNA forms duplexes with sense strand transcripts and prevents their translation. We conclude that a major role of chloroplast RNase J is RNA surveillance to prevent overaccumulation of antisense RNA, which would otherwise exert deleterious effects on chloroplast gene expression.

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“…They focus on single RNAs and do not point to ncRNAs as a more general regulatory principle (Nishimura et al, 2004;Georg et al, 2010;Hotto et al, 2010;Zghidi-Abouzid et al, 2011). One study identified 12 ncRNAs in tobacco chloroplasts (Lung et al, 2006).…”

Section: Plastid Ncrnasmentioning

confidence: 99%

“…Cyanobacteria, the chloroplast progenitors, have a plethora of noncoding RNAs at their disposal to regulate gene expression . However, only a few potentially regulatory RNAs have been reported to be transcribed from plastid genes (Nishimura et al, 2004;Georg et al, 2010;Hotto et al, 2010;Zghidi-Abouzid et al, 2011). Therefore, chloroplasts may contain additional hitherto undetected genes for regulatory ncRNAs.…”

Section: Introductionmentioning

confidence: 99%

The Primary Transcriptome of Barley Chloroplasts: Numerous Noncoding RNAs and the Dominating Role of the Plastid-Encoded RNA Polymerase

et al. 2012

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Gene expression in plastids of higher plants is dependent on two different transcription machineries, a plastid-encoded bacterial-type RNA polymerase (PEP) and a nuclear-encoded phage-type RNA polymerase (NEP), which recognize distinct types of promoters. The division of labor between PEP and NEP during plastid development and in mature chloroplasts is unclear due to a lack of comprehensive information on promoter usage. Here, we present a thorough investigation into the distribution of PEP and NEP promoters within the plastid genome of barley (Hordeum vulgare). Using a novel differential RNA sequencing approach, which discriminates between primary and processed transcripts, we obtained a genome-wide map of transcription start sites in plastids of mature first leaves. PEP-lacking plastids of the albostrians mutant allowed for the unambiguous identification of NEP promoters. We observed that the chloroplast genome contains many more promoters than genes. According to our data, most genes (including genes coding for photosynthesis proteins) have both PEP and NEP promoters. We also detected numerous transcription start sites within operons, indicating transcriptional uncoupling of genes in polycistronic gene clusters. Moreover, we mapped many transcription start sites in intergenic regions and opposite to annotated genes, demonstrating the existence of numerous noncoding RNA candidates.

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Overexpression of a natural chloroplast-encoded antisense RNA in tobacco destabilizes 5S rRNA and retards plant growth

Cited by 37 publications

References 66 publications

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

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

Chloroplast RNase J compensates for inefficient transcription termination by removal of antisense RNA

The Primary Transcriptome of Barley Chloroplasts: Numerous Noncoding RNAs and the Dominating Role of the Plastid-Encoded RNA Polymerase

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