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

Sharwood, Robert E.; Hotto, Amber M.; Bollenbach, Thomas; Stern, David B.

doi:10.1261/rna.2336611

Cited by 37 publications

(35 citation statements)

References 63 publications

(66 reference statements)

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“…and its complement in rnc3/4 may suggest that duplex formation creates a Mini-III recognition site, although the two-nucleotide overhang characteristic of Mini-III cleavage was not observed. In this way, the pncRNA could regulate RNA processing, as postulated previously for the complementary chloroplast 5S and AS5 transcripts (Hotto et al, 2010;Sharwood et al, 2011b). We have not yet found evidence for increased accumulation of other pncRNAs in rnc3/4, however, raising the possibility that the 4.5S-5S region is one of a small number of Mini-III targets.…”

Section: A Pncrna Is Processed By Mini-iiisupporting

confidence: 65%

Arabidopsis Chloroplast Mini-Ribonuclease III Participates in rRNA Maturation and Intron Recycling

et al. 2015

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RNase III proteins recognize double-stranded RNA structures and catalyze endoribonucleolytic cleavages that often regulate gene expression. Here, we characterize the functions of RNC3 and RNC4, two Arabidopsis thaliana chloroplast Mini-RNase III-like enzymes sharing 75% amino acid sequence identity. Whereas rnc3 and rnc4 null mutants have no visible phenotype, rnc3/rnc4 (rnc3/4) double mutants are slightly smaller and chlorotic compared with the wild type. In Bacillus subtilis, the RNase Mini-III is integral to 23S rRNA maturation. In Arabidopsis, we observed imprecise maturation of 23S rRNA in the rnc3/4 double mutant, suggesting that exoribonucleases generated staggered ends in the absence of specific Mini-III-catalyzed cleavages. A similar phenotype was found at the 39 end of the 16S rRNA, and the primary 4.5S rRNA transcript contained 39 extensions, suggesting that Mini-III catalyzes several processing events of the polycistronic rRNA precursor. The rnc3/4 mutant showed overaccumulation of a noncoding RNA complementary to the 4.5S-5S rRNA intergenic region, and its presence correlated with that of the extended 4.5S rRNA precursor. Finally, we found rnc3/4-specific intron degradation intermediates that are probable substrates for Mini-III and show that B. subtilis Mini-III is also involved in intron regulation. Overall, this study extends our knowledge of the key role of Mini-III in intron and noncoding RNA regulation and provides important insight into plastid rRNA maturation.

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Section: A Pncrna Is Processed By Mini-iiisupporting

confidence: 65%

Arabidopsis Chloroplast Mini-Ribonuclease III Participates in rRNA Maturation and Intron Recycling

et al. 2015

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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%

“…thaliana (Col-0) plants used for Agrobacterium infiltration were grown as described in Sharwood et al (2011). N. benthamiana plants for infiltration were germinated and grown in a greenhouse at 25°C with light provided to ensure a 14-h light/10-h dark cycle.…”

Section: Plant Materialsmentioning

confidence: 99%

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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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“…Available evidence suggests that RNase P releases the downstream tRNA Arg , and RNase II trims the intergenic region between the 5S rRNA and trnR . Alternatively and/or additionally, an endonucleolytic cleavage by either RNase E or RNase J can occur in the 5S‐ trnR intergenic region, followed by exonuclease trimming by RNase II or PNPase 129…”

Section: Processing Of Primary Transcriptsmentioning

confidence: 99%

RNA processing and decay in plastids

et al. 2013

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Plastids were derived through endosymbiosis from a cyanobacterial ancestor, whose uptake was followed by massive gene transfer to the nucleus, resulting in the compact size and modest coding capacity of the extant plastid genome. Plastid gene expression is essential for plant development, but depends on nucleus-encoded proteins recruited from cyanobacterial or host-cell origins. The plastid genome is heavily transcribed from numerous promoters, giving posttranscriptional events a critical role in determining the quantity and sizes of accumulating RNA species. The major events reviewed here are RNA editing, which restores protein conservation or creates correct open reading frames by converting C residues to U, RNA splicing, which occurs both in cis and trans, and RNA cleavage, which relies on a variety of exoribonucleases and endoribonucleases. Because the RNases have little sequence specificity, they are collectively able to remove extraneous RNAs whose ends are not protected by RNA secondary structures or sequence-specific RNA-binding proteins (RBPs). Other plastid RBPs, largely members of the helical-repeat superfamily, confer specificity to editing and splicing reactions. The enzymes that catalyze RNA processing are also the main actors in RNA decay, implying that these antagonistic roles are optimally balanced. We place the actions of RBPs and RNases in the context of a recent proteomic analysis that identifies components of the plastid nucleoid, a protein-DNA complex with multiple roles in gene expression. These results suggest that sublocalization and/or concentration gradients of plastid proteins could underpin the regulation of RNA maturation and degradation.

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

Cited by 37 publications

References 63 publications

Arabidopsis Chloroplast Mini-Ribonuclease III Participates in rRNA Maturation and Intron Recycling

Arabidopsis Chloroplast Mini-Ribonuclease III Participates in rRNA Maturation and Intron Recycling

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

RNA processing and decay in plastids

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