Mutational analysis of Arabidopsis chloroplast polynucleotide phosphorylase reveals roles for both RNase PH core domains in polyadenylation, RNA 3′‐end maturation and intron degradation

Germain, Arnaud; Herlich, Shira; Larom, Shirley; Kim, Sang Hu; Schuster, Gadi; Stern, David B.

doi:10.1111/j.1365-313x.2011.04601.x

Cited by 45 publications

(69 citation statements)

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“…Evidence from plants depleted for mitochondrial PNPase suggests that in mitochondria, this 39 / 59 exoribonuclease is responsible for eliminating these products of ''relaxed'' transcription (Holec et al 2006). However, while chloroplast PNPase mutants fail to correctly mature 39 termini, there is no evidence for widespread accumulation of antisense or non-coding transcripts not found in WT plants (Germain et al 2011). We hypothesize that in chloroplasts RNase J has assumed this surveillance role, whereas mitochondria, lacking RNase J, depend on PNPase for the same function.…”

Section: Discussionmentioning

confidence: 89%

“…PNPase has both 39 / 59 exonucleolytic activity and 59 / 39 polymerization activity (Yehudai-Resheff et al 2003) and is involved both in polyadenylation, which destabilizes chloroplast transcripts, and in transcript maturation. PNPase mutants have chlorotic young leaves but are fertile (Marchive et al 2009) and have multiple defects in 39-end maturation as well as intron degradation (Walter et al 2002;Germain et al 2011).…”

Section: Introductionmentioning

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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Section: Discussionmentioning

confidence: 89%

Section: Introductionmentioning

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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“…We investigated organelle editing in two mutants that mimic the growth phenotype of the rip1 mutant and are compromised in some aspects of organelle RNA metabolism or organelle biogenesis. Tissue was available from a mutant in the chloroplast polynucleotide phosphorylase, which has a major role in maturing mRNA and rRNA 3′ ends but also participates in RNA degradation through exonucleolytic digestion and polyadenylation (41). We obtained a second mutant that was affected in the gene encoding a chloroplast envelope membrane protein containing a putative LrgB domain, which has been reported recently to play an important role in A. thaliana chloroplast development (42).…”

Section: Resultsmentioning

confidence: 99%

RIP1, a member of an Arabidopsis protein family, interacts with the protein RARE1 and broadly affects RNA editing

Bentolila¹,

Heller²,

Sun³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

214

266

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Transcripts of plant organelle genes are modified by cytidine-touridine (C-to-U) RNA editing, often changing the encoded amino acid predicted from the DNA sequence. Members of the PLS subclass of the pentatricopeptide repeat (PPR) motif-containing family are site-specific recognition factors for either chloroplast or mitochondrial C targets of editing. However, other than PPR proteins and the cis-elements on the organelle transcripts, no other components of the editing machinery in either organelle have previously been identified. The Arabidopsis chloroplast PPR protein Required for AccD RNA Editing 1 (RARE1) specifies editing of a C in the accD transcript. RARE1 was detected in a complex of >200 kDa. We immunoprecipitated epitope-tagged RARE1, and tandem MS/MS analysis identified a protein of unknown function lacking PPR motifs; we named it RNA-editing factor interacting protein 1 (RIP1). Yeast two-hybrid analysis confirmed RIP1 interaction with RARE1, and RIP1-GFP fusions were found in both chloroplasts and mitochondria. Editing assays for all 34 known Arabidopsis chloroplast targets in a rip1 mutant revealed altered efficiency of 14 editing events. In mitochondria, 266 editing events were found to have reduced efficiency, with major loss of editing at 108 C targets. Virusinduced gene silencing of RIP1 confirmed the altered editing efficiency. Transient introduction of a WT RIP1 allele into rip1 improved the defective RNA editing. The presence of RIP1 in a protein complex along with chloroplast editing factor RARE1 indicates that RIP1 is an important component of the RNA editing apparatus that acts on many chloroplast and mitochondrial C targets.nucleoid | RNA editosome | dual targeting P osttranscriptional C-to-U RNA editing occurs in plastid and plant mitochondrial transcripts. In a typical vascular plant, ∼30 C targets in chloroplasts and over 500 C targets in mitochondria are targeted for editing (1, 2). The majority of the editing events results in encoding of a different amino acid than the one predicted from the genomic sequence. The editing-encoded amino acid is usually more conserved relative to residues present in homologous proteins in other organisms than the genomically encoded amino acid. Because there is presently no known case in which useful genetic variation results from partial editing of a transcript population, the current concept is that editing is a correction mechanism for thymidine-to-cytidine (T-to-C) mutations that have arisen in plant organelle genomes (1,3,4).Little is known about the molecular apparatus that is responsible for recognizing the correct C target for editing and converting it to U, although plant mitochondrial RNA editing was discovered over 20 y ago (5-7). cis-Elements for recognition of editing sites have been identified proximal and 5′ to the nucleotide to be modified (8-10). As few as 22 nt in sequence surrounding the C target is sufficient to specify RNA editing (9). In 2005, a pentatricopeptide repeat (PPR) motif-containing protein termed CRR4 was discovered to ...

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“…RNA gel blot analysis was performed as described in Germain et al (60). Primers used to make the probes are shown in Table S4.…”

Section: Methodsmentioning

confidence: 99%

An RNA recognition motif-containing protein is required for plastid RNA editing in Arabidopsis and maize

Sun

Germain

Giloteaux

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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136

185

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Plant RNA editing modifies cytidines (C) to uridines (U) at specific sites in the transcripts of both mitochondria and plastids. Specific targeting of particular Cs is achieved by pentatricopeptide proteins that recognize cis elements upstream of the C that is edited. Members of the RNA-editing factor interacting protein (RIP) family in Arabidopsis have recently been shown to be essential components of the plant editosome. We have identified a gene that contains a pair of truncated RIP domains (RIP-RIP). Unlike any previously described RIP family member, the encoded protein carries an RNA recognition motif (RRM) at its C terminus and has therefore been named Organelle RRM protein 1 (ORRM1). ORRM1 is an essential plastid editing factor; in Arabidopsis and maize mutants, RNA editing is impaired at particular sites, with an almost complete loss of editing for 12 sites in Arabidopsis and 9 sites in maize. Transfection of Arabidopsis orrm1 mutant protoplasts with constructs encoding a region encompassing the RIP-RIP domain or a region spanning the RRM domain of ORRM1 demonstrated that the RRM domain is sufficient for the editing function of ORRM1 in vitro. According to a yeast two-hybrid assay, ORRM1 interacts selectively with pentatricopeptide transfactors via its RIP-RIP domain. Phylogenetic analysis reveals that the RRM in ORRM1 clusters with a clade of RRM proteins that are targeted to organelles. Taken together, these results suggest that other members of the ORRM family may likewise function in RNA editing.

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Mutational analysis of Arabidopsis chloroplast polynucleotide phosphorylase reveals roles for both RNase PH core domains in polyadenylation, RNA 3′‐end maturation and intron degradation

Cited by 45 publications

References 50 publications

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

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

RIP1, a member of an Arabidopsis protein family, interacts with the protein RARE1 and broadly affects RNA editing

An RNA recognition motif-containing protein is required for plastid RNA editing in Arabidopsis and maize

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