5′ to 3′ exoribonucleolytic activity is a normal component of chloroplast mRNA decay pathways

Drager, Robert G.; Higgs, David A.; Kindle, Karen L.; Stern, David B.

doi:10.1046/j.1365-313x.1999.00546.x

Cited by 61 publications

(56 citation statements)

References 59 publications

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“…It has also been reported that a 5Ј-to 3Ј-exoribonucleolytic activity is involved in RNA degradation pathways in Chlamydomonas chloroplasts (21). Here, we present two lines of evidence strongly in favor of the involvement of a 3Ј-to 5Ј-exonuclease activity in the degradation of plant mitochondrial polyadenylated mRNAs.…”

Section: Discussionsupporting

confidence: 58%

Plant Mitochondrial Polyadenylated mRNAs Are Degraded by a 3′- to 5′-Exoribonuclease Activity, Which Proceeds Unimpeded by Stable Secondary Structures

Gagliardi¹,

Perrin²,

Maréchal-Drouard³

et al. 2001

Journal of Biological Chemistry

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Recently, we and others have reported that mRNAs may be polyadenylated in plant mitochondria, and that polyadenylation accelerates the degradation rate of mRNAs. To further characterize the molecular mechanisms involved in plant mitochondrial mRNA degradation, we have analyzed the polyadenylation and degradation processes of potato atp9 mRNAs. The overall majority of polyadenylation sites of potato atp9 mRNAs is located at or in the vicinity of their mature 3-extremities. We show that a 3-to 5-exoribonuclease activity is responsible for the preferential degradation of polyadenylated mRNAs as compared with non-polyadenylated mRNAs, and that 20 -30 adenosine residues constitute the optimal poly(A) tail size for inducing degradation of RNA substrates in vitro. The addition of as few as seven non-adenosine nucleotides 3 to the poly(A) tail is sufficient to almost completely inhibit the in vitro degradation of the RNA substrate. Interestingly, the exoribonuclease activity proceeds unimpeded by stable secondary structures present in RNA substrates. From these results, we propose that in plant mitochondria, poly(A) tails added at the 3 ends of mRNAs promote an efficient 3-to 5-degradation process.The control of mRNA stability constitutes an important aspect of the regulation of gene expression in all organisms. Polyadenylation of mRNAs is involved in the control of mRNA stability, however, with two significantly different roles depending on the organism or subcellular compartment concerned. Polyadenylation is required for stabilizing virtually all nuclear-encoded mRNAs in all eukaryotes. In contrast, polyadenylation targets mRNAs for degradation in eubacteria (1, 2), chloroplasts (3-6), plant mitochondria (7-9), and trypanosome mitochondria (10). In eubacteria and chloroplasts, degradation of mRNAs is initiated by endonucleolytic cleavages. The resulting fragments are then degraded by 3Ј-to 5Ј-exonuclease activities (1, 5). In both systems, polyadenylation targets endonucleolytic cleavage products for rapid degradation. In chloroplasts, structured mature 3Ј ends are poor substrates for polyadenylation as compared with the internal RNA fragments generated by endonuclease(s) (5, 6).In plant mitochondria, molecular mechanisms involved in mRNA stability or degradation are still poorly understood (11). For instance, no proteins involved in these processes have been formally identified, although several candidate genes have now been identified since the completion of the nuclear genome sequence of Arabidopsis thaliana. Stable secondary structures can be predicted at the 3Ј-extremities of some but not all plant mitochondrial mRNAs (12). When present, these structures appear to be involved in stabilizing the transcripts (13-15) and in the correct processing of 3Ј-extremities rather than being signals for transcription termination (15). Mature 3Ј termini of plant mitochondria mRNAs are generated by 3Ј-processing of longer pre-mRNA molecules (15), and stable mature mRNAs are not constitutively polyadenylated at their 3Ј-extremit...

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Section: Discussionsupporting

confidence: 58%

Plant Mitochondrial Polyadenylated mRNAs Are Degraded by a 3′- to 5′-Exoribonuclease Activity, Which Proceeds Unimpeded by Stable Secondary Structures

Gagliardi¹,

Perrin²,

Maréchal-Drouard³

et al. 2001

Journal of Biological Chemistry

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“…Five additional strains were created that have insertions at the ϩ25 position relative to the mature uidA mRNA 5Ј end. We have previously shown that deletions up to ϩ25 do not affect transcription rates (19) and that small mutations or a polyguanosine insertion within the ϩ1-25 region do not decrease petD RNA levels (21,28). In addition, the sequences around this position are not important for petD mRNA 5Ј end maturation (28), although any insertion into this site translationally inactivates the message for unknown reasons.…”

Section: Fig 2 Sequence Requirements For Ecs-originated Downstream mentioning

confidence: 99%

“…RNA was isolated from 10 ml of cells as previously described (21). For RNA filter hybridizations, 10 g of total RNA was fractionated in 1.2% agarose, 6% formaldehyde gels, transferred to Hybond-N (Amersham Biosciences, Inc.), and cross-linked by UV irradiation.…”

Section: Rna Isolation Filter Hybridizations and Primer Extensionmentioning

confidence: 99%

An mRNA 3′ Processing Site Targets Downstream Sequences for Rapid Degradation in Chlamydomonas Chloroplasts

Hicks¹,

Drager²,

Higgs³

et al. 2002

Journal of Biological Chemistry

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In Chlamydomonas chloroplasts, atpB pre-mRNA matures through a two-step process. Initially, endonuclease cleavage occurs 8 -10 nt downstream of the mature 3 end, which itself lies at the end of a stem-loop-forming inverted repeat (IR) sequence. This intermediate product is then trimmed by a 3 3 5 exonuclease activity. Although the initial endonucleolytic cleavage by definition generates two products, the downstream product of atpB pre-mRNA endonucleolytic processing cannot be detected, even transiently. This product thus appears to be highly unstable, and it can be hypothesized that specific mechanisms exist to prevent its accumulation. In experiments described here, the atpB 3 maturation site was placed upstream of reporter genes in vivo. Constructs containing both the IR and endonuclease cleavage site (ECS) did not accumulate the reporter gene mRNA, whereas constructs containing only the IR did accumulate the reporter mRNA. The ECS alone gave an intermediate result, suggesting that the IR and ECS act synergistically. Additional secondary structures were used to test whether 5 3 3 and/or 3 3 5 exonuclease activities mediated degradation. Because these structures did not prevent degradation, rapid endonucleolytic cleavages most likely trigger RNA destruction after ECS cleavage. On the other hand, fragments resulting from cleavage within the endogenous atpB mRNA could occasionally be detected as antisense transcripts of the adjacent reporter genes. Because endonuclease cleavages are also involved in the 5 maturation of chloroplast mRNAs, where only the downstream cleavage product accumulates, it appears that chloroplast endoribonuclease activities have evolved mechanisms to selectively stabilize different ECS products.Maturation of mRNA can involve multiple steps in both prokaryotic and eukaryotic systems, and results in functional transcripts with a stability and subcellular localization appropriate to their functions. RNA sequence and secondary structure, along with ribonucleases and a variety of accessory factors, are used to achieve these goals. Built into this process is the necessity to recognize nonfunctional RNAs and eliminate them; destruction of nonsense codon-containing mRNAs is a good example of the complexity of such surveillance mechanisms (reviewed in Ref. 1).Our laboratory has focused on 5Ј end and 3Ј end maturation of chloroplast mRNAs, using both vascular plants and the unicellular green alga Chlamydomonas reinhardtii as models. In this respect, chloroplast transcripts have mostly prokaryotic features such as the lack of a trimethylguanosine 5Ј cap, a 3Ј stem-loop-forming inverted repeat (IR) 1 structure, and they are destabilized by polyadenylation (reviewed in Refs. 2 and 3). Processing of chloroplast mRNAs, with rare exceptions, depends on nucleus-encoded proteins, several of which have been identified genetically through screens for plants or Chlamydomonas strains unable to carry out photosynthesis. The phenotypes suggest that defects in intercistronic processing of polycistronic transcripts...

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“…Thus, primary triphosphorylated transcripts are thought to be largely impervious to RNase J exonucleolytic attack. Chloroplasts are known to have a net 59 / 39 RNA degradation pathway (Drager et al 1999;Hicks et al 2002), and RNase J has also been proposed to catalyze 59 end trimming of monocistronic RNAs and polycistronic transcript derivatives, being stalled by gene-specific RNA binding proteins (see Fig. 2 in Barkan 2011).…”

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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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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5′ to 3′ exoribonucleolytic activity is a normal component of chloroplast mRNA decay pathways

Cited by 61 publications

References 59 publications

Plant Mitochondrial Polyadenylated mRNAs Are Degraded by a 3′- to 5′-Exoribonuclease Activity, Which Proceeds Unimpeded by Stable Secondary Structures

Plant Mitochondrial Polyadenylated mRNAs Are Degraded by a 3′- to 5′-Exoribonuclease Activity, Which Proceeds Unimpeded by Stable Secondary Structures

An mRNA 3′ Processing Site Targets Downstream Sequences for Rapid Degradation in Chlamydomonas Chloroplasts

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

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