<i>In vivo</i> evidence for 5′→3′ exoribonuclease degradation of an unstable chloroplast mRNA

Drager, Robert G.; Girard‐Bascou, Jacqueline; Choquet, Yves; Kindle, Karen L.; Stern, David B.

doi:10.1046/j.1365-313x.1998.00016.x

Cited by 105 publications

(100 citation statements)

References 68 publications

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“…Although we had to begin with a relatively unstable (atpB⌬26) mRNA to test added 3Ј-UTR sequences, the results clearly showed a destabilizing effect of poly(A), which was expected on the basis of previous in vitro results, and a probable stimulatory effect of poly(U), which was unexpected. Although we did not measure transcription rates here, we have shown (21,26,32) that 3Ј-UTR manipulations do not affect the atpB or petD promoters. Thus, the absence or decrease in RNA levels must be caused by instability.…”

Section: Discussionmentioning

confidence: 91%

Evidence for in vivo modulation of chloroplast RNA stability by 3′-UTR homopolymeric tails in Chlamydomonas reinhardtii

Komine

Kikis

Schuster

et al. 2002

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

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Polyadenylation of synthetic RNAs stimulates rapid degradation in vitro by using either Chlamydomonas or spinach chloroplast extracts. Here, we used Chlamydomonas chloroplast transformation to test the effects of mRNA homopolymer tails in vivo, with either the endogenous atpB gene or a version of green fluorescent protein developed for chloroplast expression as reporters. Strains were created in which, after transcription of atpB or gfp, RNase P cleavage occurred upstream of an ectopic tRNA Glu moiety, thereby exposing A28, U25A3, [A؉U]26, or A3 tails. Analysis of these strains showed that, as expected, polyadenylated transcripts failed to accumulate, with RNA being undetectable either by filter hybridization or reverse transcriptase-PCR. In accordance, neither the ATPase ␤-subunit nor green fluorescent protein could be detected. However, a U25A3 tail also strongly reduced RNA accumulation relative to a control, whereas the [A؉U] tail did not, which is suggestive of a degradation mechanism that does not specifically recognize poly(A), or that multiple mechanisms exist. With an A 3 tail, RNA levels decreased relative to a control with no added tail, but some RNA and protein accumulation was observed. We took advantage of the fact that the strain carrying a modified atpB gene producing an A28 tail is an obligate heterotroph to obtain photoautotrophic revertants. Each revertant exhibited restored atpB mRNA accumulation and translation, and seemed to act by preventing poly(A) tail exposure. This suggests that the poly(A) tail is only recognized as an instability determinant when exposed at the 3 end of a message.

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Section: Discussionmentioning

confidence: 91%

Evidence for in vivo modulation of chloroplast RNA stability by 3′-UTR homopolymeric tails in Chlamydomonas reinhardtii

Komine

Kikis

Schuster

et al. 2002

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

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“…2 were not due to a peculiarity of the reporter gene chosen, certain constructs were replicated using the aadA coding region instead of uidA. aadA is a commonly used selectable marker gene in Chlamydomonas chloroplasts and has also been used to study RNA cis elements (8,30). Fig.…”

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

confidence: 99%

“…The region spanning from 10 -89 nt downstream of the atpB stop codon contains an IR sequence predicted to form an AU-rich stem-loop structure, and the mature 3Ј end is found 3-4 nt downstream of the IR (12, 13). Deletions that destabilize this structure cause RNA instability (12); however the sequence can be functionally replaced by an IR from spinach chloroplasts (13) or by a polyguanosine tract, which forms a tertiary structure impervious to either 5Ј 3 3Ј or 3Ј 3 5Ј exonucleases (8,14).Both in vitro and in vivo approaches have shown that chloroplast IR sequences do not efficiently terminate transcription, and those tested include Chlamydomonas atpB (13,15). This implies that post-transcriptional 3Ј end maturation is required.…”

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confidence: 99%

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confidence: 99%

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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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“…For instance, analysis of chloroplast reporter gene constructs revealed that, in both C. reinhardtii and tobacco, rbcL transcripts are stabilized via elements within their 5¢ regions in vivo (Salvador 1993;Shiina et al 1998). The same applies to 5¢UTRs of psbD, petD and psbB transcripts in C. reinhardtii, which were shown to contain the target sites for nucleus-encoded factors involved in the gene-specific stabilization of the corresponding mRNAs (Nickelsen et al 1994;Drager et al 1998;Vaistij et al 2000a). For psbD and petD RNAs, site-directed mutagenesis enabled the precise localization of distinct RNA elements mediating RNA stabilization (Higgs et al 1999;Nickelsen et al 1999).…”

Section: Rbps Interacting With 5¢utrsmentioning

confidence: 92%

Chloroplast RNA-binding proteins

Nickelsen

2003

Current Genetics

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Chloroplast gene expression is regulated by nucleus-encoded factors, which mainly act at the posttranscriptional level. Plastid RNA-binding proteins (RBPs) represent good candidates for mediating these functions. The picture emerging from recent analyses is that of a great number of differentially regulated RBPs, which are organized in distinct, spatially separated supramolecular complexes. This reflects the complexity of the regulatory network that underlies the intracellular communication system between the nucleus and the chloroplast.

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In vivo evidence for 5′→3′ exoribonuclease degradation of an unstable chloroplast mRNA

Cited by 105 publications

References 68 publications

Evidence for in vivo modulation of chloroplast RNA stability by 3′-UTR homopolymeric tails in Chlamydomonas reinhardtii

Evidence for in vivo modulation of chloroplast RNA stability by 3′-UTR homopolymeric tails in Chlamydomonas reinhardtii

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

Chloroplast RNA-binding proteins

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