Mutations in nine chloroplast loci of Chlamydomonas affecting different photosynthetic functions.

Shepherd, Hurley S.; Boynton, John E.; Gillham, Nicholas W.

doi:10.1073/pnas.76.3.1353

Cited by 100 publications

(60 citation statements)

References 22 publications

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“…Extracts for in vitro assays were obtained from strain CC406. Strain CC373 (ac-u-c-2-25), which carries a deletion in the atpB gene and downstream region (18), was used for chloroplast transformations. Cells were grown under constant light in Tris-acetate-phosphate medium (19).…”

Section: Methodsmentioning

confidence: 99%

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: Methodsmentioning

confidence: 99%

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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“…In fact, the DNA filter blots in Figure 2B reveal a few additional faintly hybridizing bands, which may signify ongoing evolution of the genomes. A subtle selective pressure might be exerted by the fact that chloroplast ATPase mutants are sensitive to high light (Majeran et al, 2001), although we have maintained these strains under the same conditions that are not deleterious to the well-characterized atpB mutant CC-373 (Shepherd et al, 1979).…”

Section: Dynamic Alteration Of Cpdna In Spa Strainsmentioning

confidence: 99%

Antisense Transcript and RNA Processing Alterations Suppress Instability of Polyadenylated mRNA in Chlamydomonas Chloroplasts

et al. 2004

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In chloroplasts, the control of mRNA stability is of critical importance for proper regulation of gene expression. The Chlamydomonas reinhardtii strain D26pAtE is engineered such that the atpB mRNA terminates with an mRNA destabilizing polyadenylate tract, resulting in this strain being unable to conduct photosynthesis. A collection of photosynthetic revertants was obtained from D26pAtE, and gel blot hybridizations revealed RNA processing alterations in the majority of these suppressor of polyadenylation (spa) strains, resulting in a failure to expose the atpB mRNA 39 poly(A) tail. Two exceptions were spa19 and spa23, which maintained unusual heteroplasmic chloroplast genomes. One genome type, termed PSþ, conferred photosynthetic competence by contributing to the stability of atpB mRNA; the other, termed PSÿ, was required for viability but could not produce stable atpB transcripts. Based on strand-specific RT-PCR, S1 nuclease protection, and RNA gel blots, evidence was obtained that the PSþ genome stabilizes atpB mRNA by generating an atpB antisense transcript, which attenuates the degradation of the polyadenylated form. The accumulation of double-stranded RNA was confirmed by insensitivity of atpB mRNA from PSþ genome-containing cells to S1 nuclease digestion. To obtain additional evidence for antisense RNA function in chloroplasts, we used strain D26, in which atpB mRNA is unstable because of the lack of a 39 stem-loop structure. In this context, when a 121-nucleotide segment of atpB antisense RNA was expressed from an ectopic site, an elevated accumulation of atpB mRNA resulted. Finally, when spa19 was placed in a genetic background in which expression of the chloroplast exoribonuclease polynucleotide phosphorylase was diminished, the PSþ genome and the antisense transcript were no longer required for photosynthesis. Taken together, our results suggest that antisense RNA in chloroplasts can protect otherwise unstable transcripts from 39!59 exonuclease activity, a phenomenon that may occur naturally in the symmetrically transcribed and densely packed chloroplast genome.

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“…Chlamydomonas strain CC-373 (Shepherd et al, 1979) was transformed using the DuPont particle delivery system with 2 µg of either pDG2, pDGS pDGSpG, or pDAAD DNA as described by Stern et al (1991). Phototrophic transformants were streaked for single colonies until homoplasmic strains had been recovered, and their chloroplast genome structures were verified by appropriate DNA filter hybridizations.…”

Section: Chloroplast Transformationmentioning

confidence: 99%

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

et al. 1998

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SummaryThe acetate-requiring Chlamydomonas reinhardtii nuclear mutant F16 harbors the mutation mcd1-1 and fails to accumulate the cytochrome b6/f complex. The primary defect of mcd1-1 was determined to be the instability of petD mRNA, which encodes subunit IV of the complex. Chimeric reporter genes introduced by chloroplast transformation demonstrated that the determinant of petD mRNA instability in the mcd1-1 background is located in the 5Ј untranslated region (UTR). However, when this 5Ј UTR was present downstream of other sequences in dicistronic or chimeric transcripts, the RNAs were no longer destabilized in the mcd1-1 background. Together, these results suggest that the 5Ј end of the petD 5Ј UTR interacts with the MCD1 product. The insertion of a polyguanosine sequence into the petD 5Ј UTR fused to a reporter gene allowed accumulation of the reporter gene transcript in the mutant background. Since polyguanosine forms a structure that is known to impede exonucleases, these data provide in vivo evidence that petD mRNA can be degraded by 5Ј→3Ј exoribonuclease activity. Furthermore, the data support a model in which protein binding to the petD 5Ј UTR protects the mRNA from 5Ј→3Ј degradation in wild-type cells.

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Mutations in nine chloroplast loci of Chlamydomonas affecting different photosynthetic functions.

Abstract: Chloroplast components known to be coded by chloroplast DNA

Cited by 100 publications

References 22 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

Antisense Transcript and RNA Processing Alterations Suppress Instability of Polyadenylated mRNA in Chlamydomonas Chloroplasts

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

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