Specific sequence elements in the 5′ untranslated regions of rbcL and atpB gene mRNAs stabilize transcripts in the chloroplast of Chlamydomonas reinhardtii

Anthonisen, Inger Lill; Salvador, María Luisa Esteban; Klein, U.

doi:10.1017/s1355838201001479

Cited by 29 publications

(32 citation statements)

References 56 publications

(38 reference statements)

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“…In accordance with previous studies (Dron et al, 1982;Anthonisen et al, 2001), we found a single rbcL transcript in Chlamydomonas, and the presence of a 59 triphosphate indicates that it is not a processed form (see Supplemental Figure 3 online). It is still detectable in the mutant, consistent with our finding that the mutation prevents the stabilization of the rbcL transcript, but not its transcription.…”

Section: Molecular Target and Mechanism Of Action Of A Conserved Ppr supporting

confidence: 92%

MRL1, a Conserved Pentatricopeptide Repeat Protein, Is Required for Stabilization of rbcL mRNA in Chlamydomonas and Arabidopsis

et al. 2010

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We identify and functionally characterize MRL1, a conserved nuclear-encoded regulator of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. The nonphotosynthetic mrl1 mutant of Chlamydomonas reinhardtii lacks ribulose-1,5-bisphosphate carboxylase/oxygenase, and the resulting block in electron transfer is partially compensated by redirecting electrons toward molecular oxygen via the Mehler reaction. This allows continued electron flow and constitutive nonphotochemical quenching, enhancing cell survival during illumination in spite of photosystem II and photosystem I photoinhibition. The mrl1 mutant transcribes rbcL normally, but the mRNA is unstable. The molecular target of MRL1 is the 59 untranslated region of rbcL. MRL1 is located in the chloroplast stroma, in a high molecular mass complex. Treatment with RNase or deletion of the rbcL gene induces a shift of the complex toward lower molecular mass fractions. MRL1 is well conserved throughout the green lineage, much more so than the 10 other pentatricopeptide repeat proteins found in Chlamydomonas. Depending upon the organism, MRL1 contains 11 to 14 pentatricopeptide repeats followed by a novel MRL1-C domain. In Arabidopsis thaliana, MRL1 also acts on rbcL and is necessary for the production/stabilization of the processed transcript, presumably because it acts as a barrier to 59>39 degradation. The Arabidopsis mrl1 mutant retains normal levels of the primary transcript and full photosynthetic capacity.

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Section: Molecular Target and Mechanism Of Action Of A Conserved Ppr supporting

confidence: 92%

MRL1, a Conserved Pentatricopeptide Repeat Protein, Is Required for Stabilization of rbcL mRNA in Chlamydomonas and Arabidopsis

et al. 2010

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“…In Chlamydomonas, short nucleotide sequences required for the stable accumulation of several transcripts have been identified by site-directed mutagenesis of their 5ЈUTRs (2,30,49), such as the stability element I (nucleotides 1 to 9) that is required for stable accumulation of the petD mRNA (30). Here, we show that the first 29 nucleotides from the petD mRNA are sufficient to confer an MCD1-dependent stability to a chimeric transcript.…”

Section: Mca1 Codes For a Ppr Proteinmentioning

confidence: 76%

Molecular Identification and Function of cis- and trans-Acting Determinants for petA Transcript Stability in Chlamydomonas reinhardtii Chloroplasts

Loiselay

Gumpel

Girard‐Bascou

et al. 2008

Molecular and Cellular Biology

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In organelles, the posttranscriptional steps of gene expression are tightly controlled by nucleus-encoded factors, most often acting in a gene-specific manner. Despite the molecular identification of a growing number of factors, their mode of action remains largely unknown. In the green alga Chlamydomonas reinhardtii, expression of the chloroplast petA gene, which codes for cytochrome f, depends on two specific nucleus-encoded factors. MCA1 controls the accumulation of the transcript, while TCA1 is required for its translation. We report here the cloning of MCA1, the first pentatricopeptide repeat protein functionally identified in this organism. By chloroplast transformation with modified petA genes, we investigated the function of MCA1 in vivo. We demonstrate that MCA1 acts on the very first 21 nucleotides of the petA 5 untranslated region to protect the whole transcript from 533 degradation but does not process the 5 end of the petA mRNA. MCA1 and TCA1 recognize adjacent targets and probably interact together for efficient expression of petA mRNA. MCA1, although not strictly required for translation, shows features of a translational enhancer, presumably by assisting the binding of TCA1 to its own target. Conversely, TCA1 participates to the full stabilization of the transcript through its interaction with MCA1.Organelle genomes have retained a limited set of genes from their prokaryotic ancestor, the expression of which is tightly controlled by the nucleus. Within organelles, mRNAs may undergo cis or trans splicing, editing, endo-and exonucleolytic cleavage, and 5Ј-and 3Ј-end processing. Their stabilization, translation, and degradation are highly regulated (reviewed in references 3, 5, 23, 26, and 63). Each of these posttranscriptional steps depends on nucleus-encoded factors. Strikingly, most are gene specific, one factor being required for the expression of one or a few organelle mRNAs. Altogether, several hundreds of nucleus-encoded factors may be required for the proper expression of the organelle genome (3, 65).The green alga Chlamydomonas reinhardtii and the yeast Saccharomyces cerevisiae have been instrumental in the identification of these factors. Genetic analyses of nuclear mutants, defective for the expression of single organelle genes, have emphasized the existence of two major classes of trans-acting factors. Some are required for the proper maturation and stabilization of specific organellar transcripts (reviewed in references 3, 44, and 48), while others are required for their translation (reviewed in references 3, 23, and 78). Among the numerous nucleus-encoded factors involved in the stabilization of specific chloroplast transcripts identified in Chlamydomonas (14,16,28,33,35,36,39,45,66), only three have been cloned up to now-NAC2, MCD1, and MBB1-that, respectively, control psbD, petD, and psbB transcript stability (6,46,71). They act on the 5Ј untranslated regions (5ЈUTRs) of their target transcripts and protect them from 5Ј33Ј ribonucleolytic degradation (14,50,71), but the molecular bas...

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“…Subsequently, RT-PCR was performed using a one-step assay with primers YK11þKY09 to detect the atpB coding region or for another chloroplast gene, petD, as a control. For both genes, clear Artificially polyadenylated atpB mRNA is unstable because the 39 end is susceptible to the attack by 39/59 exoribonuclease(s), such as PNPase, whereas its 59 end would be protected from 59/39 exoribonuclease(s) by the stem-loop structure in its 59 UTR (Anthonisen et al, 2001) and possibly a protein encoded by the nuclear gene Mdb1, which is mutated in the atpB mRNA stability mutant Thm24 (Drapier et al, 1992;see text). But in spa19/ 23, artificially polyadenylated atpB sense mRNA transcribed from the PSÿ genome, and atpB antisense mRNA transcribed from the PSþ genome (top), may form a dsRNA duplex (center).…”

Section: Antisense Transcripts For Atpb Accumulate and Form Duplexes mentioning

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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Specific sequence elements in the 5′ untranslated regions of rbcL and atpB gene mRNAs stabilize transcripts in the chloroplast of Chlamydomonas reinhardtii

Cited by 29 publications

References 56 publications

MRL1, a Conserved Pentatricopeptide Repeat Protein, Is Required for Stabilization of rbcL mRNA in Chlamydomonas and Arabidopsis

MRL1, a Conserved Pentatricopeptide Repeat Protein, Is Required for Stabilization of rbcL mRNA in Chlamydomonas and Arabidopsis

Molecular Identification and Function of cis- and trans-Acting Determinants for petA Transcript Stability in Chlamydomonas reinhardtii Chloroplasts

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

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