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David B Stern

Publications and source records attributed to David B Stern.

At least 19 recordsLinked to original sources
PubMed2006

The nucleus-encoded factor MCD4 participates in degradation of nonfunctional 3' UTR sequences generated by cleavage of pre-mRNA in Chlamydomonas chloroplasts.

The 3' maturation of chloroplast pre-mRNAs in Chlamydomonas proceeds via endonucleolytic cleavage, exonucleolytic trimming of the upstream cleavage product, and rapid degradation of the downstream moiety. However, the cis elements and trans factors remain to be characterized in detail. In the case of atpB, a 300 nucleotide processing determinant (PD), consisting of an inverted repeat (IR) and endonuclease cleavage site (ECS), directs 3' maturation. To further characterize the PD, 15 variants were examined in vivo in ectopic contexts. This revealed that the IR, and nucleotides 15-37 downstream of the ECS stimulate processing. A candidate trans factor for 3' maturation was subsequently functionally analyzed. This factor is encoded by the nuclear locus MCD4, and the mcd4 mutant was known to accumulate abnormally 3'-processed chloroplast mRNAs. When the mcd4 mutation was crossed into strains containing reporter genes with insertions of several PD versions, processing was reduced in some cases. This caused accumulation of RNA sequences downstream of the PD, which are normally degraded. From these data, it can be suggested that MCD4 facilitates the endonucleolytic cleavage step in 3' end maturation of atpB and perhaps other mRNAs, by interacting with the IR, RNA downstream of the IR, or with proteins bound there.

3' Untranslated Regions↗
PubMed2006

The sulfur acclimation SAC3 kinase is required for chloroplast transcriptional repression under sulfur limitation in Chlamydomonas reinhardtii.

Sulfur (S) deprivation responses have been studied extensively in algae and land plants; however, little is known of the signals that link perception of S status to chloroplast gene expression. Here, we have compared the chloroplast S limitation response in WT vs. sac1 and sac3 sulfur acclimation mutants of the green alga Chlamydomonas reinhardtii. We provide evidence that in the WT, chloroplast transcriptional activity rapidly decreases after removal of S from the medium, leading to reduced transcript accumulation. This decrease correlates with reduced abundance of a sigma70-like factor, Sig1, which is most likely the unique chloroplast transcription specificity factor. We further show that reduced transcription activity and diminished Sig1 accumulation are mediated by the SAC3 gene product, a putative Snf1-type Ser/Thr kinase previously shown to have both positive and negative effects on nuclear gene expression. Inclusion of the protein kinase inhibitor 6-dimethylaminopurine during S limitation yielded a pattern of expression that was largely similar to that seen in the sac3 mutant, lending support to the hypothesis that Sac3 kinase activation leads to transcriptional repression and Sig1 proteolysis. The finding that Sac3 regulates chloroplast gene expression suggests that it has a previously unknown role in integrating the S limitation response in multiple subcellular compartments.

Animals↗
PubMed2006

Adaptive evolution of chloroplast genome structure inferred using a parametric bootstrap approach.

BACKGROUND: Genome rearrangements influence gene order and configuration of gene clusters in all genomes. Most land plant chloroplast DNAs (cpDNAs) share a highly conserved gene content and with notable exceptions, a largely co-linear gene order. Conserved gene orders may reflect a slow intrinsic rate of neutral chromosomal rearrangements, or selective constraint. It is unknown to what extent observed changes in gene order are random or adaptive. We investigate the influence of natural selection on gene order in association with increased rate of chromosomal rearrangement. We use a novel parametric bootstrap approach to test if directional selection is responsible for the clustering of functionally related genes observed in the highly rearranged chloroplast genome of the unicellular green alga Chlamydomonas reinhardtii, relative to ancestral chloroplast genomes. RESULTS: Ancestral gene orders were inferred and then subjected to simulated rearrangement events under the random breakage model with varying ratios of inversions and transpositions. We found that adjacent chloroplast genes in C. reinhardtii were located on the same strand much more frequently than in simulated genomes that were generated under a random rearrangement processes (increased sidedness; p < 0.0001). In addition, functionally related genes were found to be more clustered than those evolved under random rearrangements (p < 0.0001). We report evidence of co-transcription of neighboring genes, which may be responsible for the observed gene clusters in C. reinhardtii cpDNA. CONCLUSION: Simulations and experimental evidence suggest that both selective maintenance and directional selection for gene clusters are determinants of chloroplast gene order.

Adaptation, Physiological↗
PubMed2006

Chlamydomonas reinhardtii encodes a single sigma70-like factor which likely functions in chloroplast transcription.

Chlamydomonas reinhardtii EST clones encoding a protein highly similar to prokaryotic sigma factors and plant sigma-like factors (SLFs) were used to isolate a BAC clone containing the full-length gene CrRpoD. The gene is likely to be single-copy, in contrast to small gene families encoding SLFs in plants. The CrRpoD mRNA comprises 3,033 nt with an open reading frame of 2,256 nt, encoding a putative protein of 752 amino acids with a molecular mass of 80.2 kDa. The sequence contains conserved regions 2-4 typically found in sigma factors, and an unusually long amino terminal extension, which by in silico analysis has properties of a chloroplast transit peptide. Expression of CrRpoD was confirmed by immunodetection of a 85 kDa polypeptide in a preparation enriched for chloroplast proteins. To demonstrate functionality in transcription initiation, a recombinant CrRpoD-thioredoxin fusion protein was reconstituted with E. coli RNA polymerase core enzyme and tested in vitro. This chimeric holoenzyme specifically bound the spinach psbA and Chlamydomonas rrn16 promoters in gel mobility shift assays and exhibited specific transcription initiation from the same two promoters, providing evidence for the role of CrRpoD as a functional transcription factor.

Algal Proteins↗
PubMed2006

Nuclear suppressors define three factors that participate in both 5' and 3' end processing of mRNAs in Chlamydomonas chloroplasts.

Chloroplast RNA processing and degradation are orchestrated by nucleus-encoded factors. Although several transcript-specific factors have been identified, those involved in global RNA metabolism have mostly remained elusive. Using Chlamydomonas reinhardtii, we have identified three pleiotropic nuclear mutations, mcd3, mcd4 and mcd5, which cause quantitative variation between polycistronic transcripts and accumulation of transcripts with novel 3' ends. The mcd3, mcd4 and mcd5 mutants were initially isolated as photoautotrophic suppressors of the petD 5' mutants LS2 and LS6, which harbour four nucleotide linker-scanning mutations near the 5' end of the mature transcript. The LS mutants accumulate 1-3% of the wild-type (WT) petD mRNA level and no cytochrome b6/f complex subunit IV, which is the petD gene product and required for photosynthesis. Each suppressor restores approximately 15% of the WT petD mRNA and subunit IV levels. Genetic analysis showed mcd4 to be recessive, and suggested that MCD4 interacts with the petD mRNA stability factor MCD1. To assess the specificity of mcd3, mcd4 and mcd5, transcripts from 32 chloroplast genes were analysed by RNA filter hybridizations. mcd3 and mcd4 displayed aberrant transcript patterns for 17 genes, whereas only three were altered in mcd5. Since the mutations affect multiple RNAs in a variety of ways, our data suggest that MCD3, MCD4 and MCD5 may participate in a series of multiprotein complexes responsible for RNA maturation and degradation in Chlamydomonas chloroplasts.

Algal Proteins↗
PubMed2005

A spontaneous tRNA suppressor of a mutation in the Chlamydomonas reinhardtii nuclear MCD1 gene required for stability of the chloroplast petD mRNA.

Numerous nuclear gene products are required for the correct expression of organellar genes. One such gene in the green alga Chlamydomonas reinhardtii is MCD1, whose product is required for stability of the chloroplast-encoded petD mRNA. In mcd1 mutants, which are non-photosynthetic, petD mRNA is degraded by a 5'-3' exonuclease activity, resulting in a failure to synthesize its product, subunit IV of the cytochrome b (6)/f complex. Here, we report the sequence of the wild-type MCD1 gene, which encodes a large and novel putative protein. Analysis of three mutant alleles showed that two harbored large deletions, but that one allele, mcd1-2, had a single base change resulting in a nonsense codon near the N-terminus. This same mutant allele can be suppressed by a second-site mutation in the nuclear MCD2 gene, whereas mcd2-1 cannot suppress the deletion in mcd1-1 (Esposito,D. Higgs,D.C. Drager,R.G. Stern, D.B. and Girard-Bascou,J. (2001) Curr. Genet., 39, 40-48). We report the cloning of mcd2-1, and show that the mutation lies in a tRNA(Ser)(CGA), which has been modified to translate the nonsense codon in mcd1-2. We discuss how the existence of a large tRNA(Ser) gene family may permit this suppression without pleiotropic consequences.

Algal Proteins↗
PubMed2005

RNR1, a 3'-5' exoribonuclease belonging to the RNR superfamily, catalyzes 3' maturation of chloroplast ribosomal RNAs in Arabidopsis thaliana.

Arabidopsis thaliana chloroplasts contain at least two 3' to 5' exoribonucleases, polynucleotide phosphorylase (PNPase) and an RNase R homolog (RNR1). PNPase has been implicated in both mRNA and 23S rRNA 3' processing. However, the observed maturation defects do not affect chloroplast translation, suggesting that the overall role of PNPase in maturation of chloroplast rRNA is not essential. Here, we show that this role can be largely ascribed to RNR1, for which homozygous mutants germinate only on sucrose-containing media, and have white cotyledons and pale green rosette leaves. Accumulation of chloroplast-encoded mRNAs and tRNAs is unaffected in such mutants, suggesting that RNR1 activity is either unnecessary or redundant for their processing and turnover. However, accumulation of several chloroplast rRNA species is severely affected. High-resolution RNA gel blot analysis, and mapping of 5' and 3' ends, revealed that RNR1 is involved in the maturation of 23S, 16S and 5S rRNAs. The 3' extensions of the accumulating 5S rRNA precursors can be efficiently removed in vitro by purified RNR1, consistent with this view. Our data suggest that decreased accumulation of mature chloroplast ribosomal RNAs leads to a reduction in the number of translating ribosomes, ultimately compromising chloroplast protein abundance and thus plant growth and development.

Arabidopsis↗
PubMed2005

Microarray analysis confirms the specificity of a Chlamydomonas reinhardtii chloroplast RNA stability mutant.

The expression of chloroplast and mitochondrial genes depends on nucleus-encoded proteins, some of which control processing, stability, and/or translation of organellar RNAs. To test the specificity of one such RNA stability factor, we used two known Chlamydomonas reinhardtii nonphotosynthetic mutants carrying mutations in the Mcd1 nuclear gene (mcd1-1 and mcd1-2). We previously reported that these mutants fail to accumulate the chloroplast petD mRNA and its product, subunit IV of the cytochrome b6/f complex, which is essential for photosynthesis. Such mutants are generally presumed to be gene specific but are not tested rigorously. Here, we have used microarray analysis to assess changes in chloroplast, mitochondrial, and nuclear RNAs, and since few other RNAs were significantly altered in these mutants, conclude that Mcd1 is indeed specifically required for petD mRNA accumulation. In addition, a new unlinked nuclear mutation was discovered in mcd1-2, which greatly reduced chloroplast atpA mRNA accumulation. Genetic analyses showed failure to complement mda1-ncc1, where atpA-containing transcripts are similarly affected (D. Drapier, J. Girard-Bascou, D.B. Stern, F.-A. Wollman [2002] Plant J 31: 687-697), and we have named this putative new allele mda1-2. We conclude that DNA microarrays are efficient and useful for characterizing the specificity of organellar RNA accumulation mutants.

Animals↗
PubMed2005

Beyond complementation. Map-based cloning in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is an excellent model system for plant biologists because of its ease of manipulation, facile genetics, and the ability to transform the nuclear, chloroplast, and mitochondrial genomes. Numerous forward genetics studies have been performed in Chlamydomonas, in many cases to elucidate the regulation of photosynthesis. One of the resultant challenges is moving from mutant phenotype to the gene mutation causing that phenotype. To date, complementation has been the primary method for gene cloning, but this is impractical in several situations, for example, when the complemented strain cannot be readily selected or in the case of recessive suppressors that restore photosynthesis. New tools, including a molecular map consisting of 506 markers and an 8X-draft nuclear genome sequence, are now available, making map-based cloning increasingly feasible. Here we discuss advances in map-based cloning developed using the strains mcd4 and mcd5, which carry recessive nuclear suppressors restoring photosynthesis to chloroplast mutants. Tools that have not been previously applied to Chlamydomonas, such as bulked segregant analysis and marker duplexing, are being implemented to increase the speed at which one can go from mutant phenotype to gene. In addition to assessing and applying current resources, we outline anticipated future developments in map-based cloning in the context of the newly extended Chlamydomonas genome initiative.

Animals↗
PubMed2004

Antisense transcript and RNA processing alterations suppress instability of polyadenylated mRNA in chlamydomonas chloroplasts.

In chloroplasts, the control of mRNA stability is of critical importance for proper regulation of gene expression. The Chlamydomonas reinhardtii strain Delta26pAtE 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 Delta26pAtE, 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 3' poly(A) tail. Two exceptions were spa19 and spa23, which maintained unusual heteroplasmic chloroplast genomes. One genome type, termed PS+, conferred photosynthetic competence by contributing to the stability of atpB mRNA; the other, termed PS-, 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 PS+ 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 PS+ genome-containing cells to S1 nuclease digestion. To obtain additional evidence for antisense RNA function in chloroplasts, we used strain Delta26, in which atpB mRNA is unstable because of the lack of a 3' 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 PS+ 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 3'-->5' exonuclease activity, a phenomenon that may occur naturally in the symmetrically transcribed and densely packed chloroplast genome.

Animals↗
PubMed2004

Analysis of developing maize plastids reveals two mRNA stability classes correlating with RNA polymerase type.

The plastid genome is transcribed by two distinct RNA polymerases, the PEP encoded by the plastid genome and the NEP encoded in the nucleus. Initial models of plastid transcription held that the NEP is responsible for the transcription of housekeeping genes needed early in development, and that the PEP transcribes genes required for photosynthesis. Recently, this model was challenged by the discovery that all plastid genes are transcribed by NEP in PEP-deficient tobacco plastids, suggesting that mRNA turnover may have a strong role in previously observed transcription patterns. In this study, we provide evidence that the NEP enzyme level decreases as plastids mature. In contrast, production of mRNAs by NEP increases as plastids mature, yet their accumulations remain constant. These results suggest that as plastids mature NEP may become more active, and that mRNA turnover varies between transcripts synthesized by NEP and PEP.

DNA-Directed RNA Polymerases↗
PubMed2004

Short dispersed repeats in the Chlamydomonas chloroplast genome are collocated with sites for mRNA 3' end formation.

The Chlamydomonas reinhardtii chloroplast genome possesses thousands of small dispersed repeats (SDRs), which are of unknown function. Here, we used the petA gene as a model to investigate the role of SDRs in mRNA 3' end formation. In wild-type cells, petA mRNA accumulated as a major 1.3-kb transcript, whose 3' end was mapped to the distal end of a predicted stem-loop structure. To determine whether this stem-loop was required for petA mRNA stability, a series of deletions was constructed. These deletion strains accumulated a variety of petA mRNAs, for which approximate 3' ends were deduced. These 3' ends were found to flank stem-loop structures, many of which were formed partially or completely from inverted copies of SDRs. All strains accumulated wild-type levels of cytochrome f, demonstrating that alternative 3' termini are compatible with efficient translation. The ability to form alternative mRNA termini using SDRs lends additional flexibility to the chloroplast gene expression apparatus and thus could confer an evolutionary advantage.

Algal Proteins↗
PubMed2004

Cooperation of endo- and exoribonucleases in chloroplast mRNA turnover.

Chloroplasts were acquired by eukaryotic cells through endosymbiosis and have retained their own gene expression machinery. One hallmark of chloroplast gene regulation is the predominance of posttranscriptional control, which is exerted both at the gene-specific and global levels. This review focuses on how chloroplast mRNA stability is regulated, through an examination of poly(A)-dependent and independent pathways. The poly(A)-dependent pathway is catalyzed by polynucleotide phosphorylase (PNPase), which both adds and degrades destabilizing poly(A) tails, whereas RNase II and PNPase may both participate in the poly(A)-independent pathway. Each system is initiated through endonucleolytic cleavages that remove 3' stem-loop structures, which are catalyzed by the related proteins CSP41a and CSP41b and possibly an RNase E-like enzyme. Overall, chloroplasts have retained the prokaryotic endonuclease-exonuclease RNA degradation system despite evolution in the number and character of the enzymes involved. This reflects the presence of the chloroplast within a eukaryotic host and the complex responses that occur to environmental and developmental cues.

Chloroplasts↗
PubMed2003

Divalent metal-dependent catalysis and cleavage specificity of CSP41, a chloroplast endoribonuclease belonging to the short chain dehydrogenase/reductase superfamily.

CSP41 is a ubiquitous chloroplast endoribonuclease belonging to the short chain dehydrogenase/reductase (SDR) superfamily. To help elucidate the role of CSP41 in chloroplast gene regulation, the mechanisms that determine its substrate recognition and catalytic activity were investigated. A divalent metal is required for catalysis, most probably to provide a nucleophile for cleavage 5' to the phosphodiester bond, and may also participate in cleavage site selection. This requirement distinguishes CSP41 from other Rossman fold-containing proteins from the SDR superfamily, including several RNA-binding proteins and endonucleases. CSP41 is active only in the presence of MgCl2 and CaCl2. Although Mg2+- and Ca2+-activated CSP41 cleave at identical sites in the single-stranded regions of a stem-loop-containing substrate, Mg2+-activated CSP41 was also able to cleave within the double-stranded region of the stem-loop. Mixed metal experiments with Mg2+ and Ca2+ suggest that CSP41 contains a single divalent metal-binding site which is non-selective, since Mn2+, Co2+ and Zn2+ compete with Mg2+ for binding, although there is no activity in their presence. Using site-directed mutagenesis, we identified three residues, Asn71, Asp89 and Asp103, which may form the divalent metal-binding pocket. The activation constant for Mg2+ (K(A,Mg) = 2.1 +/- 0.4 mM) is of the same order of magnitude as the stromal Mg2+ concentrations, which fluctuate between 0.5 and 10 mM as a function of light and of leaf development. These changes in stromal Mg2+ concentration may regulate CSP41 activity, and thus cpRNA stability, during plant development.

Base Sequence↗
PubMed2003

Maize BMS cultured cell lines survive with massive plastid gene loss.

As part of developing an ex planta model system for the study of maize plastid and mitochondrial gene expression, a series of established Black Mexican Sweet (BMS) suspension cell lines was characterized. Although the initial assumption was that their organelle biochemistry would be similar enough to normal in planta cells to facilitate future work, each of the three lines was found to have plastid DNA (ptDNA) differing from control maize plants, in one case lacking as much as 70% of the genome. The other two BMS lines possessed either near-wild-type ptDNA or displayed an intermediate state of gene loss, suggesting that these clonal lines are rapidly evolving. Gene expression profiles of BMS cells varied dramatically from those in maize leaf chloroplasts, but resembled those of albino plants lacking plastid ribosomes. In spite of lacking most plastid gene expression and apparently mature rRNAs, BMS cells appear to import proteins from the cytoplasm in a normal manner. The regions retained in BMS ptDNAs point to a set of tRNA genes universally preserved among even highly reduced plastid genomes, whereas the other preserved regions may illuminate which plastid genes are truly indispensable for plant cell survival.

Biolistics↗
PubMed2003

Secondary structures common to chloroplast mRNA 3'-untranslated regions direct cleavage by CSP41, an endoribonuclease belonging to the short chain dehydrogenase/reductase superfamily.

CSP41 (chloroplast stem-loop-binding protein of 41 kDa), a chloroplast endonuclease belonging to the SDR superfamily, preferentially cleaves stem-loop-containing RNAs in vitro. This potentially directs it to the 3'-ends of mature chloroplast mRNAs, which generally possess such structures. To understand the basis for this discrimination, the RNA elements directing CSP41 cleavage of petD RNA in vitro were dissected. Substrates containing fully base-paired stem-loops were optimal substrates, whereas deletion of part of the stem-loop decreased activity by 100-fold, and deletion of the distal arm of the stem-loop abolished cleavage, even in substrates containing the primary CSP41 cleavage site. Competition assays showed that the decrease in activity resulted from decreased affinity for the RNA by CSP41. Mutations of the residues at the scissile bond and mutations and deletions at the terminal loop of the stem had a moderate effect on activity but no effect on cleavage site specificity, suggesting that CSP41 has no sequence specificity. Titration of ethidium bromide into the assay decreased activity to a basal level of approximately 18%, and introduction of a single base bulge into either arm of the stem-loop decreased cleavage at the primary cleavage site by up to 70%. This suggests that changing the structure of the helical stem has a mild effect on activity. Deletion analysis of CSP41 suggests that the specificity domain lies in the first 73 amino acids of the protein, a domain that also contains a putative dehydrogenaselike mononucleotide binding motif. These results are consistent with a broad role for CSP41 in the degradation of stem-loop-containing mRNAs.

3' Untranslated Regions↗
PubMed2003

In vivo evidence for the prokaryotic model of extended codon-anticodon interaction in translation initiation.

Initiation codon context is an important determinant of translation initiation rates in both prokaryotes and eukaryotes. Such sequences include the Shine- Dalgarno ribosome-binding site, as well as other motifs surrounding the initiation codon. One proposed interaction is between the base immediately preceding the initiation codon (-1 position) and the nucleotide 3' to the tRNAf(Met) anticodon, at position 37. Adenine is conserved at position 37, and a uridine at -1 has been shown in vitro to favor initiation. We have tested this model in vivo, by manipulating the chloroplast of the green alga Chlamydomonas reinhardtii, where the translational machinery is prokaryotic in nature. We show that translational defects imparted by mutations at the petA -1 position can be suppressed by compensatory mutations at position 37 of an ectopically expressed tRNA(fMet). The mutant tRNAs are fully aminoacylated and do not interfere with the translation of other proteins. Although this extended base pairing is not an absolute requirement for initiation, it may convey added specificity to transcripts carrying non-standard initiation codons, and/or preserve translational fidelity under certain stress conditions.

Algal Proteins↗