Protein-mediated protection as the predominant mechanism for defining processed mRNA termini in land plant chloroplasts

Most chloroplast mRNAs are processed from larger precursors. Several mechanisms have been proposed to mediate these processing events, including site-specific cleavage and the stalling of exonucleases by RNA structures. A protein barrier mechanism was proposed based on analysis of the pentatricopeptide repeat (PPR) protein PPR10: PPR10 binds two intercistronic regions and impedes 5′- and 3′-exonucleases, resulting in processed RNAs with PPR10 bound at the 5′- or 3′-end. In this study, we provide evidence that protein barriers are the predominant means for defining processed mRNA termini in chloroplasts. First, we map additional RNA termini whose arrangement suggests biogenesis via a PPR10-like mechanism. Second, we show that the PPR protein HCF152 binds to the immediate 5′- or 3′-termini of transcripts that require HCF152 for their accumulation, providing evidence that HCF152 defines RNA termini by blocking exonucleases. Finally, we build on the observation that the PPR10 and HCF152 binding sites accumulate as small chloroplast RNAs to infer binding sites of other PPR proteins. We show that most processed mRNA termini are represented by small RNAs whose sequences are highly conserved. We suggest that each such small RNA is the footprint of a PPR-like protein that protects the adjacent RNA from degradation

RBCS1A and RBCS3B, two major members within the Arabidopsis RBCS multigene family, function to yield sufficient Rubisco content for leaf photosynthetic capacity

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (RBCS) is encoded by a nuclear RBCS multigene family in many plant species. The contribution of the RBCS multigenes to accumulation of Rubisco holoenzyme and photosynthetic characteristics remains unclear. T-DNA insertion mutants of RBCS1A (rbcs1a-1) and RBCS3B (rbcs3b-1) were isolated among the four Arabidopsis RBCS genes, and a double mutant (rbcs1a3b-1) was generated. RBCS1A mRNA was not detected in rbcs1a-1 and rbcs1a3b-1, while the RBCS3B mRNA level was suppressed to ∼20% of the wild-type level in rbcs3b-1 and rbcs1a3b-1 leaves. As a result, total RBCS mRNA levels declined to 52, 79, and 23% of the wild-type level in rbcs1a-1, rbcs3b-1, and rbcs1a3b-1, respectively. Rubisco contents showed declines similar to total RBCS mRNA levels, and the ratio of Rubisco-nitrogen to total nitrogen was 62, 78, and 40% of the wild-type level in rbcs1a-1, rbcs3b-1, and rbcs1a3b-1, respectively. The effects of RBCS1A and RBCS3B mutations in rbcs1a3b-1 were clearly additive. The rates of CO2 assimilation at ambient CO2 of 40 Pa were reduced with decreased Rubisco contents in the respective mutant leaves. Although the RBCS composition in the Rubisco holoenzyme changed, the CO2 assimilation rates per unit of Rubisco content were the same irrespective of the genotype. These results clearly indicate that RBCS1A and RBCS3B contribute to accumulation of Rubisco in Arabidopsis leaves and that these genes work additively to yield sufficient Rubisco for photosynthetic capacity. It is also suggested that the RBCS composition in the Rubisco holoenzyme does not affect photosynthesis under the present ambient [CO2] conditions

Micro-algae come of age as a platform for recombinant protein production

A complete set of genetic tools is still being developed for the micro-alga Chlamydomonas reinhardtii. Yet even with this incomplete set, this photosynthetic single-celled plant has demonstrated significant promise as a platform for recombinant protein expression. In recent years, techniques have been developed that allow for robust expression of genes from both the nuclear and plastid genome. With these advances, many research groups have examined the pliability of this and other micro-algae as biological machines capable of producing recombinant peptides and proteins. This review describes recent successes in recombinant protein production in Chlamydomonas, including production of complex mammalian therapeutic proteins and monoclonal antibodies at levels sufficient for production at economic parity with existing production platforms. These advances have also shed light on the details of algal protein production at the molecular level, and provide insight into the next steps for optimizing micro-algae as a useful platform for the production of therapeutic and industrially relevant recombinant proteins

Importance des facteurs codés par le génome nucléaire pour l'expression du génome chloroplastique (l'exemple du gène chloroplastique petA chez Chlamydomonas reinhardtii)

L'expression des gènes chloroplastiques dépend de nombreux facteurs codés par le génome nucléaire. Chez C. reinhardtii, l'analyse de mutants a révélé deux caractéristiques majeures de ces facteurs : ils sont généralement impliqués dans les étapes post-transcriptionnelles de l'expression d'un unique gène chloroplastique. Ainsi, MCA1 et TCA1 sont deux facteurs nucléaires respectivement impliqués dans la stabilisation et la traduction de l'ARNm chloroplastique petA codant le cytochrome f. Ces deux facteurs ont été clonés : TCA1 est une protéine pionnière tandis que MCA1 est une protéine PPR (PentatricoPeptide Repeats).L'analyse de transformants exprimant des niveaux variés de MCA1 ou TCA1 a montré que ces facteurs sont limitants pour l'expression du gène petA et qu'ils peuvent jouer un rôle régulateur. Parallèlement, le mode d'action de MCA1 et TCA1 a été analysé in vivo en modifiant la région 5' non traduite de petA par mutagenèse dirigée. Nous avons ainsi montré que MCA1 reconnaît une cible localisée à l'extrémité 5' de l'ARNm et protège le transcrit d'une dégradation ribonucléolytique 5'-3'. Contrairement aux autres facteurs de stabilisation analysés jusqu'ici, MCA1 n'est pas strictement requis pour la traduction. Néanmoins, il pourrait interagir avec TCA1, dont une cible putative a été identifiée à proximité de celle de MCA1.Enfin, j'ai identifié le mutant nucléaire crp4, affecté dans la maturation de l'extrémité 3' de l'ARNm petA. CRP4 pourrait être un composant de la machinerie générale de maturation/dégradation des ARN chloroplastiques car il semble impliqué dans la maturation de nombreux transcrits ainsi que dans une voie de dégradation des ARN liée à la traduction.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Les traumatismes du genou aux urgences du CHU de Rennes (bilan à un an de la mise en place d'une filière de prise en charge)

La prise en charge de la traumatologie du genou aux urgences est une activité fréquente. Le diagnostic est difficile en aigu, il faut souvent pouvoir réévaluer le genou traumatisé à distance quand l'œdème et la douleur ont diminué. La filière de réévaluation des genoux traumatiques reçus aux urgences par le service de médecine du sport a été mise en place en septembre 2007 au CHU de Rennes, en lien avec les services d'orthopédie et imagerie. L'étude a donc consisté à: décrire la prise en charge initiale des traumatismes du genou aux urgences, étudier le devenir des patients après leur passage aux urgences, décrire la consultation de réévaluation en médecine du sport, comparer enfin l'examen clinique du genou entre les urgences et la médecine du sport. 766 patients de 16 à 55 ans reçus aux urgences pour traumatisme du genou ont été inclus dans l'étude dont 17,5% revus en médecine du sport. Aux urgences, l'examen était souvent incomplet, probablement à cause de la spécificité de l'exercice en médecine d'urgences, de l'impotence fonctionnelle et la douleur en aigu, mais aussi de la connaissance partielle, notamment des internes intervenant aux urgences concernant certaines pathologies du genou. L'étude a permis de montrer l'intérêt de la filière médecine du sport pour redresser le diagnostic notamment d'instabilité patellaire, guider les examens d'imagerie et parfois envisager un avis chirurgical. Pour améliorer la prise en charge des patients, on propose de: standardiser l'examen clinique du genou en l'adaptant à la médecine d'urgence (par une fiche), effectuer une formation continue de l'ensemble des intervenants dans la prise en charge des traumatismes du genou en urgence, réévaluer de façon continue et régulière la filière mise en place en proposant de réaliser une évaluation des pratiques professionnelles.The care of knee traumatology in emergency room is a frequent activity. Diagnosis is difficult in acute, it is often necessary to revalue injured knee remotely when œdema and pain decreased. The field of revaluation of the traumatic knees received in emergency room by the service of sports medicine was created in September, 2007 in the University hospital of Rennes, in connection with the services of orthopaedics and radiology. The study consisted of: describing the initial care of the knee injuries in emergency room, studying the future of the patients after their coming in emergency room, describing the consultation of revaluation in sports medicine, comparing the clinical examination of the knee between emergency room and sports medicine. 766 patients from 16 to 55 years old, coming in emergency room to knee injuries were included in the study, whose 17,5% revised in sports medicine. In emergency room, the medical examination was often incomplete, probably because of the specificity of the exercise of medical emergencies, the functional disability and the acute pain. But also of the partial knowledge, in particular housemen in emergency room concerning certain pathologies of the knee. The study allowed to show the interest of the field of sports medicine to right the diagnosis in particular of patellar instability, guide the imaging and sometimes envisage a surgical opinion. To improve the care of the patients, we suggest: standardizing the clinical examination of the knee by adapting it to the emergency medicine (by an index card), making an in-service training of all the medical who care the knee injuries in emergency room, revaluating on a continuous and regular way the field of sports medicine by offering to conduct an assessment of professional practices.RENNES1-BU Santé (352382103) / SudocSudocFranceF

Dual functions of the nucleus-encoded factor TDA1 in trapping and translation activation of atpA transcripts in Chlamydomonas reinhardtii chloroplasts

International audienc

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

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 5′→3′ 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