The crystal structure of Pyrococcus abyssi tRNA (uracil-54, C5)-methyltransferase provides insights into its tRNA specificity

The 5-methyluridine is invariably found at position 54 in the TΨC loop of tRNAs of most organisms. In Pyrococcus abyssi, its formation is catalyzed by the S-adenosyl-l-methionine-dependent tRNA (uracil-54, C5)-methyltransferase (PabTrmU54), an enzyme that emerged through an ancient horizontal transfer of an RNA (uracil, C5)-methyltransferase-like gene from bacteria to archaea. The crystal structure of PabTrmU54 in complex with S-adenosyl-l-homocysteine at 1.9 Å resolution shows the protein organized into three domains like Escherichia coli RumA, which catalyzes the same reaction at position 1939 of 23S rRNA. A positively charged groove at the interface between the three domains probably locates part of the tRNA-binding site of PabTrmU54. We show that a mini-tRNA lacking both the D and anticodon stem-loops is recognized by PabTrmU54. These results were used to model yeast tRNAAsp in the PabTrmU54 structure to get further insights into the different RNA specificities of RumA and PabTrmU54. Interestingly, the presence of two flexible loops in the central domain, unique to PabTrmU54, may explain the different substrate selectivities of both enzymes. We also predict that a large TΨC loop conformational change has to occur for the flipping of the target uridine into the PabTrmU54 active site during catalysis

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The DEAH-box helicase Prp43 is a key player in pre-mRNA splicing as well as the maturation of rRNAs. The exact modus operandi of Prp43 and of all other spliceosomal DEAH-box RNA helicases is still elusive. Here, we report crystal structures of Prp43 complexes in different functional states and the analysis of structure-based mutants providing insights into the unwinding and loading mechanism of RNAs. The Prp43ATP-analogRNA complex shows the localization of the RNA inside a tunnel formed by the two RecA-like and C-terminal domains. In the ATP-bound state this tunnel can be transformed into a groove prone for RNA binding by large rearrangements of the C-terminal domains. Several conformational changes between the ATP- and ADP-bound states explain the coupling of ATP hydrolysis to RNA translocation, mainly mediated by a ?-turn of the RecA1 domain containing the newly identified RF motif. This mechanism is clearly different to those of other RNA helicases

5-methylcytosine in RNA: detection, enzymatic formation and biological functions

The nucleobase modification 5-methylcytosine (m5C) is widespread both in DNA and different cellular RNAs. The functions and enzymatic mechanisms of DNA m5C-methylation were extensively studied during the last decades. However, the location, the mechanism of formation and the cellular function(s) of the same modified nucleobase in RNA still remain to be elucidated. The recent development of a bisulfite sequencing approach for efficient m5C localization in various RNA molecules puts ribo-m5C in a highly privileged position as one of the few RNA modifications whose detection is amenable to PCR-based amplification and sequencing methods. Additional progress in the field also includes the characterization of several specific RNA methyltransferase enzymes in various organisms, and the discovery of a new and unexpected link between DNA and RNA m5C-methylation. Numerous putative RNA:m5C-MTases have now been identified and are awaiting characterization, including the identification of their RNA substrates and their related cellular functions. In order to bring these recent exciting developments into perspective, this review provides an ordered overview of the detection methods for RNA methylation, of the biochemistry, enzymology and molecular biology of the corresponding modification enzymes, and discusses perspectives for the emerging biological functions of these enzymes

Proteomic and 3D structure analyses highlight the C/D box snoRNP assembly mechanism and its control

International audienceIn vitro, assembly of box C/D small nucleolar ribonucleoproteins (snoRNPs) involves the sequential recruitment of core proteins to snoRNAs. In vivo, however, assembly factors are required (NUFIP, BCD1, and the FISP90-R2TP complex), and it is unknown whether a similar sequential scheme applies. In this paper, we describe systematic quantitative stable isotope labeling by amino acids in cell culture proteomic experiments and the crystal structure of the core protein Snu 13p/15.5K bound to a fragment of the assembly factor Rsa1p/NUFIP. This revealed several unexpected features: (a) the existence of a protein-only pre-snoRNP complex containing five assembly factors and two core proteins, 15.5K and Nop58; (b) the characterization of ZNHIT3, which is present in the protein-only complex but gets released upon binding to C/D snoRNAs; (c) the dynamics of the R2TP complex, which,appears a to load/unload RuvBL AAA(+) adenosine triphosphatase from pre-snoRNPs; and (d) a potential mechanism for preventing premature activation of snoRNP catalytic activity. These data provide a framework for understanding the assembly of box C/D snoRNPs

Comparative Structural Analysis of Human DEAD-Box RNA Helicases

DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members

Native Variants of the MRB1 Complex Exhibit Specialized Functions in Kinetoplastid RNA Editing

We want to thank Kathy Kyler for editing this manuscript, Ken Stuart for supplying monoclonal antisera against RECC subunits, and Laurie K. Read for her gift of polyclonal antisera against GAP1 and RGG2. Funding: National Science Foundation Grant No. NSF1122109 (PI: J.Cruz-Reyes.). NIH/National Institute of Allergies and Infectious Diseases R01 AI088011 (PI: Blaine Mooers). Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103640. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Adaptation and survival of Trypanosoma brucei requires editing of mitochondrial mRNA by uridylate (U) insertion and deletion. Hundreds of small guide RNAs (gRNAs) direct the mRNA editing at over 3,000 sites. RNA editing is controlled during the life cycle but the regulation of substrate and stage specificity remains unknown. Editing progresses in the 3’ to 5’ direction along the pre-mRNA in blocks, each targeted by a unique gRNA. A critical editing factor is the mitochondrial RNA binding complex 1 (MRB1) that binds gRNA and transiently interacts with the catalytic RNA editing core complex (RECC). MRB1 is a large and dynamic complex that appears to be comprised of distinct but related subcomplexes (termed here MRBs). MRBs seem to share a ‘core’ complex of proteins but differ in the composition of the ‘variable’ proteins. Since some proteins associate transiently the MRBs remain imprecisely defined. MRB1 controls editing by unknown mechanisms, and the functional relevance of the different MRBs is unclear. We previously identified two distinct MRBs, and showed that they carry mRNAs that undergo editing. We proposed that editing takes place in the MRBs because MRBs stably associate with mRNA and gRNA but only transiently interact with RECC, which is RNA free. Here, we identify the first specialized functions in MRBs: 1) 3010-MRB is a major scaffold for RNA editing, and 2) REH2-MRB contains a critical trans-acting RNA helicase (REH2) that affects multiple steps of editing function in 3010-MRB. These trans effects of the REH2 include loading of unedited mRNA and editing in the first block and in subsequent blocks as editing progresses. REH2 binds its own MRB via RNA, and conserved domains in REH2 were critical for REH2 to associate with the RNA and protein components of its MRB. Importantly, REH2 associates with a ~30 kDa RNA-binding protein in a novel ~15S subcomplex in RNA-depleted mitochondria. We use these new results to update our model of MRB function and organization.Yeshttp://www.plosone.org/static/editorial#pee

Roles of local factors in the distribution and the persistence of the European beech communities (Fagus sylvatica) at the margin of the distribution range

En marge arrière d’aire de répartition, les espèces persistent ou disparaissent en réponse aux changements climatiques et autres facteurs globaux. Les refuges climatiques ont joué un rôle majeur dans la persistance de la biodiversité au cours des périodes de transitions climatiques majeures et une compréhension mécaniste claire de leur fonctionnement est primordiale pour la conservation des populations de marge arrière. En limite géographique sud-ouest de son aire de distribution, des fragments de hêtre (Fagus sylvatica) persistent dans des micro-refuges en raison de facteurs locaux. Connue pour sa biodiversité remarquable, la vallée du Ciron offre un laboratoire naturel idéal pour l'étude des refuges climatiques et la prédiction de leur évolution dans un climat régional de plus en plus chaud et sec. Cette thèse interdisciplinaire est centrée sur l’étude des populations de hêtres et des communautés associées par des approches in situ et ex situ combinant écologie des communautés et physique de l’environnement. Nos résultats mettent en évidence une forte originalité floristique des communautés du sous-bois des populations à hêtre de marge chaude, ainsi que l’existence de variations microclimatiques à fine échelle, fonction notamment de la topographie, de la présence de la rivière et de la localisation dans le paysage. Nous montrons que la régénération du hêtre dépend du microclimat et qu’elle est modulée par des interactions allélopathiques. De plus, notre étude met en avant le rôle facilitateur de la canopée forestière ainsi que l’importance du rayonnement solaire, au moins dans des conditions où ce dernier impacte peu la disponibilité en eau. L’ensemble des résultats suggèrent la persistance probable du hêtre dans la vallée du Ciron ainsi qu’une potentialité de présence plus importante dans le Bassin aquitain.In the margin of their distribution range, species can persist or disappear in response to climate change and global factors. Climate refugia have played a major part in the persistence of biodiversity during periods of fundamental climatic transitions, and a clear mechanistic understanding of their functioning is essential for the conservation of marginal populations. In the southwestern geographical limit, fragments of beech populations (Fagus sylvatica) persist in micro-refugia due to the importance of local factors. Known for its remarkable biodiversity, the Ciron valley provides an ideal natural laboratory for studying climate refugia and predicting their evolution in an increasingly hot and dry regional climate. This interdisciplinary thesis involves the study of beech populations and associated communities through in-situ and ex-situ approaches combining community ecology and environmental physics. Our results underline the strong floristic originality of the undergrowth communities of warm-margin beech populations, and reveal the existence of fine-scale microclimatic variations caused, in particular, by the local topography, the presence of the river, and location in the landscape. We show that beech regeneration depends on microclimate and that it is modulated by allelopathic interactions. Moreover, our research highlights the facilitating role of the forest canopy and the importance of solar radiation, in conditions where it has low impact on water availability. The overall results suggest the likely persistence of beech in the Ciron Valley and a greater potential presence of beech in the Aquitanian Basin

Roles of local factors in the distribution and the persistence of the European beech communities (Fagus sylvatica) at the margin of the distribution range

En marge arrière d’aire de répartition, les espèces persistent ou disparaissent en réponse aux changements climatiques et autres facteurs globaux. Les refuges climatiques ont joué un rôle majeur dans la persistance de la biodiversité au cours des périodes de transitions climatiques majeures et une compréhension mécaniste claire de leur fonctionnement est primordiale pour la conservation des populations de marge arrière. En limite géographique sud-ouest de son aire de distribution, des fragments de hêtre (Fagus sylvatica) persistent dans des micro-refuges en raison de facteurs locaux. Connue pour sa biodiversité remarquable, la vallée du Ciron offre un laboratoire naturel idéal pour l'étude des refuges climatiques et la prédiction de leur évolution dans un climat régional de plus en plus chaud et sec. Cette thèse interdisciplinaire est centrée sur l’étude des populations de hêtres et des communautés associées par des approches in situ et ex situ combinant écologie des communautés et physique de l’environnement. Nos résultats mettent en évidence une forte originalité floristique des communautés du sous-bois des populations à hêtre de marge chaude, ainsi que l’existence de variations microclimatiques à fine échelle, fonction notamment de la topographie, de la présence de la rivière et de la localisation dans le paysage. Nous montrons que la régénération du hêtre dépend du microclimat et qu’elle est modulée par des interactions allélopathiques. De plus, notre étude met en avant le rôle facilitateur de la canopée forestière ainsi que l’importance du rayonnement solaire, au moins dans des conditions où ce dernier impacte peu la disponibilité en eau. L’ensemble des résultats suggèrent la persistance probable du hêtre dans la vallée du Ciron ainsi qu’une potentialité de présence plus importante dans le Bassin aquitain.In the margin of their distribution range, species can persist or disappear in response to climate change and global factors. Climate refugia have played a major part in the persistence of biodiversity during periods of fundamental climatic transitions, and a clear mechanistic understanding of their functioning is essential for the conservation of marginal populations. In the southwestern geographical limit, fragments of beech populations (Fagus sylvatica) persist in micro-refugia due to the importance of local factors. Known for its remarkable biodiversity, the Ciron valley provides an ideal natural laboratory for studying climate refugia and predicting their evolution in an increasingly hot and dry regional climate. This interdisciplinary thesis involves the study of beech populations and associated communities through in-situ and ex-situ approaches combining community ecology and environmental physics. Our results underline the strong floristic originality of the undergrowth communities of warm-margin beech populations, and reveal the existence of fine-scale microclimatic variations caused, in particular, by the local topography, the presence of the river, and location in the landscape. We show that beech regeneration depends on microclimate and that it is modulated by allelopathic interactions. Moreover, our research highlights the facilitating role of the forest canopy and the importance of solar radiation, in conditions where it has low impact on water availability. The overall results suggest the likely persistence of beech in the Ciron Valley and a greater potential presence of beech in the Aquitanian Basin