Caractérisation moléculaire et fonctionnelle de la protéine DYW1 dans le complexe d'édition chloroplastique d'Arabidopsis thaliana

Dans les organites des plantes, l édition de l ARN consiste majoritairement en une désamination de cytidines à des sites spécifiques de l ARNm. Trente-quatre sites d édition ont été découverts dans les transcrits chloroplastiques d Arabidopsis thaliana et plus de 500 dans les transcrits mitochondriaux. Depuis 2005, beaucoup de facteurs d édition ont été trouvés. La majorité de ces protéines appartiennent à la famille des PentatricoPeptide Repeat (PPR). Parmi ces PPR, certaines contiennent un domaine DYW possédant de faibles similarités avec les cytidines désaminases (CDA), alors que d autres en sont dénuées, générant un doute sur le fait qu il ait une activité CDA. Le gène At1g47580 (DYW1) code une protéine unique chez Arabidopsis thaliana contenant seulement un domaine DYW. Il a été proposé que DYW1 puisse interagir avec les PPR ne contenant pas de domaine DYW, pour former un hétérodimère, capable d éditer spécifiquement un site. En accord avec cette hypothèse, nous avons montré que DYW1 agissait sur le même site d édition que CRR4, une PPR sans domaine DYW, et que ces protéines interagissaient in vivo. De plus, nous avons montré que DYW1 remplaçait les parties manquantes de CRR4 pour l édition. Pour obtenir plus d informations sur la fonction du domaine DYW, des mutations ont été introduites dans DYW1. Nous avons montré que la signature CDA dans les protéines DYW était essentielle à l édition de l ARN ainsi qu à l interaction avec les ions zinc. Les données sont en accord avec l hypothèse d une activité CDA dans le domaine DYW. Cependant, aucune activité CDA n a pu être mise à jour in vitro. Il est vraisemblable qu au moins un cofacteur doive encore être identifié.In plant organelles, RNA editing mostly takes the form of conversions of cytidines to uridines at specific sites in mRNAs. Thirty-four editing sites have been found in Arabidopsis thaliana chloroplast transcripts and more than 500 sites in mitochondrial transcripts. Since 2005, lots of proteins have been found to act as RNA editing factors. Most of these proteins belong to the PentatricoPeptide Repeat (PPR) family. Amongst these PPR, some contain a DYW domain with weak similarity to cytidine deaminases (CDA), whilst others lack such a domain, creating doubts about whether this domain is required for editing. The gene At1g47580 (named DYW1) encodes a protein in Arabidopsis thaliana that contains only a DYW domain. Our initial hypothesis was that DYW1 might interact with PPR proteins that lack a DYW domain, in order to form a heterodimer, able to perform site-specific editing. In accordance with this hypothesis, we discovered that DYW1 is involved in editing the same site as CRR4, a PPR lacking a DYW domain, and that these two proteins interact together in vivo. Moreover, we showed that DYW1 replaces all the missing parts of CRR4 for editing. So, other partners need to be hypothesized for other DYW-lacking editing factors if this hypothesis is to be generalized. The highly conserved residues making up the CDA signature in DYW proteins were found to be essential for RNA editing and are also required for zinc binding, which is a known characteristic of CDAs. All the data so far are consistent with the DYW domain being (part of) a CDA activity; nevertheless, no CDA activity could be detected in vitro. It is likely that at least one required cofactor remains to be identified.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

RNA Editing at Baseline and Following Endoplasmic Reticulum Stress.

RNA sequences are expected to be identical to the DNA template. However, some RNA processing steps, such as RNA editing, can lead to differences in the RNA sequence that affect the fate of the RNA transcripts or the resultant proteins. My thesis focuses on the regulation of the canonical A-to-G editing and non-canonical RNA-DNA sequence Differences (RDD). My work contributed to the identification of RDDs throughout the human transcriptome. We identified all 12 types of single base differences across multiple individuals and various tissue types. We also detected peptides matching the RDD-encoded sequences suggesting that RDDs are translated into proteins. In subsequent work, we found that the non-canonical RDDs are found in nascent RNA. Through the use of nuclear run-on assays, we found that RDD occurs within seconds of exiting the RNA polymerase complex. Chapters 4 and 5 discuss my study of canonical A-to-G editing mediated by Adenosine Deaminase Acting on RNA (ADAR). First, we found that A-to-G editing levels differ across individuals. I searched for and identified genetic variants whose alleles are associated with editing levels of sites in the same gene. These data demonstrate that ADAR editing is cis regulated and can lead to individual variability in editing levels. Furthermore, by utilizing individual variability in editing and studying the relationship between editing sites, I learned how ADAR edits multiple sites in a given transcript. My data support a model where ADAR edits multiple sites along one side of a double-stranded RNA structure. To learn about the biological significance of RNA editing, I focused on endoplasmic reticulum (ER) stress. I found that editing levels change following ER stress suggesting that these RNA processing steps play a role in the ER stress response. Together, this work sheds light on the regulation of RNA editing and RDDs in the human transcriptome and aids in the understanding of how these processes may play a role in cellular response to stress.PhDHuman GeneticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/116721/1/allirich_1.pd

Spliceosomal intron and spliceosome evolution in Giardia lamblia and other diplomonads

Spliceosomal introns interrupt protein coding genes in all characterized eukaryotic nuclear genomes and are removed by a large RNA-protein complex termed the spliceosome. Diplomonads are diverse unicellular eukaryotes that display compact genomes with few spliceosomal introns. My thesis objectives were to explore spliceosomal intron and spliceosome diversity as well as RNA processing mechanisms in the diplomonads Giardia lamblia and Spironucleus spp. Surprisingly, G. lamblia was found to contain a proportionally large number of fragmented spliceosomal introns that are spliced in trans from separate pre-mRNA molecules. Next, both evolutionarily divergent and conventional spliceosomal small nuclear RNAs were identified in G. lamblia and Spironucleus spp. and an RNA 3ʹ end motif was determined to be involved in processing of both non-coding RNAs and trans-introns in G. lamblia. These findings shed light on spliceosome and spliceosomal intron evolution in eukaryotes undergoing severe genomic reduction and potentially complete loss of their spliceosomal introns.University of Lethbridge (SGS Graduate Fellowship), Natural Sciences and Engineering Research Council of Canada (NSERC) (Discovery Grant and Alexander Graham Bell Canada Graduate Scholarships)

DNA hydrolysis and genome editing applications of GIY-YIG family homing endonucleases

The ability to manipulate complex genomes in a precise manner is essential for studying biological processes in model systems, engineering plant strains for agriculture, or advancing human cellular therapies to treat diseases. Genomic alterations are most efficient when a double-strand DNA break is introduced at the loci where the modification is desired. Different classes of naturally occurring DNA endonucleases, including homing endonucleases, have therefore been explored as candidates for genome modification studies as they target long stretches of DNA. Homing endonucleases are mobile genetic elements whose biological role is to introduce site-specific double-strand breaks into naïve genomes, ultimately resulting in the selfish propagation of their own genes. Consequently, homing endonucleases are an ideal enzymatic system whose natural properties can be exploited to manipulate genes. In the present studies, I examine the cleavage mechanism of GIY-YIG family homing endonucleases, as until now the method by which they hydrolyze DNA has remained poorly understood. Using the GIY-YIG homing endonuclease I-BmoI as a model system, I investigate the amino acid, nucleotide, and divalent metal ion requirements of the GIY-YIG nuclease domain to generate a double-strand break. I specifically test models of hydrolysis by which enzymes with a single active site could nick both strands of DNA, and determine that I-BmoI functions as a monomer throughout the reaction pathway. Furthermore, I demonstrate that the nuclease domain itself has weak binding affinity, is tethered to DNA by a high affinity binding domain, and must reposition across each strand through a series of protein and substrate conformational changes to facilitate DNA hydrolysis. To explore the relevance of GIY-YIG homing endonucleases as genome editing reagents, I fused the nuclease domain of I-TevI to three different re-targetable DNA-binding platforms utilized in the field. The engineered nucleases developed within the present studies are mechanistically distinct from established technologies, as they function as monomers and cleave DNA at a preferred sequence motif. I therefore envision that the engineered GIY-YIG nucleases may circumvent complications associated with established technologies, and provide an alternative and potentially safer set of genome editing reagents

Current Frontiers and Perspectives in Cell Biology

A numerous internationally renowned authors in the pages of this book present the views of the fields of cell biology and their own research results or review of current knowledge. Chapters are divided into five sections that are dedicated to cell structures and functions, genetic material, regulatory mechanisms, cellular biomedicine and new methods in cell biology. Multidisciplinary and often quite versatile approach by many authors have imposed restrictions of this classification, so it is certain that many chapters could belong to the other sections of this book. The current frontiers, on the manner in which they described in the book, can be a good inspiration to many readers for further improving, and perspectives which are highlighted can be seen in many areas of fundamental biology, biomedicine, biotechnology and other applications of knowledge of cell biology. The book will be very useful for beginners to gain insight into new area, as well as experts to find new facts and expanding horizons

Γενετική και μοριακή μελέτη της ετεροκαρύωσης στον φυτοπαθογόνο μύκητα verticillium

Πληθυσμιακή ανάλυση: Μελετήθηκε ένας πληθυσμός του V. dahliae από την Κρήτη, εμπλουτισμένος με διεθνή στελέχη από πολυάριθμους ξενιστές, και κατέστη εφικτή η συσχέτιση των VCGs με μορφολογικά και φυτοπαθολογικά χαρακτηριστικά των στελεχών του. Γενετική ανάλυση: Η κατάταξη σε VCGs συγκρίθηκε με τις ζώνες αντεπίδρασης (barrages, ένας ευρέως χρησιμοποιούμενος για ορισμένους μύκητες φαινότυπος ασυμβατότητας μεταξύ στελεχών φυσικού τύπου) και αποδείχθηκε ότι τα δύο φαινόμενα διαφέρουν ως προς τον γενετικό τους έλεγχο. Κυτταροβιολογική/φυσιολογική ανάλυση: Αποδείχθηκε πως τα κονίδια του V. dahliae σε φάση βλάστησης συντήκονται συχνά μεταξύ τους μέσω του σχηματισμού σωλήνων αναστόμωσης κονιδίων (CATs) υπό περιβαλλοντικές συνθήκες που διαφέρουν από τις αντίστοιχες για τους μύκητες-μοντέλα. Γονιδιωματική ανάλυση και μοριακή/λειτουργική μελέτη: Με βάση εκφυλισμένους εκκινητές για συντηρημένες περιοχές γονιδίων het εντοπίσθηκαν τμήματα ομόλογων γονιδίων het σε είδη Verticillium, κλωνοποιήθηκαν για προσδιορισμό της αλληλουχίας τους και χρησιμοποιήθηκαν ως ιχνηθέτες σε υβριδισμούς κατά Southern για τη μελέτη της κατανομής του γονιδίου het-c1, και σε σάρωση γονιδιακής βιβλιοθήκης του V. dahliae για την ανάκτηση των γονιδίων mod-E και un-24

Phytochrome genes in higher plants: Structure,expression, and evolution

© 2006 Springer. All Rights Reserved. Phytochromes play critical roles in monitoring light quantity, quality, and periodicity in plants and they relay this photosensory information to a large number of signaling pathways that regulate plant growth and development. Given these complex functions, it is not surprising that the phytochrome apoproteins are encoded by small multigene families and that different forms of phytochrome regulate different aspects of photomorphogenesis. Over the course of the last decade, progress has been made in defining the number, molecular properties, and biological activities of the photoreceptors that constitute a plant R/FR sensing system. This chapter summarizes our current understanding of the structure of the genes that encode the phytochrome apoproteins (the PHY genes), the expression patterns of those genes, the nature of the phytochrome apoprotein family, and PHY gene evolution in seed plants. Phytochrome was discovered and its basic photochemical properties were first described through physiological studies of light-sensitive seed germination and photoperiodic effects on flowering (Borthwick, et al., 1948, Borthwick, et al., 1952). The pigment itself was initially isolated from extracts of dark-grown (etiolated) plant tissue in 1959 (Butler, et al., 1959), but it was not until much later that phytochrome was purified to homogeneity in an undegraded form (Vierstra and Quail, 1983). DNA sequences of gene and cDNA clones for oat etiolated-tissue spectroscopically in planta and purified in its native form, this dark-tissue phytochrome (now called phyA) remains the most completely biochemically and spectroscopically characterized form of the receptor. At various times throughout the first 40 years of the study of the abundant etiolated-tissue phytochrome, evidence for the presence and activity of additional forms of phytochrome, often referred to as green-tissue or light-stable phytochromes, was obtained. Initially, in physiological experiments, it was sometimes not possible to correlate specific in vivo phytochrome activities with the phytochrome provided the first complete descriptions of the apoprotein (Hershey et al., 1985). Because it accumulates to levels that permit it to be assayed known spectroscopic properties of the molecule. Later, direct evidence for multiple species of phytochrome in plants and in plant extracts was obtained using both spectroscopic and immunochemical methods (reviewed in Pratt, 1995). The molecular identities of these additional phytochrome forms were ultimately deduced from cDNA clones that were isolated by nucleic acid similarity to etiolated-tissue phytochrome sequences (Sharrock and Quail, 1989). More recently, analysis of a large number of complete and partial PHY gene or cDNA sequences from a broad sampling of plant phylogenetic groups and sequencing of several plant genomes have resulted in a much clearer and more general picture of what constitutes a higher plant R/FR photoreceptor family. It is likely that the major types of long-wavelength photosensing pigments have now been identified and the challenge that lies ahead is to understand how the signalling mechanisms, expression patterns, and interactions of these molecules contribute to plant responses to the R/FR environment. Extending the investigation of phytochrome gene families and their functions to additional angiosperm and gymnosperm genera will be an integral component of this effort and of our ability to utilize this growing understanding of phytochrome function to modify the agricultural properties of plants and to better understand the history of land plants

Area-wide Integrated Pest Management

Over 98% of sprayed insecticides and 95% of herbicides reach a destination other than their target species, including non-target species, air, water and soil. The extensive reliance on insecticide use reduces biodiversity, contributes to pollinator decline, destroys habitat, and threatens endangered species. This book offers a more effective application of the Integrated Pest Management (IPM) approach, on an area-wide (AW) or population-wide (AW-IPM) basis, which aims at the management of the total population of a pest, involving a coordinated effort over often larger areas. For major livestock pests, vectors of human diseases and pests of high-value crops with low pest tolerance, there are compelling economic reasons for participating in AW-IPM. This new textbook attempts to address various fundamental components of AW-IPM, e.g. the importance of relevant problem-solving research, the need for planning and essential baseline data collection, the significance of integrating adequate tools for appropriate control strategies, and the value of pilot trials, etc. With chapters authored by 184 experts from more than 31 countries, the book includes many technical advances in the areas of genetics, molecular biology, microbiology, resistance management, and social sciences that facilitate the planning and implementing of area-wide strategies. The book is essential reading for the academic and applied research community as well as national and regional government plant and human/animal health authorities with responsibility for protecting plant and human/animal health