In all the three domains of life, most RNAs undergo post transcriptional modifications both on the bases as well as the ribose sugars of the individual ribonucleotides. 2'-O-methylation of ribose sugars and isomerization of Uridines to Pseudouridines are two most predominant modifications in rRNAs and tRNAs across all domains of life. Besides 2'-O-methylation of ribose sugars, methylation of pseudouridine (Ø) at position 54 of tRNA, producing m1Ø, is a hallmark of many archaeal species but the specific methylase involved in the formation of this modification had yet to be characterized. A comparative genomics analysis had previously identified COG1901 (DUF358), part of the SPOUT superfamily, as a candidate for this missing methylase family. To test this prediction, the COG1901 encoding gene, HVO_1989, was deleted from the Haloferax volcanii genome. Analyses of modified base contents indicated that while m1Ø was present in tRNA extracted from the wild-type strain, it was absent from tRNA extracted from the mutant strain. Expression of the gene encoding COG1901 from Halobacterium sp. NRC-1, VNG1980C, complemented the m1Ø minus phenotype of the ÄHVO_1989 strain. This in vivo validation was extended with in vitro tests. Using the COG1901 recombinant enzyme from Methanocaldococcus jannaschii (Mj1640), purified enzyme Pus10 from M. jannaschii and full-size tRNA transcripts or TØ-arm (17-mer) fragments as substrates, the sequential pathway of m1Ø54 formation in Archaea was reconstituted. The methylation reaction is AdoMet-dependent. The efficiency of the methylase reaction depended on the identity of the residue at position 55 of the TØ-loop. The presence of Ø55 allowed the efficient conversion of Ø54 to m1Ø54, whereas in the presence of C55 the reaction was rather inefficient and no methylation reaction occurred if a purine was present at this position. These results led to renaming the Archaeal COG1901 members as TrmY proteins. Another aim of this study was to investigate the mechanism of target RNA recruitment to a box C/D sRNP. From data obtained, we have made the following hypothesis- aNop5p, either alone or as a heterodimer with Fibrillarin, binds to single stranded bulges and loops of target RNA. This aNop5p bound target is then hybridized to an assembling guide sRNP complex containing the guide RNA and L7Ae or guide RNA, L7Ae and aNop5p. If the guide:target sequences are complementary to each other, they hybridize and the target nucleotide gets modified. We also think that post modification, the guide and target strands separate, the core proteins rearrange themselves on the guide RNA and then prime the guide RNA for next round of modification. Compared to the general archaeal populations, haloarchaea contain significantly fewer number of box C/D guide RNAs. In archaea, previous studies have underscored the importance of a symmetric assembly of the core proteins on the sRNA. This meant that if the core proteins were unable to bind to either the terminal box C/D or the internal box C'/D' motifs, the sRNP was not efficient to carry out the modification of the target RNA. Essentially the only two haloarchaeal box C/D sRNPs known before had a symmetric architecture. In this study we discovered the first naturally occurring asymmetric box C/D sRNP called sR-41 in Haloferax volcannii. The architecture of Haloferax volcanii sR-41 box C/D sRNP seems to be closer in conformation to eukaryal snoRNPs (eukaryal counterparts of archaeal sRNPs) in which the core proteins assemble asymmetrically on the RNA. Till date, no information regarding the catalytic mechanism of an asymmetrically arranged eukaryal box C/D snoRNPs are available, because of unavailability of any assembly systems or crystal structures. Hence, this archaeal sR-41 guide sRNP provides a unique opportunity to study mechanism of modification in an asymmetrically arranged box C/D sRNP molecule.

Les petits ARN régulateurs bactériens, généralement de 50 à 300 nt de long, agissent en appariant les bases avec des cibles d'ARNm spécifiques, affectant ainsi leur traduction et/ou leur stabilité, sont des éléments importants qui régulent divers processus. V. tasmaniensis LGP32 est un pathogène de l'huître facultatif. Un ARNs Vsr217 s'est révélé être conservé dans les vibrions et fortement régulé à la hausse lors de l'infection des huîtres. J'ai trouvé que vsr217 et le gène en aval malK (codant pour une sous-unité du transporteur principal de maltose) sont tous deux exprimés à partir d'un promoteur en amont régulé par l'activateur de maltose MalT, Vsr217 étant généré à partir de la longue 5' UTR de l'ARNm de malK. Outre un effet cis sur l’expression du malK, qui diminue chez le mutant Δvsr217, nous avons constaté que l’absence de cet ARNs entraînait, lors de la croissance de cellules dans du maltose, l’augmentation de deux enzymes importantes impliquées dans la voie de la glycolyse/néoglucogenèse, Fbp et PpsA et cet ARNm de fbp étaient une cible directe de Vsr217. J'ai également exploré la régulation de la biosynthèse des acides aminés à chaîne ramifiée (BCAA: Leucine, Valine et Isoleucine) chez V. alginolyticus, un agent pathogène des poissons et mollusques et des poissons de mer et un agent pathogène humain émergent opportuniste. Nous avons constaté que l'opéron ilvGMEDA (codant la voie principale pour la biosynthèse des BCAAs) est régulé par un peptide leader traduit. Ainsi, la traduction d'un peptide riche en BCAA codé en amont des gènes de structure fournit une réponse adaptative par un mécanisme similaire au modèle canonique de E. coli. Cette étude portant sur un organisme non modèle à Gram-négatif met en évidence la conservation mécanistique de l'atténuation de la transcription malgré l'absence de conservation de la séquence primaire. / Bacterial regulatory small RNAs, usually 50-300 nt long, act by base-pairing with specific mRNA targets, affecting their translation and/or stability, are important elements which regulate a variety of processes. V. tasmaniensis LGP32 is a facultative oyster pathogen. A sRNA Vsr217 was found to be conserved within vibrios and highly upregulated during oyster infection. I found that vsr217 and the downstream gene malK (encoding a subunit of the major maltose transporter) are both expressed from an upstream promoter regulated by the maltose activator MalT with Vsr217 being generated from the long 5' UTR of the malK mRNA. Beside a cis-effect on malK expression, which decreases in the Δvsr217 mutant, we found that the absence of this sRNA resulted, when cells grown in maltose, in the increase of two important enzymes involved in the glycolysis/neoglucogenesis pathway, Fbp and PpsA and that fbp mRNA was a direct target of Vsr217. I also explored the regulation of the biosynthesis of branched-chain amino acids (BCAAs: Leucine, Valine and Isoleucine) in V. alginolyticus, a marine fish and shellfish pathogen and an emerging opportunistic human pathogen. We found that the ilvGMEDA operon (encoding the main pathway for biosynthesis of BCAAs) is regulated by a translated leader peptide. Thus, the translation of a BCAA rich peptide encoded upstream of the structural genes provides an adaptive response by a mechanism similar to the E. coli canonical model. This study with a non-model Gram-negative organism highlights the mechanistic conservation of transcription attenuation despite the absence of primary sequence conservation.

No description available.

Knowledge of the processes of RNA interference, the regulation of gene expression by small RNAs (sRNAs), has grown at an unprecedented rate over the last 30 years. Some of the findings were literally revolutionary, as they revealed events that overturned many long-held notions. Many phenomena have been shown to be highly conserved and common to organisms of different species, but others are specific to certain lineages or have not yet been fully explored. There is also a lack of knowledge about the interconnection of numerous pathways - for example between silencing at the transcriptional (TGS, leading to the promoter methylation) and post-transcriptional levels (PTGS, affecting mRNA stability or translation). The present work summarizes the findings of two published and two unpublished works and attempts to describe some of the less known sites of RNA interference using various plant model organisms. Research on Solanum tuberosum transgenic lines has revealed the ability of 5-azacytidine to restore the expression of transcriptionally silenced transgenes at the whole plant level. De novo regeneration from leaves of such plants can lead to re-silencing of reactivated transgenes and thus serves as a selection method to exclude lines prone to spontaneous silencing. The nature of changes in the...

Introduction: In recent years, there has been growing interest in regulation of gene expression by small non-coding RNA (sRNA). The first sRNA discovered in 1960s was 6S RNA from E. coli (length ~184 nt). It took ~ 30 years to obtain meaningful insights into its function. 6S RNA binds during stationary phase to RNA polymerase (RNAP) containing sigma factor 70 (primary sigma factor), thereby preventing transcription from σ70 - dependent promoters. In our laboratory we discovered a small RNA (length ~300 nt) in stationary phase of growht in Mycobacterium smegmatis. This sRNA was named Ms 1. The function of Ms 1 is uknown and preliminary experiments indicated that Ms 1may bind to RNAP that lacks σ factor (σA ). Goals: The aim of this Diploma project is to contribute to the characterization of Ms 1. Approaches: First, by molecular cloning, affinity chromatography and in vitro transcription I prepared the tools for subsequent experiments in vitro: RNAP, σA , Ms 1 and its mutated variants. Next, these tools were used for binding experiments on native gels and for transcription experiments. Results: RNAP, σA , Ms 1 and its variants were prepared. In vitro binding assays showed that wt Ms 1 but not a mutated variant of Ms 1 binds to RNAP. Using this assays were identified areas of Ms 1 that are important...

Bacterial regulatory small RNAs (sRNAs) act as crucial regulators in central carbon metabolism by modulating translation initiation and/or degradation of target mRNAs in metabolic pathways. This work demonstrates that a noncoding sRNA, SdhX, is produced by RNase E-dependent processing from the 3 ́ untranslated region (3 ́ UTR) of an operon encoding three enzymes of the tricarboxylic acid (TCA) cycle. In Escherichia coli, SdhX negatively regulates ackA (encoding an enzyme critical for degradation of the signaling molecule acetyl phosphate) without affecting the downstream pta gene that encodes the enzyme critical for acetyl phosphate synthesis. SdhX abundance is tightly coupled to the transcription signals of the TCA cycle genes but escapes all known post-transcriptional regulation. Therefore, SdhX expression directly correlates with transcriptional input to the TCA cycle, providing an effective mechanism for the cell to link the TCA cycle with acetate metabolism pathways. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

RNA is a highly versatile molecule with functions that span from being a messenger in the transfer from DNA to protein, a catalytic molecule important for key processes in the cell to a regulator of gene expression. The post-genomic era and the use of new techniques to sequence RNAs have dramatically increased the number of regulatory RNAs during the last decade. Many of these are antisense RNAs, as for example the miRNA in eukaryotes and most sRNAs in bacteria. Antisense RNAs bind to specific targets by basepairing and thereby regulate their expression. A major step towards an understanding of the biological role of a miRNA or an sRNA is taken when one identifies which target it regulates. We have used RNA libraries to study the RNA interference pathway during development in the unicellular model organism Dictyostelium discoideum. We have also, by combining computational and experimental methods, discovered the first miRNAs in this organism and shown that they have different expression profiles during development. In parallel, we have developed a novel approach to predict targets for sRNAs in bacteria and used it to discover sRNA/target RNA interactions in the model organism Escherichia coli. We have found evidence for, and further characterized, three of these predicted sRNA/target interactions. For instance, the sRNA MicA is important for regulation of the outer membrane protein OmpA, the sRNAs OmrA and OmrB regulate the transcription factor CsgD, which is important in the sessile lifestyle of E. coli, and MicF regulates its own expression in a feed forward loop via the regulatory protein Lrp. In conclusion, we have discovered novel antisense RNAs, e.g. miRNAs in D. discoideum, developed an approach to identify targets for antisense RNAs, i.e. a target prediction program for sRNAs in bacteria, and verified and characterized some of the predicted antisense RNA interactions.

The purpose of researching bacterial gene expression is to control and prevent the diseases which are caused by bacteria. Recently researchers discovered small non-coding RNAs (ncRNA / sRNA) perform a variety of critical regulatory functions in bacteria. The genome-wide searching for sRNAs, especially the computational method, has become an effective way to predict the small non-coding RNAs because sRNAs have the consistent sequence characteristics. This article proposes a hybrid computational approach, HybridRNA, for the prediction of small non-coding RNAs, which integrates three critical techniques, including secondary structural algorithm, thermo-dynamic stability analysis and sequence conservation prediction. Relying on these computational techniques, our approach was used to search for sRNAs in Streptococcus pyogenes which is one of the most important bacteria for human health. This search led five strongest candidates of sRNA to be predicted as the key components of known regulatory pathways in S. pyogens.

Staphylococcus aureus is a highly problematic human pathogen due to its ability to cause devastating infections, paired with a capacity to withstand the action of a large fraction of available antibiotics. Both pathogenicity and antibiotic resistance are encoded by numerous genomic elements, though the expression of these factors is energetically costly and not always beneficial for cellular survival. Therefore, S. aureus has developed sophisticated regulatory networks to integrate a multitude of signals, enabling it to navigate the delicate balance between its pathogenic lifestyle and baseline needs for cellular energy homeostasis. It is thus imperative to study S. aureus behavior and its underlying regulatory circuits not as isolated entities, but rather holistically as part of an optimized, highly interconnected system. To do so, we must seek to achieve a comprehensive understanding of all encoded regulators, that is, not only historically well defined elements like transcription factors, two-component systems and σ factors, but also the lesser studied ’non-classical’ regulators like small regulatory RNAs and regulatory subunits of RNA-dependent RNA polymerase (RNAP). To this end, we describe here the identification of numerous, previously unidentified sRNAs and their incorporation into a new standardized cataloging and annotation system. The identification and annotation procedures are based on a number of RNAseq experiments performed in three different S. aureus backgrounds (MRSA252, NCTC 8325, and USA300). We then apply RNAseq to evaluate the expression patterns of these elements when grown in human serum, thus probing for possible connections between sRNAs and S. aureus pathogenicity. In addition, we characterize the role of two small RNAP subunits, δ and ω, for S. aureus RNAP function. δ is of particular interest, as it is unique to Gram-positive bacteria; deletion of the subunit results in a loss of transcriptional stringency and decreased expression of numerous virulence determinants. These alterations are accompanied by impaired survival of the δ mutant in whole human blood, increased phagocytosis by human leukocytes, and decreased survival in a murine model septicemia when compared to its parental strain. In contrast, there is no indication of direct and gene-specific transcriptional functions for ω. Rather, we describe a role for ω in the structural integrity of the RNAP complex, where its loss leads to a structural disturbance in the RNAP complex that causes altered affinities for (alternative) σ factors and the δ subunit. Overall, the findings presented here contribute to a better understanding of the intricate regulatory systems that guide the lifestyle of an organism that presents an immense burden to patients and our health care system alike.

The growth of bacteria by binary division is a key characteristic of these organisms. This growth depends on two types of factors: endogenous and exogenous. Endogenous factors make up the molecular apparatus of cells. Among important endogenous factors belong also those involved in gene expression and its regulation. Exogenous factors are external conditions such as nutrient availability, temperature, pH, various stresses or the presence of antibacterial agents. The main aim of my Thesis was to study the effects of selected endogenous and exogenous factors on bacterial growth. As endogenous factors I studied RNase J1 in Bacillus subtilis and a small RNA called Ms1 in Mycobacterium smegmatis, which are involved in regulation of gene expression at the transcriptional level. I showed that RNase J1 can, besides its role in RNA degradation, play a role in genome integrity by removing stalled RNA polymerase (RNAP) complexes from DNA. I further showed that Ms1 binds to the RNAP core and affects the level of RNAP in the cell. The results revealed new mechanistic aspects of the transcription apparatus and show how individual components or their combinations affect bacterial growth. As exogenous factors I studied the recently discovered antibacterial compounds, called lipophosphonoxins, their interaction...