Over the past years, small RNAs (smRNAs) have been identified as important molecular regulators of gene expression and specifically eukaryotic messenger RNAs (mRNAs). Small RNAs including small-interfering RNAs (siRNAs) and microRNAs (miRNAs) take part in the RNA silencing pathway and regulate various pathways in the cell including transcription, genome integrity, chromatin structure, mRNA stability, and translation. siRNAs are usually from exogenously derived molecules, while miRNAs are expressed endogenously by the genome. The RNA silencing pathway is highly conserved between organisms and plays a critical part in maintaining homeostasis, host-pathogen interaction, and disease progression. Thus, a better understanding of the RNA silencing pathway and probing of the molecules involved in the process is instrumental in developing tools that can better regulate the expression of specific genes. The viral suppressor of RNA silencing (VSRS) p19, is a 19 kDa protein that is expressed by tombusviruses and exhibits the highest reported affinity to small RNAs, including siRNA and miRNA. Further engineering of this protein acts as an interesting means to control the RNA silencing pathway and provides a platform to design novel tools to further modulate the activity of smRNAs in living systems. The ability to incorporate new and useful chemical functionality into proteins within living organisms has been greatly enhanced by technologies that expand the genetic code. These usually involve bioorthogonal transfer RNA (tRNA) /aminoacyl-tRNA synthetase (aaRS) pairs that can selectively incorporate an unnatural amino acid (UAA) site specifically into ribosomally synthesized proteins. Site-specificity is coded for by using a rare codon such as the amber stop codon. In Chapter 2, we demonstrate the engineering of p19 for the development of a Förster resonance energy transfer (FRET) reporter system for the visualization of RNA delivery and release in cells using UAAs and bioorthogonal click chemistry, which was done by incorporating azidophenylalanine (AzF). In Chapter 3, by incorporating UAAs into p19’s binding pocket, we were able to enhance its smRNA suppressing activity by covalently trapping the bound substrates. We have demonstrated the engineering of a molecular switch that contains photo-crosslinking groups that covalently trap smRNAs. In Chapter 4, incorporating a metal-ion chelating UAA (2,2′-bipyridin-5-yl) alanine (BpyAla) into p19’s binding pocket has successfully led to site-specific cleavage of small RNAs including siRNAs and endogenous miRNAs. The genetic introduction of BpyAla provides a unique method of introducing catalytic activity into proteins of interest. The developed unnatural enzyme provides a new tool for catalytic suppression of the RNA silencing pathway. These results demonstrate the power of adding new chemistries to proteins using UAAs to achieve possible, diverse applications in therapy and biotechnology.

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Dans cette thèse, j'ai étudié le rôle des sRNAs dérivés de l'hôte et du virus lors de l'infection du colza (Brassica napus, Canola) par la souche UK1 du virus de la mosaïque du navet (TuMV-UK1). En utilisant un dérivé de TuMV fusionné avec un gène codant pour la protéine fluorescente verte (TuMV-GFP), deux cultivars de colza (‘Drakkar’ et ‘Tanto’) qui diffèrent par leur susceptibilité à ce virus ont été identifiés. Le profil transcriptionnel des foyers d'infection locale, dans les feuilles de Drakkar et de Tanto, par séquençage nouvelle génération (NGS) a révélé de nombreux gènes exprimés de manière différentielle. Les mêmes échantillons d'ARN provenant de feuilles de Drakkar et de Tanto, traitées par des virus ou utilisées en contrôle, ont également servi à établir le profil NGS des sRNAs (sRNAseq) et de leurs cibles potentielles d'ARN (PAREseq). Les analyses bioinformatiques et leur validation in vivo, ont permis d’identifier les événements de clivage de transcrits impliquant des micro ARN (miRNA) connus et encore inconnus. Fait important, les résultats indiquent que TuMV détourne la voie du RNA silencing de l’hôte avec des siRNAs issus de son propre génome (vsiRNA) pour cibler les gènes de l’hôtes. Le virus déclenche également le ciblage à grande échelle des ARN messagers (ARNm) de l’hôte par l’activation de la production de siRNAs secondaires en phase, à partir de locus PHAS. À leur tour, les vsiRNAs et les siRNAs dérivés de l'hôte (hsRNAs) ciblent et clivent l'ARN viral par le complexe RISC. Ces observations éclairent le rôle des siRNAs dérivés de l'hôte et du virus dans la coordination de l'infection virale. Un autre chapitre de cette thèse est consacré à l'analyse des maladies induites par des virus en utilisant comme modèle de plante Arabidopsis, infectée par un tobamovirus, le virus de la mosaïque du colza (ORMV). De plus, ces observations ont permis de proposer un modèle dans lequel cette guérison dépend d’un adressage important de vsiRNAs secondaires antiviraux depuis leur source de production jusqu’à leurs tissus de destination, et l'établissement d'un apport en vsiRNAs capable de bloquer l'activité VSR impliquée dans la formation des feuilles symptomatiques. / In this thesis, I investigated the role of host- and virus-derived sRNAs during infection of Rapeseed (Brassica napus, Canola) by the UK1 strain of Turnip mosaic virus (TuMV-UK1). By using a TuMV derivative tagged with a gene encoding green fluorescent protein (TuMV-GFP), two rapeseed cultivars (‘Drakkar’ and ‘Tanto’) that differ in susceptibility to this virus were identified. Transcriptional profiling of local infection foci in Drakkar and Tanto leaves by next generation sequencing (NGS) revealed numerous differentially expressed genes. The same RNA samples from mock- and virus- treated Drakkar and Tanto leaves were also used for the global NGS profiling of sRNAs (sRNAseq) and their potential RNA targets (PAREseq). The bioinformatic analysis and their in vivo validation led to the identification of transcript cleavage events involving known and yet unknown miRNAs. Importantly, the results indicate that TuMV hijacks the host RNA silencing pathway with siRNAs derived from its own genome (vsiRNAs) to target host genes. The virus also triggers the widespread targeting of host messenger RNAs (mRNAs) through activation of phased, secondary siRNA production from PHAS loci. In turn, both vsiRNAs and host-derived siRNAs (hsRNAs) target and cleave the viral RNA by the RISC-mediated pathway. These observations illuminate the role of host and virus-derived sRNAs in the coordination of virus infection. Another chapter of this thesis is dedicated to the analysis of virus-induced diseases by using Arabidopsis plants infected with the Oilseed rape mosaic tobamovirus (ORMV) as a model. Initially, the infected plants develop leaves with strong disease symptoms. However, at a later stage, disease-free, “recovered” leaves start to appear. Analysis of symptoms recovery led to the identification of a mechanism in which the VSR and virus derived-siRNAs play a central role. I used Arabidopsis mutants impaired in transcriptional and post-transcriptional silencing pathways (TGS and PTGS respectively) and a plant line carrying a promoter-driven GFP transgene silenced by PTGS (Arabidopsis line 8z2). Using various techniques able to monitor virus infection, small and long viral RNA molecules, VSR activity, as well as phloem-mediated transport with in these lines, this study led to the identification of genes required for disease symptoms and disease symptom recovery. Moreover, the observations allowed to propose a model in which symptoms recovery occurs upon robust delivery of antiviral secondary vsiRNAs from source to sink tissues, and establishment of a vsiRNA dosage able to block the VSR activity involved in the formation of disease symptoms.

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Bean pod mottle virus (BPMV), a member of the genus Comovirus in the family Comoviridae, is widespread in the major soybean-growing areas in the United States. The complete nucleotide sequences of the genomic RNAs of the naturally occurring partial diploid strain IL-Cb1 were determined. Intermolecular RNA1 recombinants were isolated from strain IL-Cb1 and characterized at the molecular level. Structurally similar recombinant RNA1 was also generated after four passages in soybean derived from plants previously inoculated with a mixture of infectious RNA1 transcripts from two distinct strains. BPMV was developed as a plant viral vector that is appropriate for gene expression and virus-induced gene silencing (VIGS) in soybean. The foreign gene was inserted between the movement protein (MP) and the large coat protein (L-CP) coding regions. The recombinant BPMV constructs were stable following several serial passages in soybean and relatively high levels of protein expression were attained. Successful expression of several proteins with different biological activities was demonstrated from the BPMV vector. Double infection of soybean by BPMV and SMV triggers a synergistic interaction leading to a serious disease. To investigate the underlying mechanism, helper componentprotease (HC-Pro) genes from several SMV strains and TEV were expressed from BPMV vectors. The recombinant BPMV vectors carrying the HC-Pro genes from SMV strain G7 or TEV induced very severe symptoms on soybean whereas constructs containing the HC-Pro gene from SMV isolate P10, a mild strain with an apparent defect in synergism, induced only very mild symptoms. Transient agroinfiltration assays using GFP-transgenic Nicotiana benthamiana showed that HC-Pro from SMV isolate P10 was not a RNA silencing suppressor, whereas those of SMV strain G7 and TEV exhibited strong suppressor activities. Analysis of chimeric HC-Pro genes and point mutations indicated that a positively charged amino acid at position 144 is critical for the suppressor function of not only SMV HC-Pro but also other potyvirus HC-Pro proteins. Although amino acid substitution at position 144 resulted in changes in small RNA profile, it did not affect HC-Pro stability.

When conspecific individuals are crossed, the ensuing hybridization creates a spectrum of phenotypes in the resulting offspring. Many of hybrid traits will be additive, similar to the parental phenotypes. In some cases however, transgressive phenotypes are formed, outside the range of that of the parental phenotypes. Transgressive phenotypes can either be restricted to the F1 generation or be heritable throughout the hybrid lineage. While the mechanism behind heritable transgressive phenotyped is yet to be determined, transgressive gene expression is thought to be the root cause of their formation. Epigenetics modifications, heritable variation separate to the DNA code, can alter gene expression, persist through generations, and vary between individuals and over time. This makes them ideal candidates to be involved in the formation of transgressive phenotypes. RNA silencing is an epigenetic mechanism of gene regulation relying on 20Q24nt single stranded small RNAs (sRNAs). Small RNAs, due to their ability to set up persistent epigenetic marks at a locus, have the potential to create heritable transgressive gene expression. For example, when genetic variation from one parental genome presents novel targets to the sRNAs of the other parental genome, new epigenetic marks such as DNA methylation or secondary sRNAs can be created at target sites. In order to understand the potential of small RNAs to influence hybrid phenotype, I designed crossing experiments with Chlamydomonas reinhardtii, choosing this unicellular alga due to the genetic tools available and the haploid nature of its vegetative cells. The specific aim of the experiment was to identify transgressively expressed sRNA populations. Crossing two geographically distinct strains of C. reinhardtii, and sequencing both the genomes and sRNAomes of parents and recombinants, I was able catalogue both genetic and epigenetic variation in the parental strains providing unique insight into the inheritance of small RNAs in this alga. In this thesis, I first compare the genomes of the parental strains, identifying polymorphisms and assessing genetic variation in RNA silencing pathway components. I then describe the sRNA profiles of the parental strains, identifying differentially expressed sRNA loci. I then describe my approach to identifying transgressively expressed sRNA loci in the hybrids. While many sRNA loci in the recombinants exhibit additive sRNA expression, I found multiple transgressively expressed sRNA loci. Using the available bioinformatics tools, I identified potential miRNAs and phased secondary sRNAs within the list of transgressively expressed loci. Target analysis of one of the transgressively expressed miRNAs linked it with the transgressive expression of certain phased loci, suggesting a potential for sRNAs to be able to set up heritable epigenetic marks in recombinant C. reinhardtii cells.

ARGONAUTE proteins (AGOs) are generally accepted as key components of the post transcriptional gene silencing mechanism, also involved in plant antiviral defense. Except for reports on the antiviral roles of AGO1, AGO2 and AGO7 in Arabidopsis, the exact roles played by the individual AGOs in other plant species are largely unknown. This research focused on the identification and characterization of AGOs involved in antiviral RNAi response to various viruses in N. benthamiana. Based on the temporal and spatial distribution of AGO transcripts in 3 and 8-week old plant root, stem and leaf tissues, expressions of NbAGO mRNAs were found to vary with age and tissue specificity. Plant endogenous AGO mRNAs were knocked down through virus induced gene silencing techniques using the Tobacco rattle virus vector system and posteriorly challenged with a GFP-chimeric virus construct deficient of a silencing suppressor. Unlike in control non-silenced plants, the Tomato bushy stunt virus construct deficient of its P19 silencing suppressor was consistently seen to exhibit a strong fluorescence on N. benthamiana plants silenced for NbAGOs 2 and X. Similar results were also obtained upon silencing of NbAGO2 using hairpin vector techniques. Comparable observations were also made when Tobacco mosaic virus GFP constructs were agroinfiltrated on NbAGO2 silenced plants further hinting the antiviral defense roles played by these AGOs. Agroinfiltration of Foxtailmosaic virus, Sunnhemp mosaic virus, and Turnip crinkle virus GFP chimeric constructs on NbAGO2 silenced N. benthamiana plants, however did not result in accumulation of GFP indicating the AGO antiviral defense specificity to TBSV and TMV. The results also hinted at a role for AGO7. Collectively my findings suggest that the expression of AGOs in N. benthamiana is tissue and age dependent, and that unlike in the model plant Arabidopsis where the main antiviral AGO is thought to be AtAGO1; in N. benthamiana, NbAGOs 2 and X seem to be involved in an antiviral defense role against TBSV and TMV with other AGOs perhaps contributing.

Biological molecules are essential parts of organisms and participate in a variety of biological processes within cells. Understanding the relationship between sequence, structure, and function of biological molecules are of fundamental importance in life science and the health care industry. In this dissertation, a multi-scale approach was utilized to develop coarse-grained molecular models for protein and RNA simulations. By simplifying the atomistic representation of a biomolecular system, the coarse-grained approach enables the molecular dynamics simulations to reveal the biological processes, which occur on the time and length scales that are inaccessible to the all-atom models. For RNA, an "intermediate" coarse-grained model was proposed to provide both accuracy and efficiency for RNA 3D structure modeling and prediction. The overall potential parameters were derived based on structural statistics sampled from experimental structures. For protein, a general, transferable coarse-grain framework based on the Gay-Berne potential and electrostatic point multipole expansion was developed for polypeptide simulations. Next, an advanced atomistic model was developed to model electrostatic interaction with high resolution and incorporates electronic polarization effect that is ignored in conventional atomistic models. The last part of my thesis work involves applying all-atom molecular simulations to address important questions and problems in biophysics and structural biology. For example, the interaction between protein and miRNA, the recognition mechanism of antigen and antibody, and the structure dynamics of protein in mixed denaturants. / text

Argonautes (AGOs) are present in all domains of life. Like their eukaryotic counterparts, archaeal and eubacterial AGOs adopt a similar global architecture and bind small nucleic acids. In many eukaryotes, AGOs, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. We find that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15–18 nt DNA guides derived from the chromosomal region where replication terminates, and TtAgo complexed to short DNA guides enhances target finding and prefers to bind targets with full complementarity. Additionally, TtAgo associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of both gyrase and TtAgo activity slows growth and produces long, segmented filaments in which the individual bacteria are linked by DNA. Furthermore, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. Finally, at physiologic temperature in vitro, we find TtAgo possesses highest affinity for fully complementary targets. We propose that terminus-derived guides binding in such a fashion localize TtAgo, and that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication.

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