Orientadores: Michel Georges Albert Vincentz, Fabio Tebaldi Silveira Nogueira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T11:23:48Z (GMT). No. of bitstreams: 1 Zanca_AlmirSamuel_M.pdf: 11034884 bytes, checksum: 0545b58df6802e07009aef761dda3003 (MD5) Previous issue date: 2009 / Resumo: RNAs não-codificantes de 20-27 nucleotídeos (nt) regulam transcricionalmente ou pós-transcricionalmente a expressão de genes endógenos, modelando o transcriptoma e a produção de proteínas. Dentre estes, microRNAs (miRNAs) desempenliam papel chave no desenvolvimento vegetal, observação comprovada pela avaliação fenotípica e molecular de plantas transgênicas e de mutantes defectivos na produção de tais RNAs. MiRNAs são produzidos a partir de precursores longos (pri-miRNAs), os quais são posteriormente processados por enzimas específicas, gerando o miRNA maduro (20-22 nt). O miRNA maduro, por sua vez, guia a clivagem do mRNA de genes-alvo e bloqueia a tradução de proteínas, afetando diversos aspectos do desenvolvimento. O sequênciamento de populações de RNAs regulatórios possibilitou a identificação de miRNAs conservados e específicos em diferentes espécies vegetais, embora estudos em plantas de importância econômica sejam ainda incipientes. Atualmente, existem diversos bancos públicos de sequências ESTs disponíveis. Esses bancos possuem um grande número de sequências não-codíficantes, dentre as quais podem estar presentes pri-miRNAs, os quais são também são moléculas poliadeniladas similares a mRNAs codifícantes. O banco público de ESTs de cana-de-açúcar TIGR Gene Index foi usado como base para uma busca de miRNAs. O processo criado possibilitou a identificação de 20 precursores de miRNAs, agrupados em 15 famílias distintas. No presente trabalho desenvolveu-se também ferramenta para predição de potenciais alvos para os miRNAs encontrados. As famílias de miRNAs de cana-de-áçucar e a ferramenta de predição de genes-alvo estão integrados em banco de dados que estará disponível brevemente. Análise de expressão gênica demonstrou que precursors de miRNAs de cana-de-açúcar acumulam em níveis variáveis em distintos tecidos/órgãos. Além disso, tanto o acúmulo do miRNA maduro quanto a degradação do mRNA-alvo foram avaliados para alguns casos estudados. A caracterização de um miRNA específico de monocotiledôneas (miR528) e a confirmação de seu alvo, um gene comum em angiospermas, predito pela primeira vez neste trabalho, gera um interessante questionamento sobre a regulação desse gene via miRNA apenas em monocotiledôneas / Abstract: No-coding RNAs of 20-27 nucleotides (nt) transcriptional or posttranscriptionally regulate endogenous gene expression, affecting the cellular output of transcripts and proteins. Among these RNAs, microRNAs (miRNAs) play an important role in plant development as confirmed by phenotypic and molecular evaluation of transgenic plants and knockout mutants defective in miRNA biogenesis and function. miRNAs are produced from long precursors (pri-miRNAs), which are processed by specific enzymes into the mature miRNA (20-22 nt). The mature miRNA guides the cleavage of target genes as well as impairs protein translation, affecting several development processes. Deep sequencing of small RNAs identified conserved and species-specific miRNAs. Nevertheless, studies on crops are still in their infancy. Public ESTs databases are an important source of no-coding sequences, in which we can find miRNAs precursors, which are polyadenilated RNAs as messenger RNAs. In this work, the public sugarcane EST database TIGR Gene Index was used to search conserved miRNAs. The pipeline developed in this work made possible the identification of 20 miRNAs precursors, grouped into 15 families. It was also developed a search tool for potential miRNAs targets. Sugarcane miRNAs precursors displayed tissue/organ differential expression profiles. Moreover, a new identified miRNA target was confirmed experimentally. This new target is regulated by a monocot specific miRNA, miR528. Interestingly, this miRNA target is conserved in eudicots and monocots, even though its regulation by miRNA is not. This finding raises the question of why this gene has evolved in having a miRNA-mediated posttranscriptional regulation only in monocots / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Ce travail de thèse porte sur l’étude des ARN polymérases ARN dépendant impliquées dans le RNA silencing chez Arabidopsis thaliana. Durant ma thèse, la recherche d'interacteurs des RDR, parmi des protéines impliquées dans le RNA silencing, a permis la détection d'interaction entre RDR6 et SDE3, RDR6 et SGS3, mais aussi entre SDE3 et SGS3 en Co-IP et BiFC. Une co-localisation de ces protéines a été observée lorsqu'elles sont produites transitoirement dans des cellules épidermales de N. benthamiana.Un crible d’une banque d’ADNc d’A. thaliana par double hybride de levure, a permis d’isoler des interacteurs potentiels de RDR6. Deux interacteurs potentiels, AtUAP56-1 et U2B’’, sont impliqués dans l’épissage des précurseurs des ARNm. Un effet sur le RNA silencing dans des mutants de l’épissage en 3’ des ARNm était connu et nous avons confirmé l’interaction entre RDR6 et AtUAP56-1 par BiFC. L’étude de lignées mutantes pour AtUAP56-1 a donc été initiée.Une étude biochimique de RDR6 et de RDR2 a été réalisée. Des formes recombinantes de RDR2 et RDR6 ont été produites de façon transitoire dans des feuilles de N. benthamiana, et une étude comparative de RDR2 et RDR6 a été réalisée. Les deux RDR sont actives sur des matrices ARN et ADN, et montrent in vitro une activité amorce-indépendante. De plus, nous avons détecté pour la première fois une activité amorce-dépendante de RDR6 et RDR2. Ces résultats apportent de nouvelles données biochimiques qui sont en accord avec les études menées in vivo et enrichissent les modèles actuels du RNA silencing. / The aim of this work was to study RNA-dependent RNA polymerases involved in RNA silencing in Arabidopsis thaliana. During my thesis, the search for RDR interactors among proteins involved in RNA silencing allowed the detection of interactions between RDR6 and SDE3, RDR6 and SGS3, and also between SDE3 and SGS3 using Co-IP and BiFC. In addition, the co-localisation of these proteins was observed when produced transiently in epidermal cells of N. Benthamiana.A screen of an A. thaliana cDNA library by yeast two hybrid allowed us to identify some putative new RDR6 interactors. Two putative RDR6 interactors, AtUAP56 and U2B’’, are known to be involved in pre-miRNA splicing. Furthermore, a link between pre-mRNA 3’ splicing and RNA silencing was previously reported. We also confirmed the interaction between AtUAP56-1 and RDR6 by BiFC. An investigation of A. thaliana of AtUAP56-1 mutants has been initiated.Recombinant RDRs were produced transiently in N. Benthamiana, and a biochemical comparative study of RDR2 and RDR6 performed. We found that RDR2, like RDR6, has a de novo polymerase activity on DNA and RNA templates, and for both RDRs we also showed, for the first time, a primer-dependant synthesis of dsRNA from RNA template. These findings provide important new insights into our understanding of the molecular mechanisms of RNA silencing amplification in Arabidopsis.

Le beet necrotic yellow vein virus (BNYVV) est un phytovirus qui possède un génome segmenté à ARN de polarité positive. L’ARN3 viral renferme le domaine « core » qui contient une séquence de 20 nucléotides appelée « coremin », indispensable au mouvement systémique du virus chez Beta macrocarpa. L’ARN3 subit un processus de dégradation qui conduit à la formation d’un ARN non codant (ncRNA3) correspondant à son extrémité 3’. Ce dernier est stabilisé par la séquence « coremin » à son extrémité 5’. Grâce à l’outil génétique levure, l’exoribonucléase Xrn1 puis l’exoribonucléase XRN4 de plante ont été identifiées comme étant responsable de l’accumulation du ncRNA3 à partir d’ARN3. Nous avons démontré in vitro que l’accumulation de ncRNA3 est liée au blocage de Xrn1 par « coremin ». La protéine virale p14, un suppresseur du RNA silencing codée par l’ARN2, est aussi nécessaire au mouvement systémique du virus et interagit avec la séquence « coremin ». Nos travaux confirment que l’ARN3 est capable de complémenter partiellement un mutant allélique de p14 dans l’infection locale et systémique. Nos résultats mettent en évidence un effet de la protéine p14 sur la systémie du RNA silencing et sur une éventuelle cible cellulaire RDR6. / The beet necrotic yellow vein virus (BNYVV) is a multipartite positive-stranded RNA phytovirus. The RNA3 contains a « core » sequence in which resides the « coremin » motif of 20 nucleotides absolutely required for the viral systemic movement in Beta macrocarpa. The RNA3 undergoes a process that produces a noncoding RNA3 (ncRNA3), stabilized by « coremin » at its 5’ end. Using a yeast genetic approach, the exoribonuclease Xrn1 and plant XRN4 have been identified as being responsible for the ncRNA3 accumulation from RNA3 processing. In vitro, we showed that the ncRNA3 accumulation is due to the stalling of Xrn1 by “coremin”. The viral p14 protein, an RNA silencing suppressor encoded by the RNA2, is also required for the systemic movement and interacts with the “coremin” sequence. Our studies demonstrated the ability of RNA3 to partially complement an allelic p14 mutant in local and systemic infections. Our data highlighted an effect of the p14 protein on the RNA silencing movement and on the potential cellular target RDR6.

MicroRNAs are noncoding RNAs inducing sequence-specific posttranscriptional inhibition of gene expression and represent the major class of small endogenous RNAs in mammalian cells. Over 2,500 of human microRNAs potentially regulating more than 60% of human protein-coding genes have been identified. MicroRNAs participate in the majority of cellular processes, and their expression changes in various diseases, including cancer. Currently, there is no efficient small chemical compound available for the modulation of microRNA pathway activity. At the same time, small chemical compounds represent excellent tools for research of processes involving RNA silencing pathways, for biotechnological applications, and would have a considerable therapeutic potential. The presented work represents a part of a broader project, whose ultimate goal is: (i) to find a set of small molecules allowing for stimulation or inhibition of RNA silencing and (ii) to identify crosstalks between RNA silencing and other cellular pathways. This thesis summarizes results from the first two phases of the project, the development of high-throughput screening assays and the high- throughput screening (HTS) of available libraries of small compounds. To monitor the microRNA pathway activity, we developed and optimized one biochemical...

Résumé : Pour se propager dans les cellules de son hôte et évader les réponses immunitaires, les virus végétaux ont développé plusieurs stratégies de défense. Ici, nous avons investigué les structures génétiques du Apple stem pitting virus (ASPV). Nous avons aussi étudié la diversité moléculaire des isolats d’ASPV provenant des poires en regardant les séquences des gènes CP et TGB afin de mieux comprendre les mécanismes évolutionnaires utilisés par ASPV. Nos études ont démontré que les mutations, incluant les insertions et les délétions, la sélection purificatrice et la recombinaison furent des facteurs importants dans l’évolution du l’ASPV en Chine et possiblement mondialement. Comme tous les virus végétaux, l’ASPV se défend contre le RNA silencing de l’hôte grâce à un suppresseur de RNA silencing (VSR) et nous avons montré que le VSR de l’ASPV est la protéine de capside (CP) du virus. Nous avons aussi établi que la diversité moléculaire cause non seulement une variété de symptômes chez son hôte, Nicotiana occidentalis. Cependant elle cause aussi de la variabilité antigénique chez différents isolats, ce qui mène à des écarts de réactivité sérologique entre isolats. Les plantes ont développé plusieurs stratégies pour se défendre contre les virus. Ici, nous avons étudié comment la plante Arabidopsis se défend contre le Tobacco rattle virus (TRV) via le RNA silencing. Nous avons constaté que les phénomènes de susceptibilité, récupération et virus induced gene silencing (VIGS) sont des mécanismes séparables. Nous avons démontré que les protéines AGO2 et AGO4 sont nécessaires à la susceptibilité initiale au TRV, tandis qu’AGO1 est importante pour les VIGS, tandis que la récupération est médiée par d’autres acteurs qui n’ont pas encore été identifiés. Nos résultats suggèrent l’existence de complexes distincts ciblant différentes populations d’ARN viral et cellulaire. De plus, nous avons montré que la répression de la traduction est un mécanisme important durant la récupération de la plante suite à une infection virale, et que les complexes de décoiffage et de RNA processing jouent des rôles importants dans la dégradation des ARNs viraux. Finalement, nous avons montré que les plantes ayant une mutation dans le gène DCP2 présentent un niveaux de VIGS accrue, ainsi qu’une augmentation des niveaux d’ARN viral. Puisque DCP2 fait partie des complexes de décoiffage qui se trouvent dans des granules spécialisés nommés processing bodies (PBs), cela suggère que les PBs jouent un rôle important dans l’élimination les virus. / Abstract : To live in host cells or to escape from host immunity, plant viruses involved a series of defense strategies. Here we investigated Apple stem pitting virus (ASPV) population structures and molecular diversity of ASPV pear isolates based on its function important gene CP and TGB in China, so as to infer the evolution mechanisms of ASPV. Our study showed that mutations (including insertions or deletions), purifying selection, and recombination were important factors driving ASPV evolutions in China or maybe even in the world. And also ASPV defends against it hosts by encoding a VSR. We also showed that ASPV molecular diversity not only induced different biological properties on its herbaceous host N. occidentails but also resulted in antigenic variation of different ASPV CP isolates, which leaded to differences in serological reactivity among rCPs of different ASPV isolates. Plants have developed a series of mechanisms to defend themselves against viruses. Here we how Arabidopsis defend against. We show that virus susceptibility, recovery, and virus induced gene silencing (VIGS) appear to be separable phenomena, with AGO2 and AGO4 playing important roles in the initial susceptibility to TRV, AGO1 playing an important role in VIGS, and as yet unidentifid players mediating recovery. These results suggest the existence of distinct RNA-induced silencing complexes that target different RNA populations within the cell and over time. Furthermore, we showed that translational repression of viral RNA is likely to play an important role in virus recovery and that decapping function plays an important role in clearing viral RNA from the cell. We also showed that a decapping mutant (DCP2) displayed an increased VIGS and virus RNA accumulation, an important role for PBs in eliminating viral RNA.

L'ARN interférence est un mécanisme moléculaire conservé chez les Eucaryotes dont les principaux acteurs sont les protéines ARGONAUTE (AGO). Chez les plantes, AGO1 est une protéine essentielle à la croissance et la défense antivirale. Elle utilise des petits ARNs comme sondes pour reconnaître et réguler des ARN messagers. Les virus ont développé des suppresseurs de l'ARN interférence pour surmonter cette défense. L'un d'entre eux, P0 du virus de la mosaïque jaune du navet, est comme une protéine F-box qui détourne le complexe SCF, une ubiquitine ligase E3, et conduit AGO1 vers la protéolyse ubiquitine-dépendante. Cette dégradation utilise la vacuole au lieu du protéasome 26S, généralement associé à la dégradation ubiquitine-dépendante. Ce mécanisme de protéolyse n'est pas compris et est aussi apparent quand AGO1 est déstabilisé de manière endogène, suggérant que P0 utilise une voie déjà existante. Une protéine F-box d'Arabidopsis, FBW2, a été décrite comme impactant l'homéostasie d'AGO1 indépendamment du protéasome. Mon projet de thèse visait à caractériser l'activité F-box de FBW2 et à comprendre la relation entre AGO1 et FBW2 ainsi que ses conséquences sur l'ARN interférence. Les résultats obtenus dans ce manuscrit montrent que le complexe SCFFBW2 interagit avec AGO1 et déclenche sa dégradation via un processus indépendant de l'autophagie ou du protéasome, tout en n'affectant que faiblement l'ARN interférence. FBW2 ciblerait en fait un sous-ensemble de protéines AGO1 qui semble ne pas contenir de petits ARNs. Cette régulation jouerait un rôle de surveillance pour prévenir une activité délétère d'AGO1 en absence de petits ARNs. / RNA silencing is a conserved molecular mechanism in eukaryotes, of which the main effectors are the ARGONAUTE (AGO) proteins. In plants, AGO1 is a protein that is essential for growth and antiviral defence. It uses small RNAs as probe to recognize and regulate messenger RNAs. Viruses have developed suppressors of RNA silencing to overcome this defence. One of these, P0 from the Turnip Yellows Virus, acts as an F-box protein to hijack the SCF complex, an E3 ubiquitin ligase, and guide AGO1 to the ubiquitin-dependent proteolysis. This degradation uses the vacuole instead of the 26S proteasome, generally associated with ubiquitin-dependant proteolysis. This proteolysis mechanism is not understood and is also apparent when AGO1 is endogenously destabilized, suggesting that P0 uses an already existing pathway. An Arabidopsis F-box protein, FBW2, has been shown to impact AGO1 homeostasis independently from the proteasome. My PhD project aimed at characterizing FBW2 F-box activity and understanding the relationship between AGO1 and FBW2, as well as its consequences on the RNA silencing. The results obtained in this manuscript show that the SCFFBW2 interacts with AGO1 and triggers its degradation through an autophagy- and proteasome- independent process, while only weakly affecting the RNA silencing. FBW2 would actually target a subset of AGO1 proteins, which appears not to contain small RNAs. This regulation would play a surveillance role in order to prevent a deleterious activity of AGO1 in absence of small RNAs.

miRNAs are small regulatory RNAs, which function as post-transcriptional mRNA regulators. They direct ribonucleoprotein complexes to cognate mRNA to repress them by translational inhibition and degradation. miRNAs regulate thousands of mRNAs in mammals and have been recognized as regulatory factors in most cellular and developmental processes. Dysregulation of the miRNA pathway can lead to severe defects and diseases. Interestingly, a unique situation exists in mouse oocytes, where all the miRNA pathway components are present, yet the pathway is dispensable and nonfunctional, the molecular foundation of this phenomenon and its significance still remain unclear. In spite of the pronounced effects of the miRNA pathway in gene regulation in somatic cells, study strategies of the pathway bare limitations. Current methods for studying the activity of the miRNA pathway employ corelative studies (such as NGS) or reporter assays, which have relatively low throughput and are prone to artifacts. Here, I present design and development of a new strategy for directly monitor global miRNA pathway activity and integrity in near physiological conditions in living cells, which could also be employed in vivo for studies of mouse oocytes. The strategy is based on fluorescently tagged endogenous proteins of the...

Orientadores: Iscia Teresinha Lopes-Cendes, Rovilson Gilioli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T01:47:49Z (GMT). No. of bitstreams: 1 Grippo_MariangelaCarnivalli_D.pdf: 1241429 bytes, checksum: 5f05623ad1e884a0014d2eca9109fb9a (MD5) Previous issue date: 2006 / Resumo: A interferência do RNA (RNAi) pode ser usada como uma ferramenta eficaz no silenciamento gênico específico mediado por moléculas de dupla fita de RNA (dsRNAs). Nesse contexto possui uma variedade de aplicações biológicas, incluindo o combate a patógenos infecciosos de importância biomédica. O objetivo do estudo foi determinar a eficiência e a especificidade da técnica de RNAi em eliminar o vírus da hepatite murina tipo 3 (MIN-3) in vitro. MHVs são vírus envelopados, cujo genoma é formado por uma cadeia de RNA fita simples (+) pertecentes a família Coronaviridae. Seu genoma codifica quatro proteínas estruturais: S (proteína da espícula); M (glicoproteína da transmembrana), N (proteína do nucleocapsídeo) e E (proteína associada à membrana) . Neste trabalho foi escolhido como alvo para o silenciamento gênico a proteína N, tendo sido produzidas moléculas de dsRNA complementares a sua seqüência genômica (GenBank AF 201929). Foram obtidas duas moléculas siRNAs transcritas por T7 RNA polimerase e uma terceira molécula interferente sintetizada comercialmente. Foi observado que os siRNAs produzidos pela transcrição in vitro, induziram uma resposta antiviral não específica. Além disso demonstrou-se que este efeito foi mediado através de substâncias secretadas no meio de cultura celular, provavelmente interferons (IFNs). Este efeito foi eficientemente eliminado após tratamento dos siRNAs com fosfatase alcalina. Observou-se também que a técnica de RNAi in vitro, tendo como alvo a proteína N de MHV-3, foi um tratamento eficaz e específico na infecção viral, confirmados através de estudos fenotípicos e moleculares. Desse modo, concluímos que experiências que utilizam RNAi contra alvos virais devem ser cuidadosamente monitoradas devido aos efeitos não específicos que podem ser induzidos por moléculas de dsRNA / Abstract: RNA Interference (RNAi) can be used as a powerful tool for post transcriptional gene-silencing mediated by double stranded RNA (dsRNAs) molecules. RNAi has a variety of biological applications including the combat against pathogens of biomedical importance. The objective of our study was to determine the efficiency and specificity of this new technique in eliminating mouse hepatitis virus type 3 (MIN-3) in vitro. MIN-3 is a subtype of enveloped viroses with a large plus-stranded RNA genome belonging to the Coronavirus family. Its genome codifies four structural proteins: S (spike protein); M (membrane protein); E (transmembrane glycoprotein); N (nucleocapsid protein). In the present study we target protein N by designing and producing dsRNA molecules complementary to its genomic sequence (GenBank AF 201929). We obtained three small interfering RNAs (siRNA) by in house T7 polymerase in vitro transcription and a fourth siRNA molecule that was commercially synthetized. We identified that siRNAs produced by in vitro transcription triggered a potent and sequence-unspecificied antiviral response. In addition, we demonstrated that this antiviral effect was mediated through molecules that were secreted in medium culture, probably interferons (IFNs). This unspecific effect was efficient1y suppressed when siRNAs were treated with aIkaline phosphatase prior to in vitro experiments. We also observed that RNAi targeting the N protein ofMIN-3 was a potent and specific treatment against in vitro infection, showing significant phenotypic protection and molecular evidence of specific gene-silencing. We concluded that experiments using RNAi against viral targets, although efficient, must be carefully controlled and monitored against possible sequence-unspecific effects triggered by dsRNA molecules / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Oncolytic viruses are novel candidates for cancer therapy and their efficacy relies on their capacity to overcome the host’s anti-viral barriers. In mammalian cells, the anti-viral response involves a protein-signaling cascade known as the interferon pathway, which alerts the immune system and limits the propagation of infection. Given that most cancer cells have defects in this pathway, they are susceptible to viral infection and responsive to oncolytic virotherapy. For reasons that remain unknown, many cancers are still refractory to oncolytic viruses, which suggests the existence of additional antiviral mechanisms. In this study, we investigate the potential involvement of an alternative antiviral pathway in cancer cells. Given that insects and plants rely on the RNA silencing pathway for their anti-viral protection, we investigated the presence of a similar mechanism in cancer cells. We found viral genome-derived small RNAs in various cancer cell lines upon infection, which is indicative of an RNA-mediated antiviral response. Also, various viruses encode suppressors of the RNA interference pathway. To determine if an oncolytic virus could benefit from such a factor, we engineered an oncolytic virus variant to encode the Nodamura virus B2 protein, a known inhibitor of RNA silencing-mediated immune responses. Using this virus, we observed enhanced cytotoxicity in 33 out of the 38 human cancer cell lines tested. Furthermore, our results show inhibition of viral genome cleavage and altered microRNA processing by our B2-expressing oncolytic virus. Taken together, our data suggests the blockade of RNA silencing antiviral pathways and/or antiviral microRNA processing improves the efficacy of our B2-encoding virus in a cell-line specific manner. Overall, our results establish the improved potential of our novel virus therapy and demonstrate for the first time the involvement of RNA pathways in the antiviral defense of cancer cells.

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.