221 research outputs found

    Antagonistic and cooperative AGO2-PUM interactions in regulating mRNAs.

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    Approximately 1500 RNA-binding proteins (RBPs) profoundly impact mammalian cellular function by controlling distinct sets of transcripts, often using sequence-specific binding to 3' untranslated regions (UTRs) to regulate mRNA stability and translation. Aside from their individual effects, higher-order combinatorial interactions between RBPs on specific mRNAs have been proposed to underpin the regulatory network. To assess the extent of such co-regulatory control, we took a global experimental approach followed by targeted validation to examine interactions between two well-characterized and highly conserved RBPs, Argonaute2 (AGO2) and Pumilio (PUM1 and PUM2). Transcriptome-wide changes in AGO2-mRNA binding upon PUM knockdown were quantified by CLIP-seq, and the presence of PUM binding on the same 3'UTR corresponded with cooperative and antagonistic effects on AGO2 occupancy. In addition, PUM binding sites that overlap with AGO2 showed differential, weakened binding profiles upon abrogation of AGO2 association, indicative of cooperative interactions. In luciferase reporter validation of candidate 3'UTR sites where AGO2 and PUM colocalized, three sites were identified to host antagonistic interactions, where PUM counteracts miRNA-guided repression. Interestingly, the binding sites for the two proteins are too far for potential antagonism due to steric hindrance, suggesting an alternate mechanism. Our data experimentally confirms the combinatorial regulatory model and indicates that the mostly repressive PUM proteins can change their behavior in a context-dependent manner. Overall, the approach underscores the importance of further elucidation of complex interactions between RBPs and their transcriptome-wide extent

    RNA Binding Proteins in the miRNA pathway

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    © 2015 by the authors; licensee MDPI, Basel, Switzerland. microRNAs (miRNAs) are short ~22 nucleotides (nt) ribonucleic acids which post-transcriptionally regulate gene expression. miRNAs are key regulators of all cellular processes, and the correct expression of miRNAs in an organism is crucial for proper development and cellular function. As a result, the miRNA biogenesis pathway is highly regulated. In this review, we outline the basic steps of miRNA biogenesis and miRNA mediated gene regulation focusing on the role of RNA binding proteins (RBPs). We also describe multiple mechanisms that regulate the canonical miRNA pathway, which depends on a wide range of RBPs. Moreover, we hypothesise that the interaction between miRNA regulation and RBPs is potentially more widespread based on the analysis of available high-throughput datasets

    Nucleotide Complementarity Features in the Design of Effective Artificial miRNAs

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    L'importance du miARN dans la régulation des gènes a bien été établie. Cependant, le mécanisme précis du processus de reconnaissance des cibles n'est toujours pas complètement compris. Parmi les facteurs connus, la complémentarité en nucléotides, l'accessibilité des sites cibles, la concentration en espèces d'ARN et la coopérativité des sites ont été jugées importantes. En utilisant ces règles connues, nous avons précédemment conçu des miARN artificiels qui inhibent la croissance des cellules cancéreuses en réprimant l'expression de plusieurs gènes. De telles séquences guides ont été délivrées dans les cellules sous forme de shARN. Le VIH étant un virus à ARN, nous avons conçu et testé des ARN guides qui inhibent sa réplication en ciblant directement le génome viral et les facteurs cellulaires nécessaires au virus dans le cadre de mon premier projet. En utilisant une version mise à jour du programme de conception, mirBooking, nous devenons capables de prédire l'effet de concentration des espèces à ARN avec plus de précision. Les séquences guides conçues fournissaient aux cellules une résistance efficace à l'infection virale, égale ou meilleure que celles ciblant directement le génome viral par une complémentarité quasi-parfaite. Cependant, les niveaux de répression des facteurs viraux et cellulaires ne pouvaient pas être prédits avec précision. Afin de mieux comprendre les règles de reconnaissance des cibles miARN, les règles de couplage des bases au-delà du « seed » ont été approfondies dans mon deuxième projet. En concevant des séquences guides correspondant partiellement à la cible et en analysant le schéma de répression, nous avons établi un modèle unificateur de reconnaissance de cible par miARN via la protéine Ago2. Il montre qu'une fois que le « seed » est appariée avec l'ARN cible, la formation d'un duplex d'ARN est interrompue au niveau de la partie centrale du brin guide mais reprend plus loin en aval de la partie centrale en suivant un ordre distinct. L'implémentation des règles découvertes dans un programme informatique, MicroAlign, a permis d'améliorer la conception de miARN artificiels efficaces. Dans cette étude, nous avons non seulement confirmé la contribution des nucléotides non-germes à l'efficacité des miARN, mais également défini de manière quantitative la manière dont ils fonctionnent. Le point de vue actuellement répandu selon lequel les miARN peuvent cibler efficacement tous les gènes de manière égale, avec uniquement des correspondances de semences, peut nécessiter un réexamenThe importance of miRNA in gene regulation has been well established; however, the precise mechanism of its target recognition process is still not completely understood. Among the known factors, nucleotide complementarity, accessibility of the target sites, and the concentration of the RNA species, and site cooperativity were deemed important. Using these known rules, we previously designed artificial miRNAs that inhibit cancer cell growth by repressing the expression of multiple genes. Such guide sequences were delivered into the cells in the form of shRNAs. HIV is an RNA virus. We designed and tested guide RNAs that inhibit its replication by directly targeting the viral genome and cellular factors that the virus requires in my first project. Using an updated version of the design program, mirBooking, we become capable to predict the concentration effect of RNA species more accurately. Designed guide sequences provided cells with effective resistance against viral infection. The protection was equal or better than those that target the viral genome directly via near-perfect complementarity. However, the repression levels of the viral and cellular factors could not be precisely predicted. In order to gain further insights on the rules of miRNA target recognition, the rules of base pairing beyond the seed was further investigated in my second project. By designing guide sequences that partially match the target and analysing the repression pattern, we established a unifying model of miRNA target recognition via Ago2 protein. It shows that once the seed is base-paired with the target RNA, the formation of an RNA duplex is interrupted at the central portion of the guide strand but resumes further downstream of the central portion following a distinct order. The implementation of the discovered rules in a computer program, MicroAlign, enhanced the design of efficient artificial miRNAs. In this study, we not only confirmed the contribution of non-seed nucleotides to the efficiency of miRNAs, but also quantitatively defined the way through which they work. The currently popular view that miRNAs can effectively target all genes equally with only seed matches may require careful re-examination

    Regulation of gene expression by RNA binding proteins and microRNAs

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    Regulation of gene expression is essential to life. Post-transcriptional regulation of gene expression is a complex process with many inputs that lead to changes in localization, translation and stability of mRNAs. The translation and stability of many mRNAs is regulated by cis-elements, such as mRNA-structure or codon optimality; and by trans-acting factors such as RBPs and miRNAs. Here I report on the complex interactions between RBPs, miRNAs and characteristics of their target mRNAs in respect to effects on translation and RNA stability. Using a reporter based approach we studied modulation of microRNA-mediated repression by various mRNA characteristics. We observed the influence of codon optimality, 5’UTR structure, uORFs and translation efficiency on the magnitude of miRNA-mediated repression. To study functional interactions between RBPs and miRNAs, we developed a new method: PTRE-seq. This method utilizes a massively parallel reporter library to study the individual and combined effects of RBPs and miRNAs on translation and RNA stability. Using PTRE-seq we observed epistatic interactions between AU-rich elements and miRNA binding sites. In addition to PTRE-seq, we developed a novel method for immunoprecipitation of mRNAs that will facilitate the identification of miRNAs and RBPs bound to mRNAs of interest

    Ciphers and Executioners: How 3′-Untranslated Regions Determine the Fate of Messenger RNAs

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    The sequences and structures of 3′-untranslated regions (3′UTRs) of messenger RNAs govern their stability, localization, and expression. 3′UTR regulatory elements are recognized by a wide variety of trans-acting factors that include microRNAs (miRNAs), their associated machinery, and RNA-binding proteins (RBPs). In turn, these factors instigate common mechanistic strategies to execute the regulatory programs encoded by 3′UTRs. Here, we review classes of factors that recognize 3′UTR regulatory elements and the effector machineries they guide toward mRNAs to dictate their expression and fate. We outline illustrative examples of competitive, cooperative, and coordinated interplay such as mRNA localization and localized translation. We further review the recent advances in the study of mRNP granules and phase transition, and their possible significance for the functions of 3′UTRs. Finally, we highlight some of the most recent strategies aimed at deciphering the complexity of the regulatory codes of 3′UTRs, and identify some of the important remaining challenges

    Cooperativity in Mammalian RNA Silencing: A Dissertation

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    Argonaute proteins are the core component of an RNA silencing complex. The human genome encodes four Argonaute paralogs –Ago1, Ago2, Ago3 and Ago4– proteins that are guided to target mRNAs by microRNAs. More than 500 miRNAs are conserved between mammals, and each microRNA can repress hundreds of genes, regulating almost every cellular process. We still do not fully understand the molecular mechanisms by which miRNAs regulate gene expression. Although we understand many aspects of microRNA biogenesis and formation of the RNA-induced silencing complex, much less is known about the subsequent steps leading to target mRNA regulation. Mammalian microRNAs rarely have complete complementarity to their target mRNAs so, instead of endonucleolytic cleavage by Ago2, microRNAs destabilize or repress translation of target mRNAs. Here I explored the functional limits of Argonaute proteins bound to their targets directly and indirectly through microRNAs in mammalian cells. I revealed the different abilities for Argonaute proteins bound at multiple sites in a target to generate cooperativity in silencing based on the extent of pairing between the microRNA and target mRNA. Further, I harnessed the endogenous microRNA silencing mechanism to repress an mRNA that is not a direct target of the microRNA by tethering the RNA-induced silencing complex to the 3´ UTR of an mRNA. This strategy allows tissue-specific gene silencing due to the limited endogenous expression profile of the recruited microRNA. Efforts made herein further our mechanistic knowledge of microRNA-induced gene silencing in mammalian cells and advance microRNA-based strategies toward treating human disease

    Practical Aspects of microRNA Target Prediction

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    microRNAs (miRNAs) are endogenous non-coding RNAs that control gene expression at the posttranscriptional level. These small regulatory molecules play a key role in the majority of biological processes and their expression is also tightly regulated. Both the deregulation of genes controlled by miRNAs and the altered miRNA expression have been linked to many disorders, including cancer, cardiovascular, metabolic and neurodegenerative diseases. Therefore, it is of particular interest to reliably predict potential miRNA targets which might be involved in these diseases. However, interactions between miRNAs and their targets are complex and very often there are numerous putative miRNA recognition sites in mRNAs. Many miRNA targets have been computationally predicted but only a limited number of these were experimentally validated. Although a variety of miRNA target prediction algorithms are available, results of their application are often inconsistent. Hence, finding a functional miRNA target is still a challenging task. In this review, currently available and frequently used computational tools for miRNA target prediction, i.e., PicTar, TargetScan, DIANA-microT, miRanda, rna22 and PITA are outlined and various practical aspects of miRNA target analysis are extensively discussed. Moreover, the performance of three algorithms (PicTar, TargetScan and DIANA-microT) is both demonstrated and evaluated by performing an in-depth analysis of miRNA interactions with mRNAs derived from genes triggering hereditary neurological disorders known as trinucleotide repeat expansion diseases (TREDs), such as Huntington’s disease (HD), a number of spinocerebellar ataxias (SCAs), and myotonic dystrophy type 1 (DM1)
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