Conformational dynamics in microRNAs : the example of miR-34a targeting Sirt1 mRNA

In biology, regulatory mechanisms are essential to achieve complex tasks, as virtually every process can be positively or negatively modulated in its outcome, upon different cues. In humans, microRNAs (miRNAs) constitute a fundamental layer of post-transcriptional gene expression regulation. This class of molecules finely tune protein expression, by downregulating messenger RNAs (mRNA) levels and their translation. The mechanism by which miRNAs find and act upon their targets primarily relies on their nucleotide sequence, relative to the corresponding binding site on the mRNA. The development of an exhaustive miRNA–mRNA interactome is particularly attractive because of the profound implication for basic biology as well as for diagnostics and therapeutics in human health. However, computational prediction of target sites and associated downregulation levels, using the limited sequence determinants available, is still an outstanding challenge in the field. In this thesis, we bring forward the hypothesis that modeling of miRNA–mRNA pairs might benefit from considering the inherent structural flexibility of these complexes, at the molecular level. In the introductory chapter, we present the structural features of RNAs with a focus on their conformational dynamics and NMR spectroscopy as a tool to investigate these motions. The molecular details of miRNA biogenesis and function are later introduced to contextualize the results of Paper I. Finally, the challenges associated with RNA sample preparation are discussed in light of the work presented in Paper II. In Paper I, we show that a miRNA–mRNA pair involved in a cancer-regulating pathway exploits its flexibility to toggle between lower and higher target repression states. This study shows that suboptimal structures of a given miRNA–mRNA pair, that are overlooked by computational prediction and that often elude experimental detection, can be functionally relevant and are essential to draw a mechanistic picture of miRNA function. The methods used in Paper I for RNA sample preparation and molecular simulation are described in Paper II and II, respectively. While these methods were essential to achieve the results of Paper I, they also find widespread application in the RNA field

Structural study of CUG-repeating small RNAs complexed with silencing suppressor P19

The study is focused on structural aspects of interaction between silencing suppressor p19 and CUG-repeating small RNAs. The work involves crystal structure determination of a protein-unbound RNA form and RNA fragments of various lengths (19, 20, 21 nucleotides) complexed with p19-suppressor. Results prove the ability of silencing suppressor p19 to bind CUG-repeating small RNAs, as well as reveal features of U•U mismatches flanked by Watson-Crick C•G base pairs in p19-bound and p19-unbound states. In addition, structural data reveal a p19 specific site for anchoring extra nucleotides in small RNAs. In general, the study extends our knowledge about the mechanism of small RNA recognition by silencing suppressor p19

From Neurons to Nucleic Acids: Spatio-temporal Emergent Behaviors of Complex Biological Systems

Biological systems, from the molecular to the organismal level, demonstrate emergent behaviors that form fundamental characteristics of the system. Many biological phenomena are difficult to observe experimentally because of technical limitations. Computational models are a useful tool for interpretation of behaviors of complex biological systems. This dissertation examines models for two different types of emergent behaviors: cortical state and RNA structure. In Chapter 2, I use a computational neural model to understand the effects of neurons with long-range projections and propagation delays. I find that propagation delays cause a local network to exhibit a variety of metastable network states. Application of transcranial alternating current stimulation enables the switching of a network to a different metastable state. These emergent behaviors of a network of modeled neurons are a simplified version of neocortical states, and the results provide a foundation for future research on the effects of stimulation on cortical behavior. In Chapter 3, I examine the structure of the 5′ UTR of the human tumor suppressor gene RB1 using an experimentally-directed RNA structural model. The 5′ UTR adopts three distinct structures with similar frequencies. Two disease-associated mutations each collapse the structural ensemble into a single structure, and also affect translation efficiency. By creating structural models of two homologous UTRs, I find that the ability to adopt multiple conformations is a conserved feature of this UTR and that RNA structure regulates this transcript. In Chapter 4, I model RNA structure in Sindbis virus (SINV). SINV is a single-stranded RNA virus, with known functional elements within its RNA genome. I created experimentally-directed structural models for highly structured portions of the genome. By disrupting these structures through systematic mutational design, I identified regulatory RNA elements within the genome. Most structures within the genome are not conserved in related species of virus, indicating that this virus is highly structurally divergent and utilizes its evolutionary space to create new structures. These three projects present three different ways of using computational models to characterize complex biological systems. Informed by biological data, computational models provide further insight into the role of these emergent behaviors within a system.Doctor of Philosoph

RNA Interference by the Numbers: Explaining Biology Through Enzymology: A Dissertation

Small silencing RNAs function in almost every aspect of cellular biology. Argonaute proteins bind small RNA and execute gene silencing. The number of Argonaute paralogs range from 5 in Drosophila melanogaster , 8 in Homo sapiens to an astounding 27 in Caenorhabditis elegans. This begs several questions: Do Argonaute proteins have different small RNA repertoires? Do Argonaute proteins behave differently? And if so, how are they functionally and mechanistically distinct? To address these questions, we examined the thermodynamic, kinetic and functional properties of fly Argonaute1 (dAgo1), fly Argonaute2 (dAgo2) and mouse Argonaute2 (mAGO2). Our studies reveal that in fly, small RNA duplexes sort into Argonaute proteins based on their intrinsic structures: extensively paired siRNA duplex is preferentially sorted into dAgo2 while imperfectly paired miRNA duplex is channeled into dAgo1. The sorting of small RNA is uncoupled from its biogenesis. This is exemplified by mir-277, which is born a miRNA but its extensive duplex structure licenses its entry into dAgo2. In the Argonaute protein, the small RNA guide partitions into functional domains: anchor, seed, central, 3\u27 supplementary and tail. Of these domains, the seed initiates binding to target. Both dAgo2 and mAGO2 (more closely related to and a surrogate for dAgo1 in our studies) bind targets at astonishing diffusion-limited rates (~107–108 M−1s−1). The dissociation kinetics between dAgo2 and mAGO2 from their targets, however, are different. For a fully paired target, dAgo2 dissociates slowly (t½ ~2 hr) but for a seed-matched target, dAgo2 dissociates rapidly (t½ ~20 s). In comparison, mAGO2 does not discriminate between either targets and demonstrates an equivalent dissociation rate (t½ ~20 min). Regardless, both dAgo2 and mAGO2 demonstrate high binding affinity to perfect targets with equilibrium dissociation constants, KD ~4–20 pM. Functionally, we also showed that dAgo1 but not dAgo2 silence a centrally bulged target. By contrast, dAgo2 cleaved and destroyed perfectly paired targets 43-fold faster than dAgo1. In target cleavage, dAgo2 can tolerate mismatches, bulged and internal loop in the target but at the expense of reduced target binding affinities and cleavage rates. Taken together, our studies indicate that small RNAs are actively sorted into different Argonaute proteins with distinct thermodynamic, kinetic and functional behaviors. Our quantitative biochemical analysis also allows us to model how Argonaute proteins find, bind and regulate their targets

Molecular characterization of animal microRNAs : sequence, expression, and stability

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2004.Includes bibliographical references.(cont.) miRNAs. These cells lines may also be useful for other functional studies, such as validation of putative mRNA target genes.Multicellular organisms possess natural gene-regulatory pathways that employ small RNAs to negatively regulate gene expression. In nematodes, the small temporal RNAs (stRNAs), lin-4 and let-7, negatively regulate genes important in specifying developmental timing. A gene-silencing pathway present in plants, fungi and animals called RNA interference, involves the conversion of long double-stranded RNA into short interfering RNAs, which can serve to negatively regulate endogenous genes or suppress the replication of viruses and transposons. To investigate how wide a role small RNAs play in regulating gene expression in animals, we developed a RNA cloning procedure and first applied it to the cloning of small RNAs from the nematode, Caenorhabditis elegans. In addition to cloning lin-4 and let-7 sequences, our study revealed a large number of conserved and highly expressed small RNAs with features reminiscent of stRNAs. Because not all of these small RNAs were expressed in temporal fashion, we and others have referred to this novel class of tiny RNAs as microRNAs. We completed an extensive census of microRNA (miRNA) genes in C.elegans by cloning and bioinformatics searches to lay the groundwork for future functional studies. Our census marked the detection of nearly 90 C.elegans miRNAs, estimated an upper-bound of about 120 miRNA genes in C.elegans, and detailed the conservation and clustering of miRNA sequences. We also determined the high molecular abundance of several miRNAs in C.elegans and Hela cells. In an effort to understand the reason for the high molecular abundance of miRNAs, we constructed an inducible miRNA-expressing cell line to measure the stability of animal miRNAs. Time course measurements suggested a long (>24 hours) half-life for twoby Nelson C. Lau.Ph.D

Evolution, role and mechanism of prokaryotic Argonaute proteins

cum laude graduatio

Improved Workflows for RNA Homology Search

Non-coding RNAs are the most abundant class of RNAs found throughout genomes. These RNAs are key players of gene regulation and thus, the func- tion of whole organisms. Numerous methods have been developed so far for detecting novel classes of ncRNAs or finding homologs to the known ones. Because of their abundance, the sequence availability of these RNAs is rapidly increasing, as is the case for example for microRNAs. However, for classes of them, still only incomplete information is available, invertebrates 7SK snRNA for instance. Consequently, a lot of false positive outputs are produced in the former case, and more accurate annotation methods are needed for the latter cases to improve derivable knowledge. This makes the accuracy of gathering correct homologs a challenging task and it leads directly to a not less important problem, the curation of these data. Finding solutions for the aforementioned problems is more complex than one would expect as these RNAs are characterized not only by sequences informa- tion but also structure information, in addition to distinct biological features. In this work, data curation methods and sensitive homology search are shown as complementary methods to solve these problems. A careful curation and annotation method revealed new structural information in the invertebrates 7SK snRNA, which pushes the investigation in the area forward. This has been reflected by detecting new high potential 7SK RNA genes in different invertebrates groups. Moreover, the gaps between homology search and well- curated data on the one side, and between experimental and computational outputs on the other side, are closed. These gaps were bridged by a curation method applied to the microRNA data, which was then turned into a com- prehensive workflow implemented into an automated pipeline. MIRfix is a microRNA curation pipeline considering the detailed sequence and structure information of the metazoan microRNAs, together with biological features related to the microRNA biogenesis. Moreover, this pipeline can be integrated into existing methods and tools related to microRNA homology search and data curation. The application of this pipeline on the biggest open source microRNA database revealed its high capacity in detecting wrong annotated pre-miRNA, eventually improving alignment quality of the majority of the available data. Additionally, it was tested with artificial datasets highlighting the high accuracy in predicting the pre-miRNA components, miRNA and miRNA*.:Chapter 1: Introduction Chapter 2: Biological and Computational background 2.1 Biology 2.1.1 Non-coding RNAs 2.1.2 RNA secondary structure 2.1.3 Homology versus similarity 2.1.4 Evolution 2.2 The role of computational biology 2.2.1 Alignment 2.2.1.1 Pairwise alignment 2.2.1.2 Multiple sequence alignment (MSA) 2.2.2 Homology search 2.2.2.1 Sequence-based 2.2.2.2 Structure-based 2.2.3 RNA secondary structure prediction Chapter 3: Careful curation for snRNA 3.1 Biological background 3.2 Introduction to the problem 3.3 Methods 3.3.1 Initial seeds and models construction 3.3.2 Models anatomy then merging 3.4 Results 3.4.1 Refined model of arthropod 7SK RNA 3.4.1.1 5’ Stem 3.4.1.2 Extension of Stem A 3.4.1.3 Novel stem B in invertebrates 3.4.1.4 3’ Stem 3.4.2 Invertebrates model conserves the HEXIM1 binding site 3.4.3 Computationally high potential 7SK RNA candidate . 3.4.4 Sensitivity of the final proposed model 3.5 Conclusion Chapter 4: Behind the scenes of microRNA driven regulation 4.1 Biological background 4.2 Databases and problems 4.3 MicroRNA detection and curation approaches Chapter 5: Initial microRNA curation 5.1 Introduction 5.2 Methods 5.2.1 Data pre-processing 5.2.2 Initial seeds creation 5.2.3 Main course 5.3 Results and discussion 5.4 Conclusion Chapter 6: MIRfix pipeline 6.1 Introduction 6.2 Methods 6.2.1 Inputs and Outputs 6.2.2 Prediction of the mature sequences 6.2.3 The original precursor and its alternative 6.2.4 The validation of the precursor 6.2.5 Alignment processing 6.3 Results and statistics 6.4 Applications 6.4.1 Real life examples and artificial data tests 6.4.2 miRNA and miRNA* prediction 6.4.3 Covariance models 6.5 Conclusion Chapter 7: Discussio

From cell penetrating peptides to peptoids and polyamines as novel artificial molecular transporters

In recent years, RNA interference has gained a lot of importance as a tool for posttranscriptional silencing of genes due to its high specificity, efficiency and ease of application. In mammalian cells RNAi is triggered by the application of 21 bp short dsRNAs, so-called short interfering RNAs (siRNAs). Numerous studies indicate the great potential of RNAi in the therapy of viral infections and inherited diseases. Cell-penetrating peptides can be used to apply siRNAs to primary cells, non-dividing cells or fully grown organisms, that are diffcult to transfect. These short positively charged peptides are internalized by cells. They can be detected in the endosomes, lysosomes but also in the cytosol. If attached to CPPs, large cargo molecules can be taken up with a high efficiency that surpasses that of most conventional transfection methods. In the work presented here, peptide-coupled siRNAs (pepsiRNAs) have been developed as a novel tool for transient RNAi in mammalian cells. The peptides were attached to the siRNA by a disulfide bond, that is cleaved under the reducing conditions of the cytosol and thus releases the siRNA cargo. PepsiRNAs are readily taken up by many cell types that are difficult to address by conventional transfection methods. An siRNA-induced downregulation of the targeted genes was observed at concentrations between 10 and 100 nM. SiRNAs with a 5´-thiol modification upon their sense-strand were generated by in vitro transcription, for which a thiol-modified nucleotide was synthesized via an optimized route. The cell-penetrating peptides Penetratin and Tat were recombinantly expressed as fusion-proteins with glutathione-S-transferase (GST) and purified with a modified procedure to overcome the strong membrane interaction of the GST-tagged CPPs. Recombinant TEV protease was expressed to cleave the CPPs from the fusion tag, and the cleavage activity was assessed by comparison with commercially obtained TEV protease. Thus, alternative routes to the building blocks for pepsiRNAs have been provided to scale up the amount of pepsiRNAs. Finally, small molecules with cell-penetrating properties have been developed as a future replacement of the peptide moiety. Fluorescently labeled peptoids of differing length with amine-functionalized side chains have been shown to enter different mammalian cells lines at concentrations in the lower micromolar range by an endocytosis dependent mechanism. Cationic molecules as small as spermine attached to fluorescein are taken up by an endocytosis-related mechanism. By the same approach porphyrins were delivered into the interior of the cells, where they exhibited a cytotoxic effect upon illumination, so that spermine-coupled porphyrins may be developed into a novel drug for photodynamic therapy.Entwicklung neuer Delivery-Strategien für siRNAs - Von zellpenetrierenden Peptiden zu Peptoiden und Polyaminen als neuartige molekulare Transporter In den vergangenen Jahren, hat die RNA Interferenz (RNAi) aufgrund ihrer Spezifität, Effizienz und einfachen Anwendbarkeit eine große Bedeutung als Technik für das posttranskriptionale Gene-Silencing gewonnen. In Säugerzellen wird RNAi durch die Gabe von 21 bp kurzen doppelsträngigen RNAs, sogenannten short interfering RNAs (siRNAs), ausgelöst. Zahlreiche Studien belegen das große Potential dieser Methode für die Therapie viraler Infektionen und Erbkrankheiten. Um siRNAs auch in schlecht transfizierbare Systeme wie primäre und nicht-teilende Zellen bzw. ausgewachsene Organismen einzubringen, können zellpenetrierende Peptide (CPPs) verwendet werden. Diese kurzen positiv geladenen Peptide werden von Zellen aufgenommen, wo sie in Endosomen, Phagosomen, aber auch im Zytosol detektierbar sind. Verknüpft mit CPPs werden große Moleküle mit hoher Effizienz von Zellen aufgenommen, die die der meisten konventionellen Transfektionsmethoden übertrifft. In dieser Arbeit wurden peptidgekuppelte siRNAs (pepsiRNAs) als neue Methode zur transienten RNAi in Säugerzellen entwickelt. Hierzu wurden die Peptide über eine Disulfidbrücke mit den siRNAs verknüpft, unter den reduzierenden Bedingungen des Zytosols gespalten wird und , so dass die siRNA im Innern der Zelle freigesetzt wird. PepsiRNAs werden von einer Reihe von Zelltypen aufgenommen, die mit konventionellen Techniken nur schlecht behandelbar sind. Eine deutliche Herunterregulation der Ziel-Genprodukte wurde in einem Konzentrationsbereich von 10 -100 nM beobachtet. SiRNAs mit einer 5´-Thiolmodifikation am sense-Strang wurden durch in vitro Transkription gewonnen, für die ein thiolmodifizierten Nucleotid synthetisiert und dessen Synthese optimiert wurde. Die zellpenetrierenden Peptide Penetratin und Tat wurden rekombinant als Fusionproteine mit Gluatathion-S-Transferase (GST) exprimiert und mit modifizierten Verfahren gereinigt, um die starken Wechselwirkungen der GST-CPPs mit den Zellmembranen zu überwinden. Um die CPPs vom GST-Fusionstag zu trennen, wurde rekombinante TEV-Protease exprimiert und die Aktivität mit der von kommerziell erhältlicher TEV-Protease verglichen. Somit stehen nun Alternativen zur Festphasensynthese zur Verfügung, um die Bestandteile der pepsiRNAs in größeren Mengen herzustellen. Schließlich wurden kleine Moleküle mit zellpenetrierenden Eigenschaften als Ersatz für die Peptideinheit entwickelt. Es konnte gezeigt werden, dass fluoreszenzmarkierte Peptoide verschiedener Länge mit aminofunktionalisierten Seitenketten in Konzentrationen von einigen 10 µM von verschiedenen Säugerzelllinien durch einen endozytoseabhängigen Mechanismus aufgenommen werden. Selbst kleine positiv geladene Moleküle wie fluoresceinmarkiertes Spermin werden über einen endozytoseartigen Mechanismus aufgenommen. Auf diese Weise konnten auch Porphyrine ins Innere der Zellen gebracht werden, wo sie bei Belichtung ihre zytotoxische Wirkung entfalten, so dass sie als neuartiger Wirkstoff für die Photodynamische Therapie weiterentwickelt werden können

Investigating the coding region determinant binding protein-RNA interaction in vitro, in cells and in silico.

The Coding Region-Determinant Binding-Protein (CRDBP) belongs to a family of RNA-binding proteins involved in embryogenesis and oncogenesis. Accumulative evidence suggests that the physical interaction with target mRNAs is a critical determinant for CRDBP function. The goal of this study is to better understand the CRDBP-RNA interaction. The role of the glycine-X-X-glycine motif in each of the KH domains of CRDBP for RNA binding in vitro, in cells and in vivo was investigated. Smaller RNA fragments of c-myc, MITF, MAPK4 and β-TrCP1 bound by CRDBP were determined and used for in silico analysis. Overall, the results showed that the G-X-X-G motif in the KH domains of CRDBP are critical for RNA binding, and that the KH domains play differential roles in binding different RNA molecules. The mapped smaller RNA regions aid in discovering inhibitory molecules and in silico analysis leading to improved approach for understanding the CRDBP RNA interaction. --Leaf ii.The original print copy of this thesis may be available here: http://wizard.unbc.ca/record=b195337