Publications by authors named "François Major"

49 Publications

CAMAP: Artificial neural networks unveil the role of codon arrangement in modulating MHC-I peptides presentation.

PLoS Comput Biol 2021 10 22;17(10):e1009482. Epub 2021 Oct 22.

Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Canada.

MHC-I associated peptides (MAPs) play a central role in the elimination of virus-infected and neoplastic cells by CD8 T cells. However, accurately predicting the MAP repertoire remains difficult, because only a fraction of the transcriptome generates MAPs. In this study, we investigated whether codon arrangement (usage and placement) regulates MAP biogenesis. We developed an artificial neural network called Codon Arrangement MAP Predictor (CAMAP), predicting MAP presentation solely from mRNA sequences flanking the MAP-coding codons (MCCs), while excluding the MCC per se. CAMAP predictions were significantly more accurate when using original codon sequences than shuffled codon sequences which reflect amino acid usage. Furthermore, predictions were independent of mRNA expression and MAP binding affinity to MHC-I molecules and applied to several cell types and species. Combining MAP ligand scores, transcript expression level and CAMAP scores was particularly useful to increase MAP prediction accuracy. Using an in vitro assay, we showed that varying the synonymous codons in the regions flanking the MCCs (without changing the amino acid sequence) resulted in significant modulation of MAP presentation at the cell surface. Taken together, our results demonstrate the role of codon arrangement in the regulation of MAP presentation and support integration of both translational and post-translational events in predictive algorithms to ameliorate modeling of the immunopeptidome.
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http://dx.doi.org/10.1371/journal.pcbi.1009482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8577786PMC
October 2021

From transient recognition to efficient silencing: a RISCky business.

Authors:
François Major

Nat Struct Mol Biol 2020 06;27(6):519-520

Institute for Research in Immunology and Cancer and Department of Computer Science and Operations Research, Université de Montréal, Montreal, QC, Canada.

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http://dx.doi.org/10.1038/s41594-020-0451-3DOI Listing
June 2020

A transcriptome-based approach to identify functional modules within and across primary human immune cells.

PLoS One 2020 29;15(5):e0233543. Epub 2020 May 29.

Centre de recherche, Institut de cardiologie de Montréal, Montréal, Québec, Canada.

Genome-wide transcriptomic analyses have provided valuable insight into fundamental biology and disease pathophysiology. Many studies have taken advantage of the correlation in the expression patterns of the transcriptome to infer a potential biologic function of uncharacterized genes, and multiple groups have examined the relationship between co-expression, co-regulation, and gene function on a broader scale. Given the unique characteristics of immune cells circulating in the blood, we were interested in determining whether it was possible to identify functional co-expression modules in human immune cells. Specifically, we sequenced the transcriptome of nine immune cell types from peripheral blood cells of healthy donors and, using a combination of global and targeted analyses of genes within co-expression modules, we were able to determine functions for these modules that were cell lineage-specific or shared among multiple cell lineages. In addition, our analyses identified transcription factors likely important for immune cell lineage commitment and/or maintenance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0233543PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259617PMC
August 2020

RNA-Puzzles Round IV: 3D structure predictions of four ribozymes and two aptamers.

RNA 2020 08 5;26(8):982-995. Epub 2020 May 5.

Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305, USA.

RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA 3D structure prediction. With agreement from crystallographers, the RNA structures are predicted by various groups before the publication of the crystal structures. We now report the prediction of 3D structures for six RNA sequences: four nucleolytic ribozymes and two riboswitches. Systematic protocols for comparing models and crystal structures are described and analyzed. In these six puzzles, we discuss (i) the comparison between the automated web servers and human experts; (ii) the prediction of coaxial stacking; (iii) the prediction of structural details and ligand binding; (iv) the development of novel prediction methods; and (v) the potential improvements to be made. We show that correct prediction of coaxial stacking and tertiary contacts is essential for the prediction of RNA architecture, while ligand binding modes can only be predicted with low resolution and simultaneous prediction of RNA structure with accurate ligand binding still remains out of reach. All the predicted models are available for the future development of force field parameters and the improvement of comparison and assessment tools.
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http://dx.doi.org/10.1261/rna.075341.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7373991PMC
August 2020

Apoptotic endothelial cells release small extracellular vesicles loaded with immunostimulatory viral-like RNAs.

Sci Rep 2019 05 10;9(1):7203. Epub 2019 May 10.

Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, QC, H3C 3J7, Canada.

Endothelial cells have multifaceted interactions with the immune system, both as initiators and targets of immune responses. In vivo, apoptotic endothelial cells release two types of extracellular vesicles upon caspase-3 activation: apoptotic bodies and exosome-like nanovesicles (ApoExos). Only ApoExos are immunogenic: their injection causes inflammation and autoimmunity in mice. Based on deep sequencing of total RNA, we report that apoptotic bodies and ApoExos are loaded with divergent RNA cargos that are not released by healthy endothelial cells. Apoptotic bodies, like endothelial cells, contain mainly ribosomal RNA whereas ApoExos essentially contain non-ribosomal non-coding RNAs. Endogenous retroelements, bearing viral-like features, represented half of total ApoExos RNA content. ApoExos also contained several copies of unedited Alu repeats and large amounts of non-coding RNAs with a demonstrated role in autoimmunity such as U1 RNA and Y RNA. Moreover, ApoExos RNAs had a unique nucleotide composition and secondary structure characterized by strong enrichment in U-rich motifs and unstably folded RNAs. Globally, ApoExos were therefore loaded with RNAs that can stimulate a variety of RIG-I-like receptors and endosomal TLRs. Hence, apoptotic endothelial cells selectively sort in ApoExos a diversified repertoire of immunostimulatory "self RNAs" that are tailor-made for initiation of innate immune responses and autoimmunity.
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http://dx.doi.org/10.1038/s41598-019-43591-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510763PMC
May 2019

The sequence features that define efficient and specific hAGO2-dependent miRNA silencing guides.

Nucleic Acids Res 2018 09;46(16):8181-8196

Institut de recherche en immunologie et en cancérologie (IRIC), Université de Montréal, Montréal, Québec H3C 3J7, Canada.

MicroRNAs (miRNAs) are ribonucleic acids (RNAs) of ∼21 nucleotides that interfere with the translation of messenger RNAs (mRNAs) and play significant roles in development and diseases. In bilaterian animals, the specificity of miRNA targeting is determined by sequence complementarity involving the seed. However, the role of the remaining nucleotides (non-seed) is only vaguely defined, impacting negatively on our ability to efficiently use miRNAs exogenously to control gene expression. Here, using reporter assays, we deciphered the role of the base pairs formed between the non-seed region and target mRNA. We used molecular modeling to reveal that this mechanism corresponds to the formation of base pairs mediated by ordered motions of the miRNA-induced silencing complex. Subsequently, we developed an algorithm based on this distinctive recognition to predict from sequence the levels of mRNA downregulation with high accuracy (r2 > 0.5, P-value < 10-12). Overall, our discovery improves the design of miRNA-guide sequences used to simultaneously downregulate the expression of multiple predetermined target genes.
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http://dx.doi.org/10.1093/nar/gky546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6144789PMC
September 2018

Corrigendum: RNA-MoIP: prediction of RNA secondary structure and local 3D motifs from sequence data.

Nucleic Acids Res 2017 07;45(W1):W573

School of Computer Science, McGill University, 3480 University Street, Montreal, QC H3A 0E9, Canada.

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http://dx.doi.org/10.1093/nar/gkx575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570209PMC
July 2017

RNA-MoIP: prediction of RNA secondary structure and local 3D motifs from sequence data.

Nucleic Acids Res 2017 07;45(W1):W440-W444

School of Computer Science, McGill University, 3480 University Street, Montreal, QC H3A 0E9, Canada.

RNA structures are hierarchically organized. The secondary structure is articulated around sophisticated local three-dimensional (3D) motifs shaping the full 3D architecture of the molecule. Recent contributions have identified and organized recurrent local 3D motifs, but applications of this knowledge for predictive purposes is still in its infancy. We recently developed a computational framework, named RNA-MoIP, to reconcile RNA secondary structure and local 3D motif information available in databases. In this paper, we introduce a web service using our software for predicting RNA hybrid 2D-3D structures from sequence data only. Optionally, it can be used for (i) local 3D motif prediction or (ii) the refinement of user-defined secondary structures. Importantly, our web server automatically generates a script for the MC-Sym software, which can be immediately used to quickly predict all-atom RNA 3D models. The web server is available at http://rnamoip.cs.mcgill.ca.
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http://dx.doi.org/10.1093/nar/gkx429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5793723PMC
July 2017

RNA-Puzzles Round III: 3D RNA structure prediction of five riboswitches and one ribozyme.

RNA 2017 05 30;23(5):655-672. Epub 2017 Jan 30.

Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Poland.

RNA-Puzzles is a collective experiment in blind 3D RNA structure prediction. We report here a third round of RNA-Puzzles. Five puzzles, 4, 8, 12, 13, 14, all structures of riboswitch aptamers and puzzle 7, a ribozyme structure, are included in this round of the experiment. The riboswitch structures include biological binding sites for small molecules (-adenosyl methionine, cyclic diadenosine monophosphate, 5-amino 4-imidazole carboxamide riboside 5'-triphosphate, glutamine) and proteins (YbxF), and one set describes large conformational changes between ligand-free and ligand-bound states. The Varkud satellite ribozyme is the most recently solved structure of a known large ribozyme. All puzzles have established biological functions and require structural understanding to appreciate their molecular mechanisms. Through the use of fast-track experimental data, including multidimensional chemical mapping, and accurate prediction of RNA secondary structure, a large portion of the contacts in 3D have been predicted correctly leading to similar topologies for the top ranking predictions. Template-based and homology-derived predictions could predict structures to particularly high accuracies. However, achieving biological insights from de novo prediction of RNA 3D structures still depends on the size and complexity of the RNA. Blind computational predictions of RNA structures already appear to provide useful structural information in many cases. Similar to the previous RNA-Puzzles Round II experiment, the prediction of non-Watson-Crick interactions and the observed high atomic clash scores reveal a notable need for an algorithm of improvement. All prediction models and assessment results are available at http://ahsoka.u-strasbg.fr/rnapuzzles/.
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http://dx.doi.org/10.1261/rna.060368.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393176PMC
May 2017

Exploring Alternative RNA Structure Sets Using MC-Flashfold and db2cm.

Methods Mol Biol 2016 ;1490:237-51

Department of Computer Science and Operations Research, Institute for Research in Immunology and Cancer, Université de Montréal, 6128, Downtown Station, Montréal, QC, Canada, H3C 3J7.

We created an accelerated version of MC-Fold called MC-Flashfold that allows us to compute large numbers of competing secondary structures including noncanonical base pairs. We visualize the base pairs in these sets using high quality intuitive dot plots and arc plots. Our new tools allow us to explore RNA dynamics by visualizing the competing structures in free energy bands. Here we describe how to use these tools to generate dot plots that reveal the postulated anti-terminator stem in the E. coli trp operon leader sequence. These plots show the anti-terminator hairpin loop during transcription and as a minor population of the full-length leader sequence. This is a case of switching RNA structure that had been originally postulated based on short dyad inverted repeats. Other switching RNA sequences can be analyzed by using our method.
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http://dx.doi.org/10.1007/978-1-4939-6433-8_15DOI Listing
January 2018

Structural dynamics control the MicroRNA maturation pathway.

Nucleic Acids Res 2016 Nov 19;44(20):9956-9964. Epub 2016 Sep 19.

Institute for Research in Immunology and Cancer, and Department of Computer Science and Operations Research, Université de Montréal, PO Box 6128, Downtown Station, Montréal, Québec H3C 3J7, Canada

MicroRNAs (miRNAs) are crucial gene expression regulators and first-order suspects in the development and progression of many diseases. Comparative analysis of cancer cell expression data highlights many deregulated miRNAs. Low expression of miR-125a was related to poor breast cancer prognosis. Interestingly, a single nucleotide polymorphism (SNP) in miR-125a was located within a minor allele expressed by breast cancer patients. The SNP is not predicted to affect the ground state structure of the primary transcript or precursor, but neither the precursor nor mature product is detected by RT-qPCR. How this SNP modulates the maturation of miR-125a is poorly understood. Here, building upon a model of RNA dynamics derived from nuclear magnetic resonance studies, we developed a quantitative model enabling the visualization and comparison of networks of transient structures. We observed a high correlation between the distances between networks of variants with that of their respective wild types and their relative degrees of maturation to the latter, suggesting an important role of transient structures in miRNA homeostasis. We classified the human miRNAs according to pairwise distances between their networks of transient structures.
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http://dx.doi.org/10.1093/nar/gkw793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5175353PMC
November 2016

MiRBooking simulates the stoichiometric mode of action of microRNAs.

Nucleic Acids Res 2015 Aug 18;43(14):6730-8. Epub 2015 Jun 18.

Institute for Research in Immunology and Cancer, and Department of Computer Science and Operations Research, Université de Montréal, PO Box 6128, Downtown Station, Montréal, Québec H3C 3J7, Canada

In eucaryotes, gene expression is regulated by microRNAs (miRNAs) which bind to messenger RNAs (mRNAs) and interfere with their translation into proteins, either by promoting their degradation or inducing their repression. We study the effect of miRNA interference on each gene using experimental methods, such as microarrays and RNA-seq at the mRNA level, or luciferase reporter assays and variations of SILAC at the protein level. Alternatively, computational predictions would provide clear benefits. However, no algorithm toward this task has ever been proposed. Here, we introduce a new algorithm to predict genome-wide expression data from initial transcriptome abundance. The algorithm simulates the miRNA and mRNA hybridization competition that occurs in given cellular conditions, and derives the whole set of miRNA::mRNA interactions at equilibrium (microtargetome). Interestingly, solving the competition improves the accuracy of miRNA target predictions. Furthermore, this model implements a previously reported and fundamental property of the microtargetome: the binding between a miRNA and a mRNA depends on their sequence complementarity, but also on the abundance of all RNAs expressed in the cell, i.e. the stoichiometry of all the miRNA sites and all the miRNAs given their respective abundance. This model generalizes the miRNA-induced synchronistic silencing previously observed, and described as sponges and competitive endogenous RNAs.
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http://dx.doi.org/10.1093/nar/gkv619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4538818PMC
August 2015

RNA-Puzzles Round II: assessment of RNA structure prediction programs applied to three large RNA structures.

RNA 2015 Jun 16;21(6):1066-84. Epub 2015 Apr 16.

Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, 02-109 Warsaw, Poland.

This paper is a report of a second round of RNA-Puzzles, a collective and blind experiment in three-dimensional (3D) RNA structure prediction. Three puzzles, Puzzles 5, 6, and 10, represented sequences of three large RNA structures with limited or no homology with previously solved RNA molecules. A lariat-capping ribozyme, as well as riboswitches complexed to adenosylcobalamin and tRNA, were predicted by seven groups using RNAComposer, ModeRNA/SimRNA, Vfold, Rosetta, DMD, MC-Fold, 3dRNA, and AMBER refinement. Some groups derived models using data from state-of-the-art chemical-mapping methods (SHAPE, DMS, CMCT, and mutate-and-map). The comparisons between the predictions and the three subsequently released crystallographic structures, solved at diffraction resolutions of 2.5-3.2 Å, were carried out automatically using various sets of quality indicators. The comparisons clearly demonstrate the state of present-day de novo prediction abilities as well as the limitations of these state-of-the-art methods. All of the best prediction models have similar topologies to the native structures, which suggests that computational methods for RNA structure prediction can already provide useful structural information for biological problems. However, the prediction accuracy for non-Watson-Crick interactions, key to proper folding of RNAs, is low and some predicted models had high Clash Scores. These two difficulties point to some of the continuing bottlenecks in RNA structure prediction. All submitted models are available for download at http://ahsoka.u-strasbg.fr/rnapuzzles/.
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http://dx.doi.org/10.1261/rna.049502.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4436661PMC
June 2015

Noncoding regions of C. elegans mRNA undergo selective adenosine to inosine deamination and contain a small number of editing sites per transcript.

RNA Biol 2015 ;12(2):162-74

a Medical Sciences Program ; Indiana University ; Bloomington , IN USA.

ADARs (Adenosine deaminases that act on RNA) "edit" RNA by converting adenosines to inosines within double-stranded regions. The primary targets of ADARs are long duplexes present within noncoding regions of mRNAs, such as introns and 3' untranslated regions (UTRs). Because adenosine and inosine have different base-pairing properties, editing within these regions can alter splicing and recognition by small RNAs. However, despite numerous studies identifying multiple editing sites in these genomic regions, little is known about the extent to which editing sites co-occur on individual transcripts or the functional output of these combinatorial editing events. To begin to address these questions, we performed an ultra-deep sequencing analysis of 4 Caenorhabditis elegans 3' UTRs that are known ADAR targets. Synchronous editing events were determined for the long duplexes in vivo. Furthermore, the validity of each editing event was confirmed by sequencing the same regions of mRNA from worms that lack A-to-I editing. This analysis identified a large number of editing sites that can occur within each 3' UTR, but interestingly, each individual transcript contained only a small fraction of these A-to-I editing events. In addition, editing patterns were not random, indicating that an editing event can affect the efficiency of editing at subsequent adenosines. Furthermore, we identified specific sites that can be both positively and negatively correlated with additional sites leading to mutually exclusive editing patterns. These results suggest that editing in noncoding regions is selective and hyper-editing of cellular RNAs is rare.
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http://dx.doi.org/10.1080/15476286.2015.1017220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615841PMC
December 2015

Computational identification of RNA functional determinants by three-dimensional quantitative structure-activity relationships.

Nucleic Acids Res 2014 8;42(17):11261-71. Epub 2014 Sep 8.

Institute for Research in Immunology and Cancer, Université de Montréal, PO Box 6128, Downtown Station, Montréal, Québec H3C 3J7, Canada Department of Computer Science and Operations Research, Université de Montréal, PO Box 6128, Downtown Station, Montréal, Québec H3C 3J7, Canada

Anti-infection drugs target vital functions of infectious agents, including their ribosome and other essential non-coding RNAs. One of the reasons infectious agents become resistant to drugs is due to mutations that eliminate drug-binding affinity while maintaining vital elements. Identifying these elements is based on the determination of viable and lethal mutants and associated structures. However, determining the structure of enough mutants at high resolution is not always possible. Here, we introduce a new computational method, MC-3DQSAR, to determine the vital elements of target RNA structure from mutagenesis and available high-resolution data. We applied the method to further characterize the structural determinants of the bacterial 23S ribosomal RNA sarcin-ricin loop (SRL), as well as those of the lead-activated and hammerhead ribozymes. The method was accurate in confirming experimentally determined essential structural elements and predicting the viability of new SRL variants, which were either observed in bacteria or validated in bacterial growth assays. Our results indicate that MC-3DQSAR could be used systematically to evaluate the drug-target potentials of any RNA sites using current high-resolution structural data.
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http://dx.doi.org/10.1093/nar/gku816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176186PMC
February 2015

Autism-related deficits via dysregulated eIF4E-dependent translational control.

Nature 2013 Jan 21;493(7432):371-7. Epub 2012 Nov 21.

Department of Biochemistry & Goodman Cancer Research Centre, McGill University, Montreal, Quebec H3A 1A3, Canada.

Hyperconnectivity of neuronal circuits due to increased synaptic protein synthesis is thought to cause autism spectrum disorders (ASDs). The mammalian target of rapamycin (mTOR) is strongly implicated in ASDs by means of upstream signalling; however, downstream regulatory mechanisms are ill-defined. Here we show that knockout of the eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2)-an eIF4E repressor downstream of mTOR-or eIF4E overexpression leads to increased translation of neuroligins, which are postsynaptic proteins that are causally linked to ASDs. Mice that have the gene encoding 4E-BP2 (Eif4ebp2) knocked out exhibit an increased ratio of excitatory to inhibitory synaptic inputs and autistic-like behaviours (that is, social interaction deficits, altered communication and repetitive/stereotyped behaviours). Pharmacological inhibition of eIF4E activity or normalization of neuroligin 1, but not neuroligin 2, protein levels restores the normal excitation/inhibition ratio and rectifies the social behaviour deficits. Thus, translational control by eIF4E regulates the synthesis of neuroligins, maintaining the excitation-to-inhibition balance, and its dysregulation engenders ASD-like phenotypes.
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http://dx.doi.org/10.1038/nature11628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4133997PMC
January 2013

Towards 3D structure prediction of large RNA molecules: an integer programming framework to insert local 3D motifs in RNA secondary structure.

Bioinformatics 2012 Jun;28(12):i207-14

School of Computer Science & McGill Centre for Bioinformatics, McGill University, Montréal, Canada.

Motivation: The prediction of RNA 3D structures from its sequence only is a milestone to RNA function analysis and prediction. In recent years, many methods addressed this challenge, ranging from cycle decomposition and fragment assembly to molecular dynamics simulations. However, their predictions remain fragile and limited to small RNAs. To expand the range and accuracy of these techniques, we need to develop algorithms that will enable to use all the structural information available. In particular, the energetic contribution of secondary structure interactions is now well documented, but the quantification of non-canonical interactions-those shaping the tertiary structure-is poorly understood. Nonetheless, even if a complete RNA tertiary structure energy model is currently unavailable, we now have catalogues of local 3D structural motifs including non-canonical base pairings. A practical objective is thus to develop techniques enabling us to use this knowledge for robust RNA tertiary structure predictors.

Results: In this work, we introduce RNA-MoIP, a program that benefits from the progresses made over the last 30 years in the field of RNA secondary structure prediction and expands these methods to incorporate the novel local motif information available in databases. Using an integer programming framework, our method refines predicted secondary structures (i.e. removes incorrect canonical base pairs) to accommodate the insertion of RNA 3D motifs (i.e. hairpins, internal loops and k-way junctions). Then, we use predictions as templates to generate complete 3D structures with the MC-Sym program. We benchmarked RNA-MoIP on a set of 9 RNAs with sizes varying from 53 to 128 nucleotides. We show that our approach (i) improves the accuracy of canonical base pair predictions; (ii) identifies the best secondary structures in a pool of suboptimal structures; and (iii) predicts accurate 3D structures of large RNA molecules.

Availability: RNA-MoIP is publicly available at: http://csb.cs.mcgill.ca/RNAMoIP.
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http://dx.doi.org/10.1093/bioinformatics/bts226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3371858PMC
June 2012

Determining RNA three-dimensional structures using low-resolution data.

J Struct Biol 2012 Sep 23;179(3):252-60. Epub 2012 Feb 23.

Biochemistry Department, The University of Chicago, 929 E. 57th Street, Chicago, IL 60637, USA.

Knowing the 3-D structure of an RNA is fundamental to understand its biological function. Nowadays X-ray crystallography and NMR spectroscopy are systematically applied to newly discovered RNAs. However, the application of these high-resolution techniques is not always possible, and thus scientists must turn to lower resolution alternatives. Here, we introduce a pipeline to systematically generate atomic resolution 3-D structures that are consistent with low-resolution data sets. We compare and evaluate the discriminative power of a number of low-resolution experimental techniques to reproduce the structure of the Escherichia coli tRNA(VAL) and P4-P6 domain of the Tetrahymena thermophila group I intron. We test single and combinations of the most accessible low-resolution techniques, i.e. hydroxyl radical footprinting (OH), methidiumpropyl-EDTA (MPE), multiplexed hydroxyl radical cleavage (MOHCA), and small-angle X-ray scattering (SAXS). We show that OH-derived constraints are accurate to discriminate structures at the atomic level, whereas EDTA-based constraints apply to global shape determination. We provide a guide for choosing which experimental techniques or combination of thereof is best in which context. The pipeline represents an important step towards high-throughput low-resolution RNA structure determination.
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http://dx.doi.org/10.1016/j.jsb.2011.12.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3423474PMC
September 2012

RNA-Puzzles: a CASP-like evaluation of RNA three-dimensional structure prediction.

RNA 2012 Apr 23;18(4):610-25. Epub 2012 Feb 23.

Architecture et Réactivité de l'ARN, Université de Strasbourg, IBMC-CNRS, F-67084 Strasbourg, France.

We report the results of a first, collective, blind experiment in RNA three-dimensional (3D) structure prediction, encompassing three prediction puzzles. The goals are to assess the leading edge of RNA structure prediction techniques; compare existing methods and tools; and evaluate their relative strengths, weaknesses, and limitations in terms of sequence length and structural complexity. The results should give potential users insight into the suitability of available methods for different applications and facilitate efforts in the RNA structure prediction community in ongoing efforts to improve prediction tools. We also report the creation of an automated evaluation pipeline to facilitate the analysis of future RNA structure prediction exercises.
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http://dx.doi.org/10.1261/rna.031054.111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3312550PMC
April 2012

Structural messenger RNA contains cytokeratin polymerization and depolymerization signals.

Cell Tissue Res 2011 Nov 11;346(2):209-22. Epub 2011 Oct 11.

Department of Surgery, The Methodist Hospital and The Methodist Hospital Research Institute, 6565 Fannin Street, Houston, TX 77030, USA.

We have previously shown that VegT mRNA plays a structural (translation-independent) role in the organization of the cytokeratin cytoskeleton in Xenopus oocytes. The depletion of VegT mRNA causes the fragmentation of the cytokeratin network in the vegetal cortex of Xenopus oocytes. This effect can be rescued by the injection of synthetic VegT RNA into the oocyte. Here, we show that the structural function of VegT mRNA in Xenopus oocyte depends on its combinatory signals for the induction or facilitation and for the maintenance of the depolymerization vs. polymerization status of cytokeratin filaments and that the 300-nucleotide fragment of VegT RNA isolated from the context of the entire molecule induces and maintains the depolymerization of cytokeratin filaments when injected into Xenopus oocytes. A computational analysis of three homologous Xenopus VegT mRNAs has revealed the presence, within this 300-nucleotide region, of a conserved base-pairing (hairpin) configuration that might function in RNA/protein interactions.
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http://dx.doi.org/10.1007/s00441-011-1255-xDOI Listing
November 2011

NMR structure of a 4 x 4 nucleotide RNA internal loop from an R2 retrotransposon: identification of a three purine-purine sheared pair motif and comparison to MC-SYM predictions.

RNA 2011 Sep 21;17(9):1664-77. Epub 2011 Jul 21.

Department of Chemistry, University of Rochester, Rochester, New York 14627, USA.

The NMR solution structure is reported of a duplex, 5'GUGAAGCCCGU/3'UCACAGGAGGC, containing a 4 × 4 nucleotide internal loop from an R2 retrotransposon RNA. The loop contains three sheared purine-purine pairs and reveals a structural element found in other RNAs, which we refer to as the 3RRs motif. Optical melting measurements of the thermodynamics of the duplex indicate that the internal loop is 1.6 kcal/mol more stable at 37°C than predicted. The results identify the 3RRs motif as a common structural element that can facilitate prediction of 3D structure. Known examples include internal loops having the pairings: 5'GAA/3'AGG, 5'GAG/3'AGG, 5'GAA/3'AAG, and 5'AAG/3'AGG. The structural information is compared with predictions made with the MC-Sym program.
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http://dx.doi.org/10.1261/rna.2641911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3162332PMC
September 2011

RNA G-Quadruplexes in the model plant species Arabidopsis thaliana: prevalence and possible functional roles.

Nucleic Acids Res 2010 Dec 21;38(22):8149-63. Epub 2010 Sep 21.

Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802-5302, USA.

Tandem stretches of guanines can associate in hydrogen-bonded arrays to form G-quadruplexes, which are stabilized by K(+) ions. Using computational methods, we searched for G-Quadruplex Sequence (GQS) patterns in the model plant species Arabidopsis thaliana. We found ∼ 1200 GQS with a G(3) repeat sequence motif, most of which are located in the intergenic region. Using a Markov modeled genome, we determined that GQS are significantly underrepresented in the genome. Additionally, we found ∼ 43,000 GQS with a G(2) repeat sequence motif; notably, 80% of these were located in genic regions, suggesting that these sequences may fold at the RNA level. Gene Ontology functional analysis revealed that GQS are overrepresented in genes encoding proteins of certain functional categories, including enzyme activity. Conversely, GQS are underrepresented in other categories of genes, notably those for non-coding RNAs such as tRNAs and rRNAs. We also find that genes that are differentially regulated by drought are significantly more likely to contain a GQS. CD-detected K(+) titrations performed on representative RNAs verified formation of quadruplexes at physiological K(+) concentrations. Overall, this study indicates that GQS are present at unique locations in Arabidopsis and that folding of RNA GQS may play important roles in regulating gene expression.
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http://dx.doi.org/10.1093/nar/gkq804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3001093PMC
December 2010

RNA structure determination using SAXS data.

J Phys Chem B 2010 Aug;114(31):10039-48

Department of Biochemistry and Molecular Biology, 929 East 57th Street, University of Chicago, Chicago, Illinois 60637, USA.

Exploiting the experimental information from small-angle X-ray solution scattering (SAXS) in conjunction with structure prediction algorithms can be advantageous in the case of ribonucleic acids (RNA), where global restraints on the 3D fold are often lacking. Traditional usage of SAXS data often starts by attempting to reconstruct the molecular shape ab initio, which is subsequently used to assess the quality of a model. Here, an alternative strategy is explored whereby the models from a very large decoy set are directly sorted according to their fit to the SAXS data. For rapid computation of SAXS patterns, the method developed here makes use of a coarse-grained representation of RNA. It also accounts for the explicit treatment of the contribution to the scattering of water molecules and ions surrounding the RNA. The method, called Fast-SAXS-RNA, is first calibrated using a tRNA (tRNA-val) and then tested on the P4-P6 fragment of group I intron (P4-P6). Fast-SAXS-RNA is then used as a filter for decoy models generated by the MC-Fold and MC-Sym pipeline, a suite of RNA 3D all-atom structure algorithms that encode and exploit RNA 3D architectural principles. The ability of Fast-SAXS-RNA to discriminate native folds is tested against three widely used RNA molecules in molecular modeling benchmarks: the tRNA, the P4-P6, and a synthetic hairpin suspected to assemble into a homodimer. For each molecule, a large pool of decoys are generated, scored, and ranked using Fast-SAXS-RNA. The method is able to identify low-rmsd models among top ranking structures, for both tRNA and P4-P6. For the hairpin, the approach correctly identifies the dimeric state as the solution structure over the monomeric state and alternative secondary structures. The method offers a powerful strategy for recognizing native RNA conformations as well as multimeric assemblies and alternative secondary structures, thus enabling high-throughput RNA structure determination using SAXS data.
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http://dx.doi.org/10.1021/jp1057308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3164809PMC
August 2010

RKB: a Semantic Web knowledge base for RNA.

J Biomed Semantics 2010 Jun 22;1 Suppl 1:S2. Epub 2010 Jun 22.

Department of Biology, Carleton University 1125 Colonel By Drive, K1S5B6, Ottawa, Canada .

Increasingly sophisticated knowledge about RNA structure and function requires an inclusive knowledge representation that facilitates the integration of independently -generated information arising from such efforts as genome sequencing projects, microarray analyses, structure determination and RNA SELEX experiments. While RNAML, an XML-based representation, has been proposed as an exchange format for a select subset of information, it lacks domain-specific semantics that are essential for answering questions that require expert knowledge. Here, we describe an RNA knowledge base (RKB) for structure-based knowledge using RDF/OWL Semantic Web technologies. RKB extends a number of ontologies and contains basic terminology for nucleic acid composition along with context/model-specific structural features such as sugar conformations, base pairings and base stackings. RKB (available at http://semanticscience.org/projects/rkb) is populated with PDB entries and MC-Annotate structural annotation. We show queries to the RKB using description logic reasoning, thus opening the door to question answering over independently-published RNA knowledge using Semantic Web technologies.
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http://dx.doi.org/10.1186/2041-1480-1-S1-S2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2903721PMC
June 2010

Designing small multiple-target artificial RNAs.

Nucleic Acids Res 2010 Jul 7;38(13):e140. Epub 2010 May 7.

Département de Biochimie, Université de Montréal, Montréal, QC H3C 3J7 Canada.

MicroRNAs (miRNAs) are naturally occurring small RNAs that regulate the expression of several genes. MiRNAs' targeting rules are based on sequence complementarity between their mature products and targeted genes' mRNAs. Based on our present understanding of those rules, we developed an algorithm to design artificial miRNAs to target simultaneously a set of predetermined genes. To validate in silico our algorithm, we tested different sets of genes known to be targeted by a single miRNA. The algorithm finds the seed of the corresponding miRNA among the solutions, which also include the seeds of new artificial miRNA sequences potentially capable of targeting these genes as well. We also validated the functionality of some artificial miRNAs designed to target simultaneously members of the E2F family. These artificial miRNAs reproduced the effects of E2Fs inhibition in both normal human fibroblasts and prostate cancer cells where they inhibited cell proliferation and induced cellular senescence. We conclude that the current miRNA targeting rules based on the seed sequence work to design multiple-target artificial miRNAs. This approach may find applications in both research and therapeutics.
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http://dx.doi.org/10.1093/nar/gkq354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2910070PMC
July 2010

Recognition and coupling of A-to-I edited sites are determined by the tertiary structure of the RNA.

Nucleic Acids Res 2009 Nov 8;37(20):6916-26. Epub 2009 Sep 8.

Department of Molecular Biology and Functional Genomics, Stockholm University, S-106 91 Stockholm, Sweden.

Adenosine-to-inosine (A-to-I) editing has been shown to be an important mechanism that increases protein diversity in the brain of organisms from human to fly. The family of ADAR enzymes converts some adenosines of RNA duplexes to inosines through hydrolytic deamination. The adenosine recognition mechanism is still largely unknown. Here, to investigate it, we analyzed a set of selectively edited substrates with a cluster of edited sites. We used a large set of individual transcripts sequenced by the 454 sequencing technique. On average, we analyzed 570 single transcripts per edited region at four different developmental stages from embryogenesis to adulthood. To our knowledge, this is the first time, large-scale sequencing has been used to determine synchronous editing events. We demonstrate that edited sites are only coupled within specific distances from each other. Furthermore, our results show that the coupled sites of editing are positioned on the same side of a helix, indicating that the three-dimensional structure is key in ADAR enzyme substrate recognition. Finally, we propose that editing by the ADAR enzymes is initiated by their attraction to one principal site in the substrate.
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http://dx.doi.org/10.1093/nar/gkp731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2777444PMC
November 2009

New metrics for comparing and assessing discrepancies between RNA 3D structures and models.

RNA 2009 Oct 26;15(10):1875-85. Epub 2009 Aug 26.

Institute for Research in Immunology and Cancer, Department of Computer Science and Operations Research, Université de Montréal, Montréal, Québec, Canada.

To benchmark progress made in RNA three-dimensional modeling and assess newly developed techniques, reliable and meaningful comparison metrics and associated tools are necessary. Generally, the average root-mean-square deviations (RMSDs) are quoted. However, RMSD can be misleading since errors are spread over the whole molecule and do not account for the specificity of RNA base interactions. Here, we introduce two new metrics that are particularly suitable to RNAs: the deformation index and deformation profile. The deformation index is calibrated by the interaction network fidelity, which considers base-base-stacking and base-base-pairing interactions within the target structure. The deformation profile highlights dissimilarities between structures at the nucleotide scale for both intradomain and interdomain interactions. Our results show that there is little correlation between RMSD and interaction network fidelity. The deformation profile is a tool that allows for rapid assessment of the origins of discrepancies.
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http://dx.doi.org/10.1261/rna.1700409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2743038PMC
October 2009

Molecular basis of TRAP-5'SL RNA interaction in the Bacillus subtilis trp operon transcription attenuation mechanism.

RNA 2009 Jan 25;15(1):55-66. Epub 2008 Nov 25.

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.

Expression of the Bacillus subtilis trpEDCFBA operon is regulated by the interaction of tryptophan-activated TRAP with 11 (G/U)AG trinucleotide repeats that lie in the leader region of the nascent trp transcript. Bound TRAP prevents folding of an antiterminator structure and favors formation of an overlapping intrinsic terminator hairpin upstream of the trp operon structural genes. A 5'-stem-loop (5'SL) structure that forms just upstream of the triplet repeat region increases the affinity of TRAP-trp RNA interaction, thereby increasing the efficiency of transcription termination. Single-stranded nucleotides in the internal loop and in the hairpin loop of the 5'SL are important for TRAP binding. We show here that altering the distance between these two loops suggests that G7, A8, and A9 from the internal loop and A19 and G20 from the hairpin loop constitute two structurally discrete TRAP-binding regions. Photochemical cross-linking experiments also show that the hairpin loop of the 5'SL is in close proximity to the flexible loop region of TRAP during TRAP-5'SL interaction. The dimensions of B. subtilis TRAP and of a three-dimensional model of the 5'SL generated using the MC-Sym and MC-Fold pipeline imply that the 5'SL binds the protein in an orientation where the helical axis of the 5'SL is perpendicular to the plane of TRAP. This interaction not only increases the affinity of TRAP-trp leader RNA interaction, but also orients the downstream triplet repeats for interaction with the 11 KKR motifs that lie on TRAP's perimeter, increasing the likelihood that TRAP will bind in time to promote termination.
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http://dx.doi.org/10.1261/rna.1314409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2612762PMC
January 2009

The MC-Fold and MC-Sym pipeline infers RNA structure from sequence data.

Nature 2008 Mar;452(7183):51-5

Institute for Research in Immunology and Cancer, Department of Computer Science and Operations Research, Université de Montréal, PO Box 6128, Downtown Station, Montréal, Québec H3C 3J7, Canada.

The classical RNA secondary structure model considers A.U and G.C Watson-Crick as well as G.U wobble base pairs. Here we substitute it for a new one, in which sets of nucleotide cyclic motifs define RNA structures. This model allows us to unify all base pairing energetic contributions in an effective scoring function to tackle the problem of RNA folding. We show how pipelining two computer algorithms based on nucleotide cyclic motifs, MC-Fold and MC-Sym, reproduces a series of experimentally determined RNA three-dimensional structures from the sequence. This demonstrates how crucial the consideration of all base-pairing interactions is in filling the gap between sequence and structure. We use the pipeline to define rules of precursor microRNA folding in double helices, despite the presence of a number of presumed mismatches and bulges, and to propose a new model of the human immunodeficiency virus-1 -1 frame-shifting element.
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http://dx.doi.org/10.1038/nature06684DOI Listing
March 2008

Role of SLV in SLI substrate recognition by the Neurospora VS ribozyme.

RNA 2008 Apr 26;14(4):736-48. Epub 2008 Feb 26.

Département de Biochimie, Université de Montréal, Montréal, H3C 3J7 Canada.

Substrate recognition by the VS ribozyme involves a magnesium-dependent loop/loop interaction between the SLI substrate and the SLV hairpin from the catalytic domain. Recent NMR studies of SLV demonstrated that magnesium ions stabilize a U-turn loop structure and trigger a conformational change for the extruded loop residue U700, suggesting a role for U700 in SLI recognition. Here, we kinetically characterized VS ribozyme mutants to evaluate the contribution of U700 and other SLV loop residues to SLI recognition. To help interpret the kinetic data, we structurally characterized the SLV mutants by NMR spectroscopy and generated a three-dimensional model of the SLI/SLV complex by homology modeling with MC-Sym. We demonstrated that the mutation of U700 by A, C, or G does not significantly affect ribozyme activity, whereas deletion of U700 dramatically impairs this activity. The U700 backbone is likely important for SLI recognition, but does not appear to be required for either the structural integrity of the SLV loop or for direct interactions with SLI. Thus, deletion of U700 may affect other aspects of SLI recognition, such as magnesium ion binding and SLV loop dynamics. As part of our NMR studies, we developed a convenient assay based on detection of unusual (31)P and (15)N N7 chemical shifts to probe the formation of U-turn structures in RNAs. Our model of the SLI/SLV complex, which is compatible with biochemical data, leads us to propose novel interactions at the loop I/loop V interface.
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http://dx.doi.org/10.1261/rna.824308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2271362PMC
April 2008
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