Publications by authors named "Rory Johnson"

55 Publications

Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs.

Genome Res 2021 Mar 11;31(3):461-471. Epub 2021 Feb 11.

Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010 Bern, Switzerland.

CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double-strand breaks (DSBs), is removed during nonhomologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false-negative results. By using an endogenous reporter system, we show that repression of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-an early step in NHEJ-yields substantial increases in DNA deletion. This is observed across diverse cell lines, gene delivery methods, commercial inhibitors, and guide RNAs, including those that otherwise display negligible activity. We further show that DNA-PKcs inhibition can be used to boost the sensitivity of pooled functional screens and detect true-positive hits that would otherwise be overlooked. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion.
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http://dx.doi.org/10.1101/gr.265736.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919447PMC
March 2021

Annotation of Full-Length Long Noncoding RNAs with Capture Long-Read Sequencing (CLS).

Methods Mol Biol 2021 ;2254:133-159

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Catalonia, Spain.

Metazoan genomes produce thousands of long-noncoding RNAs (lncRNAs), of which just a small fraction have been well characterized. Understanding their biological functions requires accurate annotations, or maps of the precise location and structure of genes and transcripts in the genome. Current lncRNA annotations are limited by compromises between quality and size, with many gene models being fragmentary or uncatalogued. To overcome this, the GENCODE consortium has developed RNA capture long-read sequencing (CLS), an approach combining targeted RNA capture with third-generation long-read sequencing. CLS provides accurate annotations at high-throughput rates. It eliminates the need for noisy transcriptome assembly from short reads, and requires minimal manual curation. The full-length transcript models produced are of quality comparable to present-day manually curated annotations. Here we describe a detailed CLS protocol, from probe design through long-read sequencing to creation of final annotations.
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http://dx.doi.org/10.1007/978-1-0716-1158-6_9DOI Listing
March 2021

GENCODE 2021.

Nucleic Acids Res 2021 01;49(D1):D916-D923

European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK.

The GENCODE project annotates human and mouse genes and transcripts supported by experimental data with high accuracy, providing a foundational resource that supports genome biology and clinical genomics. GENCODE annotation processes make use of primary data and bioinformatic tools and analysis generated both within the consortium and externally to support the creation of transcript structures and the determination of their function. Here, we present improvements to our annotation infrastructure, bioinformatics tools, and analysis, and the advances they support in the annotation of the human and mouse genomes including: the completion of first pass manual annotation for the mouse reference genome; targeted improvements to the annotation of genes associated with SARS-CoV-2 infection; collaborative projects to achieve convergence across reference annotation databases for the annotation of human and mouse protein-coding genes; and the first GENCODE manually supervised automated annotation of lncRNAs. Our annotation is accessible via Ensembl, the UCSC Genome Browser and https://www.gencodegenes.org.
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http://dx.doi.org/10.1093/nar/gkaa1087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7778937PMC
January 2021

Human vtRNA1-1 Levels Modulate Signaling Pathways and Regulate Apoptosis in Human Cancer Cells.

Biomolecules 2020 04 16;10(4). Epub 2020 Apr 16.

Department of Chemistry and Biochemistry, University of Bern, 3012 Bern, Switzerland.

Regulatory non-protein coding RNAs perform a remarkable variety of complex biological functions. Previously, we demonstrated a role of the human non-coding vault RNA1-1 (vtRNA1-1) in inhibiting intrinsic and extrinsic apoptosis in several cancer cell lines. Yet on the molecular level, the function of the vtRNA1-1 is still not fully clear. Here, we created HeLa knock-out cell lines revealing that prolonged starvation triggers elevated levels of apoptosis in the absence of vtRNA1-1 but not in vtRNA1-3 knock-out cells. Next-generation deep sequencing of the mRNome identified the PI3K/Akt pathway and the ERK1/2 MAPK cascade, two prominent signaling axes, to be misregulated in the absence of vtRNA1-1 during starvation-mediated cell death conditions. Expression of vtRNA1-1 mutants identified a short stretch of 24 nucleotides of the vtRNA1-1 central domain as being essential for successful maintenance of apoptosis resistance. This study describes a cell signaling-dependent contribution of the human vtRNA1-1 to starvation-induced programmed cell death.
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http://dx.doi.org/10.3390/biom10040614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226377PMC
April 2020

Analyses of non-coding somatic drivers in 2,658 cancer whole genomes.

Nature 2020 02 5;578(7793):102-111. Epub 2020 Feb 5.

Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA.

The discovery of drivers of cancer has traditionally focused on protein-coding genes. Here we present analyses of driver point mutations and structural variants in non-coding regions across 2,658 genomes from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). For point mutations, we developed a statistically rigorous strategy for combining significance levels from multiple methods of driver discovery that overcomes the limitations of individual methods. For structural variants, we present two methods of driver discovery, and identify regions that are significantly affected by recurrent breakpoints and recurrent somatic juxtapositions. Our analyses confirm previously reported drivers, raise doubts about others and identify novel candidates, including point mutations in the 5' region of TP53, in the 3' untranslated regions of NFKBIZ and TOB1, focal deletions in BRD4 and rearrangements in the loci of AKR1C genes. We show that although point mutations and structural variants that drive cancer are less frequent in non-coding genes and regulatory sequences than in protein-coding genes, additional examples of these drivers will be found as more cancer genomes become available.
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http://dx.doi.org/10.1038/s41586-020-1965-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054214PMC
February 2020

Cancer LncRNA Census reveals evidence for deep functional conservation of long noncoding RNAs in tumorigenesis.

Commun Biol 2020 02 5;3(1):56. Epub 2020 Feb 5.

Department of Medical Oncology, Inselspital, University Hospital and University of Bern, 3010, Bern, Switzerland.

Long non-coding RNAs (lncRNAs) are a growing focus of cancer genomics studies, creating the need for a resource of lncRNAs with validated cancer roles. Furthermore, it remains debated whether mutated lncRNAs can drive tumorigenesis, and whether such functions could be conserved during evolution. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, we introduce the Cancer LncRNA Census (CLC), a compilation of 122 GENCODE lncRNAs with causal roles in cancer phenotypes. In contrast to existing databases, CLC requires strong functional or genetic evidence. CLC genes are enriched amongst driver genes predicted from somatic mutations, and display characteristic genomic features. Strikingly, CLC genes are enriched for driver mutations from unbiased, genome-wide transposon-mutagenesis screens in mice. We identified 10 tumour-causing mutations in orthologues of 8 lncRNAs, including LINC-PINT and NEAT1, but not MALAT1. Thus CLC represents a dataset of high-confidence cancer lncRNAs. Mutagenesis maps are a novel means for identifying deeply-conserved roles of lncRNAs in tumorigenesis.
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http://dx.doi.org/10.1038/s42003-019-0741-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002399PMC
February 2020

CASPR, an analysis pipeline for single and paired guide RNA CRISPR screens, reveals optimal target selection for long non-coding RNAs.

Bioinformatics 2020 03;36(6):1673-1680

Department of Medical Oncology, Inselspital, Bern University Hospital.

Motivation: CRISPR-Cas9 loss-of-function (LOF) pooled screening promises to identify which long non-coding RNAs (lncRNAs), amongst the many thousands to have been annotated so far, are capable of mediating cellular functions. The two principal LOF perturbations, CRISPR-inhibition and CRISPR-deletion, employ one and two guide RNAs, respectively. However, no software solution has the versatility to identify hits across both modalities, and the optimal design parameters for such screens remain poorly understood.

Results: Here, we present CRISPR Analysis for Single and Paired RNA-guides (CASPR), a user-friendly, end-to-end screen analysis tool. CASPR is compatible with both CRISPRi and CRISPR-del screens, and balances sensitivity and specificity by generating consensus predictions from multiple algorithms. Benchmarking on ground-truth sets of cancer-associated lncRNAs demonstrates CASPR's improved sensitivity with respect to existing methods. Applying CASPR to published screens, we identify two parameters that predict lncRNA hits: expression and annotation quality of the transcription start site. Thus, CASPR is a versatile and complete solution for lncRNA CRISPR screen analysis, and reveals principles for including lncRNAs in screening libraries.

Availability And Implementation: https://judithbergada.github.io/CASPR/.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz811DOI Listing
March 2020

LnCompare: gene set feature analysis for human long non-coding RNAs.

Nucleic Acids Res 2019 07;47(W1):W523-W529

Institute of Computational Medicine, School of Artificial Intelligence, Hebei University of Technology, Tianjin 300401, China.

Interest in the biological roles of long noncoding RNAs (lncRNAs) has resulted in growing numbers of studies that produce large sets of candidate genes, for example, differentially expressed between two conditions. For sets of protein-coding genes, ontology and pathway analyses are powerful tools for generating new insights from statistical enrichment of gene features. Here we present the LnCompare web server, an equivalent resource for studying the properties of lncRNA gene sets. The Gene Set Feature Comparison mode tests for enrichment amongst a panel of quantitative and categorical features, spanning gene structure, evolutionary conservation, expression, subcellular localization, repetitive sequences and disease association. Moreover, in Similar Gene Identification mode, users may identify other lncRNAs by similarity across a defined range of features. Comprehensive results may be downloaded in tabular and graphical formats, in addition to the entire feature resource. LnCompare will empower researchers to extract useful hypotheses and candidates from lncRNA gene sets.
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http://dx.doi.org/10.1093/nar/gkz410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6602513PMC
July 2019

Global Positioning System: Understanding Long Noncoding RNAs through Subcellular Localization.

Mol Cell 2019 03;73(5):869-883

Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland; Department for BioMedical Research, University of Bern, Bern, Switzerland. Electronic address:

The localization of long noncoding RNAs (lncRNAs) within the cell is the primary determinant of their molecular functions. LncRNAs are often thought of as chromatin-restricted regulators of gene transcription and chromatin structure. However, a rich population of cytoplasmic lncRNAs has come to light, with diverse roles including translational regulation, signaling, and respiration. RNA maps of increasing resolution and scope are revealing a subcellular world of highly specific localization patterns and hint at sequence-based address codes specifying lncRNA fates. We propose a new framework for analyzing sequencing-based data, which suggests that numbers of cytoplasmic lncRNA molecules rival those in the nucleus. New techniques promise to create high-resolution, transcriptome-wide maps associated with all organelles of the mammalian cell. Given its intimate link to molecular roles, subcellular localization provides a means of unlocking the mystery of lncRNA functions.
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http://dx.doi.org/10.1016/j.molcel.2019.02.008DOI Listing
March 2019

Hacking the Cancer Genome: Profiling Therapeutically Actionable Long Non-coding RNAs Using CRISPR-Cas9 Screening.

Cancer Cell 2019 04 28;35(4):545-557. Epub 2019 Feb 28.

Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland; Department for BioMedical Research, University of Bern, Bern, Switzerland. Electronic address:

Long non-coding RNAs (lncRNAs) represent a huge reservoir of potential cancer targets. Such "onco-lncRNAs" have resisted traditional RNAi methods, but CRISPR-Cas9 genome editing now promises functional screens at high throughput and low cost. The unique biology of lncRNAs demands screening strategies distinct from protein-coding genes. The first such screens have identified hundreds of onco-lncRNAs promoting cell proliferation and drug resistance. Ongoing developments will further improve screen performance and translational relevance. This Review aims to highlight the potential of CRISPR screening technology for discovering new onco-lncRNAs, and to guide molecular oncologists wishing to apply it to their cancer of interest.
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http://dx.doi.org/10.1016/j.ccell.2019.01.019DOI Listing
April 2019

Ancient exapted transposable elements promote nuclear enrichment of human long noncoding RNAs.

Genome Res 2019 02 26;29(2):208-222. Epub 2018 Dec 26.

Department for BioMedical Research (DBMR), University of Bern, 3008 Bern, Switzerland.

The sequence domains underlying long noncoding RNA (lncRNA) activities, including their characteristic nuclear enrichment, remain largely unknown. It has been proposed that these domains can originate from neofunctionalized fragments of transposable elements (TEs), otherwise known as RIDLs (repeat insertion domains of lncRNA), although just a handful have been identified. It is challenging to distinguish functional RIDL instances against a numerous genomic background of neutrally evolving TEs. We here show evidence that a subset of TE types experience evolutionary selection in the context of lncRNA exons. Together these comprise an enrichment group of 5374 TE fragments in 3566 loci. Their host lncRNAs tend to be functionally validated and associated with disease. This RIDL group was used to explore the relationship between TEs and lncRNA subcellular localization. By using global localization data from 10 human cell lines, we uncover a dose-dependent relationship between nuclear/cytoplasmic distribution and evolutionarily conserved L2b, MIRb, and MIRc elements. This is observed in multiple cell types and is unaffected by confounders of transcript length or expression. Experimental validation with engineered transgenes shows that these TEs drive nuclear enrichment in a natural sequence context. Together these data reveal a role for TEs in regulating the subcellular localization of lncRNAs.
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http://dx.doi.org/10.1101/gr.229922.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6360812PMC
February 2019

GENCODE reference annotation for the human and mouse genomes.

Nucleic Acids Res 2019 01;47(D1):D766-D773

European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK.

The accurate identification and description of the genes in the human and mouse genomes is a fundamental requirement for high quality analysis of data informing both genome biology and clinical genomics. Over the last 15 years, the GENCODE consortium has been producing reference quality gene annotations to provide this foundational resource. The GENCODE consortium includes both experimental and computational biology groups who work together to improve and extend the GENCODE gene annotation. Specifically, we generate primary data, create bioinformatics tools and provide analysis to support the work of expert manual gene annotators and automated gene annotation pipelines. In addition, manual and computational annotation workflows use any and all publicly available data and analysis, along with the research literature to identify and characterise gene loci to the highest standard. GENCODE gene annotations are accessible via the Ensembl and UCSC Genome Browsers, the Ensembl FTP site, Ensembl Biomart, Ensembl Perl and REST APIs as well as https://www.gencodegenes.org.
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http://dx.doi.org/10.1093/nar/gky955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6323946PMC
January 2019

Towards a complete map of the human long non-coding RNA transcriptome.

Nat Rev Genet 2018 09;19(9):535-548

Department of Medical Oncology, Inselspital, University Hospital and University of Bern, Bern, Switzerland.

Gene maps, or annotations, enable us to navigate the functional landscape of our genome. They are a resource upon which virtually all studies depend, from single-gene to genome-wide scales and from basic molecular biology to medical genetics. Yet present-day annotations suffer from trade-offs between quality and size, with serious but often unappreciated consequences for downstream studies. This is particularly true for long non-coding RNAs (lncRNAs), which are poorly characterized compared to protein-coding genes. Long-read sequencing technologies promise to improve current annotations, paving the way towards a complete annotation of lncRNAs expressed throughout a human lifetime.
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http://dx.doi.org/10.1038/s41576-018-0017-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451964PMC
September 2018

Capturing a Long Look at Our Genetic Library.

Cell Syst 2018 Feb;6(2):153-155

Department of Medical Oncology, Inselspital, University Hospital and University of Bern, 3010 Bern, Switzerland; Department of Biomedical Research (DBMR), University of Bern, 3008 Bern, Switzerland. Electronic address:

Long-read sequencing, coupled to cDNA capture, provides an unrivaled view of the transcriptome of chromosome 21, revealing surprises about the splicing of long noncoding RNAs.
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http://dx.doi.org/10.1016/j.cels.2018.02.003DOI Listing
February 2018

High-throughput annotation of full-length long noncoding RNAs with capture long-read sequencing.

Nat Genet 2017 Dec 6;49(12):1731-1740. Epub 2017 Nov 6.

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain.

Accurate annotation of genes and their transcripts is a foundation of genomics, but currently no annotation technique combines throughput and accuracy. As a result, reference gene collections remain incomplete-many gene models are fragmentary, and thousands more remain uncataloged, particularly for long noncoding RNAs (lncRNAs). To accelerate lncRNA annotation, the GENCODE consortium has developed RNA Capture Long Seq (CLS), which combines targeted RNA capture with third-generation long-read sequencing. Here we present an experimental reannotation of the GENCODE intergenic lncRNA populations in matched human and mouse tissues that resulted in novel transcript models for 3,574 and 561 gene loci, respectively. CLS approximately doubled the annotated complexity of targeted loci, outperforming existing short-read techniques. Full-length transcript models produced by CLS enabled us to definitively characterize the genomic features of lncRNAs, including promoter and gene structure, and protein-coding potential. Thus, CLS removes a long-standing bottleneck in transcriptome annotation and generates manual-quality full-length transcript models at high-throughput scales.
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http://dx.doi.org/10.1038/ng.3988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709232PMC
December 2017

LncATLAS database for subcellular localization of long noncoding RNAs.

RNA 2017 07 6;23(7):1080-1087. Epub 2017 Apr 6.

Department of Clinical Research, University of Bern, 3008 Bern, Switzerland.

The subcellular localization of long noncoding RNAs (lncRNAs) holds valuable clues to their molecular function. However, measuring localization of newly discovered lncRNAs involves time-consuming and costly experimental methods. We have created "lncATLAS," a comprehensive resource of lncRNA localization in human cells based on RNA-sequencing data sets. Altogether, 6768 GENCODE-annotated lncRNAs are represented across various compartments of 15 cell lines. We introduce relative concentration index (RCI) as a useful measure of localization derived from ensemble RNA-seq measurements. LncATLAS is accessible through an intuitive and informative webserver, from which lncRNAs of interest are accessed using identifiers or names. Localization is presented across cell types and organelles, and may be compared to the distribution of all other genes. Publication-quality figures and raw data tables are automatically generated with each query, and the entire data set is also available to download. LncATLAS makes lncRNA subcellular localization data available to the widest possible number of researchers. It is available at lncatlas.crg.eu.
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http://dx.doi.org/10.1261/rna.060814.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473142PMC
July 2017

Scalable Design of Paired CRISPR Guide RNAs for Genomic Deletion.

PLoS Comput Biol 2017 03 2;13(3):e1005341. Epub 2017 Mar 2.

Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, Barcelona, Spain.

CRISPR-Cas9 technology can be used to engineer precise genomic deletions with pairs of single guide RNAs (sgRNAs). This approach has been widely adopted for diverse applications, from disease modelling of individual loci, to parallelized loss-of-function screens of thousands of regulatory elements. However, no solution has been presented for the unique bioinformatic design requirements of CRISPR deletion. We here present CRISPETa, a pipeline for flexible and scalable paired sgRNA design based on an empirical scoring model. Multiple sgRNA pairs are returned for each target, and any number of targets can be analyzed in parallel, making CRISPETa equally useful for focussed or high-throughput studies. Fast run-times are achieved using a pre-computed off-target database. sgRNA pair designs are output in a convenient format for visualisation and oligonucleotide ordering. We present pre-designed, high-coverage library designs for entire classes of protein-coding and non-coding elements in human, mouse, zebrafish, Drosophila melanogaster and Caenorhabditis elegans. In human cells, we reproducibly observe deletion efficiencies of ≥50% for CRISPETa designs targeting an enhancer and exonic fragment of the MALAT1 oncogene. In the latter case, deletion results in production of desired, truncated RNA. CRISPETa will be useful for researchers seeking to harness CRISPR for targeted genomic deletion, in a variety of model organisms, from single-target to high-throughput scales.
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http://dx.doi.org/10.1371/journal.pcbi.1005341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333799PMC
March 2017

Discovery of Cancer Driver Long Noncoding RNAs across 1112 Tumour Genomes: New Candidates and Distinguishing Features.

Sci Rep 2017 01 27;7:41544. Epub 2017 Jan 27.

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain.

Long noncoding RNAs (lncRNAs) represent a vast unexplored genetic space that may hold missing drivers of tumourigenesis, but few such "driver lncRNAs" are known. Until now, they have been discovered through changes in expression, leading to problems in distinguishing between causative roles and passenger effects. We here present a different approach for driver lncRNA discovery using mutational patterns in tumour DNA. Our pipeline, ExInAtor, identifies genes with excess load of somatic single nucleotide variants (SNVs) across panels of tumour genomes. Heterogeneity in mutational signatures between cancer types and individuals is accounted for using a simple local trinucleotide background model, which yields high precision and low computational demands. We use ExInAtor to predict drivers from the GENCODE annotation across 1112 entire genomes from 23 cancer types. Using a stratified approach, we identify 15 high-confidence candidates: 9 novel and 6 known cancer-related genes, including MALAT1, NEAT1 and SAMMSON. Both known and novel driver lncRNAs are distinguished by elevated gene length, evolutionary conservation and expression. We have presented a first catalogue of mutated lncRNA genes driving cancer, which will grow and improve with the application of ExInAtor to future tumour genome projects.
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http://dx.doi.org/10.1038/srep41544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5269722PMC
January 2017

CAIPIRINHA accelerated SPACE enables 10-min isotropic 3D TSE MRI of the ankle for optimized visualization of curved and oblique ligaments and tendons.

Eur Radiol 2017 Sep 23;27(9):3652-3661. Epub 2017 Jan 23.

Russell H. Morgan Department of Radiology and Radiological Science, Section of Musculoskeletal Radiology, Johns Hopkins University School of Medicine, 601 N. Caroline Street, JHOC 3140A, Baltimore, MD, 21287, USA.

Objectives: To test the hypothesis that a fourfold CAIPIRINHA accelerated, 10-min, high-resolution, isotropic 3D TSE MRI prototype protocol of the ankle derives equal or better quality than a 20-min 2D TSE standard protocol.

Methods: Following internal review board approval and informed consent, 3-Tesla MRI of the ankle was obtained in 24 asymptomatic subjects including 10-min 3D CAIPIRINHA SPACE TSE prototype and 20-min 2D TSE standard protocols. Outcome variables included image quality and visibility of anatomical structures using 5-point Likert scales. Non-parametric statistical testing was used. P values ≤0.001 were considered significant.

Results: Edge sharpness, contrast resolution, uniformity, noise, fat suppression and magic angle effects were without statistical difference on 2D and 3D TSE images (p > 0.035). Fluid was mildly brighter on intermediate-weighted 2D images (p < 0.001), whereas 3D images had substantially less partial volume, chemical shift and no pulsatile-flow artifacts (p < 0.001). Oblique and curved planar 3D images resulted in mildly-to-substantially improved visualization of joints, spring, bifurcate, syndesmotic, collateral and sinus tarsi ligaments, and tendons (p < 0.001, respectively).

Conclusions: 3D TSE MRI with CAIPIRINHA acceleration enables high-spatial resolution oblique and curved planar MRI of the ankle and visualization of ligaments, tendons and joints equally well or better than a more time-consuming anisotropic 2D TSE MRI.

Key Points: • High-resolution 3D TSE MRI improves visualization of ankle structures. • Limitations of current 3D TSE MRI include long scan times. • 3D CAIPIRINHA SPACE allows now a fourfold-accelerated data acquisition. • 3D CAIPIRINHA SPACE enables high-spatial-resolution ankle MRI within 10 min. • 10-min 3D CAIPIRINHA SPACE produces equal-or-better quality than 20-min 2D TSE.
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http://dx.doi.org/10.1007/s00330-017-4734-yDOI Listing
September 2017

A Point Mutation in a lincRNA Upstream of GDNF Is Associated to a Canine Insensitivity to Pain: A Spontaneous Model for Human Sensory Neuropathies.

PLoS Genet 2016 Dec 29;12(12):e1006482. Epub 2016 Dec 29.

CNRS, UMR 6290, Institut de Génétique et Développement de Rennes, Rennes, France.

Human Hereditary Sensory Autonomic Neuropathies (HSANs) are characterized by insensitivity to pain, sometimes combined with self-mutilation. Strikingly, several sporting dog breeds are particularly affected by such neuropathies. Clinical signs appear in young puppies and consist of acral analgesia, with or without sudden intense licking, biting and severe self-mutilation of the feet, whereas proprioception, motor abilities and spinal reflexes remain intact. Through a Genome Wide Association Study (GWAS) with 24 affected and 30 unaffected sporting dogs using the Canine HD 170K SNP array (Illumina), we identified a 1.8 Mb homozygous locus on canine chromosome 4 (adj. p-val = 2.5x10-6). Targeted high-throughput sequencing of this locus in 4 affected and 4 unaffected dogs identified 478 variants. Only one variant perfectly segregated with the expected recessive inheritance in 300 sporting dogs of known clinical status, while it was never present in 900 unaffected dogs from 130 other breeds. This variant, located 90 kb upstream of the GDNF gene, a highly relevant neurotrophic factor candidate gene, lies in a long intergenic non-coding RNAs (lincRNA), GDNF-AS. Using human comparative genomic analysis, we observed that the canine variant maps onto an enhancer element. Quantitative RT-PCR of dorsal root ganglia RNAs of affected dogs showed a significant decrease of both GDNF mRNA and GDNF-AS expression levels (respectively 60% and 80%), as compared to unaffected dogs. We thus performed gel shift assays (EMSA) that reveal that the canine variant significantly alters the binding of regulatory elements. Altogether, these results allowed the identification in dogs of GDNF as a relevant candidate for human HSAN and insensitivity to pain, but also shed light on the regulation of GDNF transcription. Finally, such results allow proposing these sporting dog breeds as natural models for clinical trials with a double benefit for human and veterinary medicine.
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http://dx.doi.org/10.1371/journal.pgen.1006482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5198995PMC
December 2016

Cytoplasmic long noncoding RNAs are frequently bound to and degraded at ribosomes in human cells.

RNA 2016 06 18;22(6):867-82. Epub 2016 Apr 18.

Centre for Genomic Regulation (CRG), 08003 Barcelona, Spain Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), 08003 Barcelona, Spain.

Recent footprinting studies have made the surprising observation that long noncoding RNAs (lncRNAs) physically interact with ribosomes. However, these findings remain controversial, and the overall proportion of cytoplasmic lncRNAs involved is unknown. Here we make a global, absolute estimate of the cytoplasmic and ribosome-associated population of stringently filtered lncRNAs in a human cell line using polysome profiling coupled to spike-in normalized microarray analysis. Fifty-four percent of expressed lncRNAs are detected in the cytoplasm. The majority of these (70%) have >50% of their cytoplasmic copies associated with polysomal fractions. These interactions are lost upon disruption of ribosomes by puromycin. Polysomal lncRNAs are distinguished by a number of 5' mRNA-like features, including capping and 5'UTR length. On the other hand, nonpolysomal "free cytoplasmic" lncRNAs have more conserved promoters and a wider range of expression across cell types. Exons of polysomal lncRNAs are depleted of endogenous retroviral insertions, suggesting a role for repetitive elements in lncRNA localization. Finally, we show that blocking of ribosomal elongation results in stabilization of many associated lncRNAs. Together these findings suggest that the ribosome is the default destination for the majority of cytoplasmic long noncoding RNAs and may play a role in their degradation.
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http://dx.doi.org/10.1261/rna.053561.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4878613PMC
June 2016

Erratum to: 'DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs'.

BMC Genomics 2016 Mar 9;17:215. Epub 2016 Mar 9.

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003, Barcelona, Spain.

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http://dx.doi.org/10.1186/s12864-016-2544-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784307PMC
March 2016

DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs.

BMC Genomics 2015 Oct 23;16:846. Epub 2015 Oct 23.

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003, Barcelona, Spain.

Background: CRISPR genome-editing technology makes it possible to quickly and cheaply delete non-protein-coding regulatory elements. We present a vector system adapted for this purpose called DECKO (Double Excision CRISPR Knockout), which applies a simple two-step cloning to generate lentiviral vectors expressing two guide RNAs (gRNAs) simultaneously. The key feature of DECKO is its use of a single 165 bp starting oligonucleotide carrying the variable sequences of both gRNAs, making it fully scalable from single-locus studies to complex library cloning.

Results: We apply DECKO to deleting the promoters of one protein-coding gene and two oncogenic lncRNAs, UCA1 and the highly-expressed MALAT1, focus of many previous studies employing RNA interference approaches. DECKO successfully deleted genomic fragments ranging in size from 100 to 3000 bp in four human cell lines. Using a clone-derivation workflow lasting approximately 20 days, we obtained 9 homozygous and 17 heterozygous promoter knockouts in three human cell lines. Frequent target region inversions were observed. These clones have reductions in steady-state MALAT1 RNA levels of up to 98 % and display reduced proliferation rates.

Conclusions: We present a dual CRISPR tool, DECKO, which is cloned using a single starting oligonucleotide, thereby affording simplicity and scalability to CRISPR knockout studies of non-coding genomic elements, including long non-coding RNAs.
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http://dx.doi.org/10.1186/s12864-015-2086-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4619085PMC
October 2015

CARMEN, a human super enhancer-associated long noncoding RNA controlling cardiac specification, differentiation and homeostasis.

J Mol Cell Cardiol 2015 Dec 28;89(Pt A):98-112. Epub 2015 Sep 28.

Experimental Cardiology Unit, Department of Medicine, University of Lausanne Medical School, Lausanne, Switzerland. Electronic address:

Long noncoding RNAs (lncRNAs) are emerging as important regulators of developmental pathways. However, their roles in human cardiac precursor cell (CPC) remain unexplored. To characterize the long noncoding transcriptome during human CPC cardiac differentiation, we profiled the lncRNA transcriptome in CPCs isolated from the human fetal heart and identified 570 lncRNAs that were modulated during cardiac differentiation. Many of these were associated with active cardiac enhancer and super enhancers (SE) with their expression being correlated with proximal cardiac genes. One of the most upregulated lncRNAs was a SE-associated lncRNA that was named CARMEN, (CAR)diac (M)esoderm (E)nhancer-associated (N)oncoding RNA. CARMEN exhibits RNA-dependent enhancing activity and is upstream of the cardiac mesoderm-specifying gene regulatory network. Interestingly, CARMEN interacts with SUZ12 and EZH2, two components of the polycomb repressive complex 2 (PRC2). We demonstrate that CARMEN knockdown inhibits cardiac specification and differentiation in cardiac precursor cells independently of MIR-143 and -145 expression, two microRNAs located proximal to the enhancer sequences. Importantly, CARMEN expression was activated during pathological remodeling in the mouse and human hearts, and was necessary for maintaining cardiac identity in differentiated cardiomyocytes. This study demonstrates therefore that CARMEN is a crucial regulator of cardiac cell differentiation and homeostasis.
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http://dx.doi.org/10.1016/j.yjmcc.2015.09.016DOI Listing
December 2015

RING Dimerization Links Higher-Order Assembly of TRIM5α to Synthesis of K63-Linked Polyubiquitin.

Cell Rep 2015 Aug 23;12(5):788-97. Epub 2015 Jul 23.

Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229, USA. Electronic address:

Members of the tripartite motif (TRIM) protein family of RING E3 ubiquitin (Ub) ligases promote innate immune responses by catalyzing synthesis of polyubiquitin chains linked through lysine 63 (K63). Here, we investigate the mechanism by which the TRIM5α retroviral restriction factor activates Ubc13, the K63-linkage-specific E2. Structural, biochemical, and functional characterization of the TRIM5α:Ubc13-Ub interactions reveals that activation of the Ubc13-Ub conjugate requires dimerization of the TRIM5α RING domain. Our data explain how higher-order oligomerization of TRIM5α, which is promoted by the interaction with the retroviral capsid, enhances the E3 Ub ligase activity of TRIM5α and contributes to its antiretroviral function. This E3 mechanism, in which RING dimerization is transient and depends on the interaction of the TRIM protein with the ligand, is likely to be conserved in many members of the TRIM family and may have evolved to facilitate recognition of repetitive epitope patterns associated with infection.
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http://dx.doi.org/10.1016/j.celrep.2015.06.072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4526822PMC
August 2015

Evolution of selenophosphate synthetases: emergence and relocation of function through independent duplications and recurrent subfunctionalization.

Genome Res 2015 Sep 20;25(9):1256-67. Epub 2015 Jul 20.

Bioinformatics and Genomics Programme, Centre for Genomic Regulation (CRG), 08003 Barcelona, Catalonia, Spain; Universitat Pompeu Fabra (UPF), 08003 Barcelona, Catalonia, Spain; Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), 08003 Barcelona, Catalonia, Spain;

Selenoproteins are proteins that incorporate selenocysteine (Sec), a nonstandard amino acid encoded by UGA, normally a stop codon. Sec synthesis requires the enzyme Selenophosphate synthetase (SPS or SelD), conserved in all prokaryotic and eukaryotic genomes encoding selenoproteins. Here, we study the evolutionary history of SPS genes, providing a map of selenoprotein function spanning the whole tree of life. SPS is itself a selenoprotein in many species, although functionally equivalent homologs that replace the Sec site with cysteine (Cys) are common. Many metazoans, however, possess SPS genes with substitutions other than Sec or Cys (collectively referred to as SPS1). Using complementation assays in fly mutants, we show that these genes share a common function, which appears to be distinct from the synthesis of selenophosphate carried out by the Sec- and Cys- SPS genes (termed SPS2), and unrelated to Sec synthesis. We show here that SPS1 genes originated through a number of independent gene duplications from an ancestral metazoan selenoprotein SPS2 gene that most likely already carried the SPS1 function. Thus, in SPS genes, parallel duplications and subsequent convergent subfunctionalization have resulted in the segregation to different loci of functions initially carried by a single gene. This evolutionary history constitutes a remarkable example of emergence and evolution of gene function, which we have been able to trace thanks to the singular features of SPS genes, wherein the amino acid at a single site determines unequivocally protein function and is intertwined to the evolutionary fate of the entire selenoproteome.
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http://dx.doi.org/10.1101/gr.190538.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561486PMC
September 2015

Human genomics. The human transcriptome across tissues and individuals.

Science 2015 May;348(6235):660-5

Center for Genomic Regulation (CRG), Barcelona, Catalonia, Spain. Universitat Pompeu Fabra (UPF), Barcelona, Catalonia, Spain. Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Barcelona, Catalonia, Spain. Joint CRG-Barcelona Super Computing Center (BSC)-Institut de Recerca Biomedica (IRB) Program in Computational Biology, Barcelona, Catalonia, Spain.

Transcriptional regulation and posttranscriptional processing underlie many cellular and organismal phenotypes. We used RNA sequence data generated by Genotype-Tissue Expression (GTEx) project to investigate the patterns of transcriptome variation across individuals and tissues. Tissues exhibit characteristic transcriptional signatures that show stability in postmortem samples. These signatures are dominated by a relatively small number of genes—which is most clearly seen in blood—though few are exclusive to a particular tissue and vary more across tissues than individuals. Genes exhibiting high interindividual expression variation include disease candidates associated with sex, ethnicity, and age. Primary transcription is the major driver of cellular specificity, with splicing playing mostly a complementary role; except for the brain, which exhibits a more divergent splicing program. Variation in splicing, despite its stochasticity, may play in contrast a comparatively greater role in defining individual phenotypes.
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http://dx.doi.org/10.1126/science.aaa0355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547472PMC
May 2015

Functional importance of cardiac enhancer-associated noncoding RNAs in heart development and disease.

J Mol Cell Cardiol 2014 Nov 19;76:55-70. Epub 2014 Aug 19.

Experimental Cardiology Unit, Department of Medicine, University of Lausanne Medical School, Lausanne, Switzerland. Electronic address:

The key information processing units within gene regulatory networks are enhancers. Enhancer activity is associated with the production of tissue-specific noncoding RNAs, yet the existence of such transcripts during cardiac development has not been established. Using an integrated genomic approach, we demonstrate that fetal cardiac enhancers generate long noncoding RNAs (lncRNAs) during cardiac differentiation and morphogenesis. Enhancer expression correlates with the emergence of active enhancer chromatin states, the initiation of RNA polymerase II at enhancer loci and expression of target genes. Orthologous human sequences are also transcribed in fetal human hearts and cardiac progenitor cells. Through a systematic bioinformatic analysis, we identified and characterized, for the first time, a catalog of lncRNAs that are expressed during embryonic stem cell differentiation into cardiomyocytes and associated with active cardiac enhancer sequences. RNA-sequencing demonstrates that many of these transcripts are polyadenylated, multi-exonic long noncoding RNAs. Moreover, knockdown of two enhancer-associated lncRNAs resulted in the specific downregulation of their predicted target genes. Interestingly, the reactivation of the fetal gene program, a hallmark of the stress response in the adult heart, is accompanied by increased expression of fetal cardiac enhancer transcripts. Altogether, these findings demonstrate that the activity of cardiac enhancers and expression of their target genes are associated with the production of enhancer-derived lncRNAs.
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http://dx.doi.org/10.1016/j.yjmcc.2014.08.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445080PMC
November 2014

The RIDL hypothesis: transposable elements as functional domains of long noncoding RNAs.

RNA 2014 Jul 21;20(7):959-76. Epub 2014 May 21.

Centre for Genomic Regulation (CRG), 08003 Barcelona, Spain Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), 08003 Barcelona, Spain.

Our genome contains tens of thousands of long noncoding RNAs (lncRNAs), many of which are likely to have genetic regulatory functions. It has been proposed that lncRNA are organized into combinations of discrete functional domains, but the nature of these and their identification remain elusive. One class of sequence elements that is enriched in lncRNA is represented by transposable elements (TEs), repetitive mobile genetic sequences that have contributed widely to genome evolution through a process termed exaptation. Here, we link these two concepts by proposing that exonic TEs act as RNA domains that are essential for lncRNA function. We term such elements Repeat Insertion Domains of LncRNAs (RIDLs). A growing number of RIDLs have been experimentally defined, where TE-derived fragments of lncRNA act as RNA-, DNA-, and protein-binding domains. We propose that these reflect a more general phenomenon of exaptation during lncRNA evolution, where inserted TE sequences are repurposed as recognition sites for both protein and nucleic acids. We discuss a series of genomic screens that may be used in the future to systematically discover RIDLs. The RIDL hypothesis has the potential to explain how functional evolution can keep pace with the rapid gene evolution observed in lncRNA. More practically, TE maps may in the future be used to predict lncRNA function.
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http://dx.doi.org/10.1261/rna.044560.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4114693PMC
July 2014

Genome-wide profiling of the cardiac transcriptome after myocardial infarction identifies novel heart-specific long non-coding RNAs.

Eur Heart J 2015 Feb 30;36(6):353-68a. Epub 2014 Apr 30.

Experimental Cardiology Unit, Department of Medicine, University of Lausanne Medical School, CH-1011 Lausanne, Switzerland

Aim: Heart disease is recognized as a consequence of dysregulation of cardiac gene regulatory networks. Previously, unappreciated components of such networks are the long non-coding RNAs (lncRNAs). Their roles in the heart remain to be elucidated. Thus, this study aimed to systematically characterize the cardiac long non-coding transcriptome post-myocardial infarction and to elucidate their potential roles in cardiac homoeostasis.

Methods And Results: We annotated the mouse transcriptome after myocardial infarction via RNA sequencing and ab initio transcript reconstruction, and integrated genome-wide approaches to associate specific lncRNAs with developmental processes and physiological parameters. Expression of specific lncRNAs strongly correlated with defined parameters of cardiac dimensions and function. Using chromatin maps to infer lncRNA function, we identified many with potential roles in cardiogenesis and pathological remodelling. The vast majority was associated with active cardiac-specific enhancers. Importantly, oligonucleotide-mediated knockdown implicated novel lncRNAs in controlling expression of key regulatory proteins involved in cardiogenesis. Finally, we identified hundreds of human orthologues and demonstrate that particular candidates were differentially modulated in human heart disease.

Conclusion: These findings reveal hundreds of novel heart-specific lncRNAs with unique regulatory and functional characteristics relevant to maladaptive remodelling, cardiac function and possibly cardiac regeneration. This new class of molecules represents potential therapeutic targets for cardiac disease. Furthermore, their exquisite correlation with cardiac physiology renders them attractive candidate biomarkers to be used in the clinic.
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http://dx.doi.org/10.1093/eurheartj/ehu180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320320PMC
February 2015