UCSF RNA Journal Club

A newsletter announcing the next presenter for RNA Journal Club

Leonardo Ramos Ferreira

Nucleic acid detection with CRISPR-Cas13a/C2c2
Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F.
Science. 2017 Apr 28;356(6336):438-442. doi: 10.1126/science.aam9321. Epub 2017 Apr 13.
April 28, 2017
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA. Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA. Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Center for Systems Biology, Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA. Department of Immunology and Infectious Disease, Harvard School of Public Health, Boston, MA 02115, USA. Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. [email protected] [email protected]
Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.
Date: 
May 3, 2017
Where: 
HSW 1057 at noon

Roman Camarda

UV Irradiation Induces a Non-coding RNA that Functionally Opposes the Protein Encoded by the Same Gene
Williamson L, Saponaro M, Boeing S, East P, Mitter R, Kantidakis T, Kelly GP, Lobley A, Walker J, Spencer-Dene B, Howell M, Stewart A, Svejstrup JQ.
Cell. 2017 Feb 23;168(5):843-855.e13. doi: 10.1016/j.cell.2017.01.019. Epub 2017 Feb 16.
February 23, 2017
Mechanisms of Transcription Laboratory, The Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK. Mechanisms of Transcription Laboratory, The Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK; Institute of Cancer and Genomic Sciences, University of Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TT, UK. Mechanisms of Transcription Laboratory, The Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK; Bioinformatics and Biostatistics, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Bioinformatics and Biostatistics, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Experimental Histopathology, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. High Throughput Screening Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Mechanisms of Transcription Laboratory, The Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK. Electronic address: [email protected]
The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal ∼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage.
Date: 
April 26, 2017
Where: 
HSW 1057 at noon

Hui Li

Visualizing the secondary and tertiary architectural domains of lncRNA RepA
Liu F1,2, Somarowthu S1, Pyle AM1,2,3.
Nat Chem Biol. 2017 Mar;13(3):282-289. doi: 10.1038/nchembio.2272. Epub 2017 Jan 9.
March 1, 2017
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. Department of Chemistry, Yale University, New Haven, Connecticut, USA.
Long noncoding RNAs (lncRNAs) are important for gene expression, but little is known about their structures. RepA is a 1.6-kb mouse lncRNA comprising the same sequence as the 5' region of Xist, including A and F repeats. It has been proposed to facilitate the initiation and spread of X-chromosome inactivation, although its exact role is poorly understood. To gain insight into the molecular mechanism of RepA and Xist, we determined a complete phylogenetically validated secondary-structural map of RepA through SHAPE and DMS chemical probing of a homogeneously folded RNA in vitro. We combined UV-cross-linking experiments with RNA modeling methods to produce a three-dimensional model of RepA functional domains demonstrating that tertiary architecture exists within lncRNA molecules and occurs within specific functional modules. This work provides a foundation for understanding of the evolution and functional properties of RepA and Xist and offers a framework for exploring architectural features of other lncRNAs.
Date: 
April 19, 2017
Where: 
HSW 1057 at noon

Gabriel Eades

Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions
Kyuho Han, Edwin E Jeng, Gaelen T Hess, David W Morgens, Amy Li & Michael C Bassik
Nature Biotechnology
March 20, 2017
Department of Genetics, Stanford University, Stanford, California, USA. Program in Cancer Biology, Stanford University, Stanford, California, USA. Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford University, Stanford, California, USA.
Identification of effective combination therapies is critical to address the emergence of drug-resistant cancers, but direct screening of all possible drug combinations is infeasible. Here we introduce a CRISPR-based double knockout (CDKO) system that improves the efficiency of combinatorial genetic screening using an effective strategy for cloning and sequencing paired single guide RNA (sgRNA) libraries and a robust statistical scoring method for calculating genetic interactions (GIs) from CRISPR-deleted gene pairs. We applied CDKO to generate a large-scale human GI map, comprising 490,000 double-sgRNAs directed against 21,321 pairs of drug targets in K562 leukemia cells and identified synthetic lethal drug target pairs for which corresponding drugs exhibit synergistic killing. These included the BCL2L1 and MCL1 combination, which was also effective in imatinib-resistant cells. We further validated this system by identifying known and previously unidentified GIs between modifiers of ricin toxicity. This work provides an effective strategy to screen synergistic drug combinations in high-throughput and a CRISPR-based tool to dissect functional GI networks.
Date: 
April 12, 2017
Where: 
HSW 1057 at noon

Bin Zhang

Intragenic DNA methylation prevents spurious transcription initiation
Neri F1,2, Rapelli S3, Krepelova A1,3, Incarnato D1, Parlato C1, Basile G1, Maldotti M1,3, Anselmi F1,3, Oliviero S1,3.
Nature. 2017 Mar 2;543(7643):72-77. doi: 10.1038/nature21373. Epub 2017 Feb 22.
March 2, 2017
Human Genetics Foundation (HuGeF), via Nizza 52, 10126 Torino, Italy. Leibniz Institute on Aging - Fritz Lipmann Institute (FLI), Beutenbergstrasse 11, 07745 Jena, Germany. Dartimento di Scienze della Vita e Biologia dei Sistemi, Università di Torino, via Accademia Albertina 13, 10123 Torino, Italy.
In mammals, DNA methylation occurs mainly at CpG dinucleotides. Methylation of the promoter suppresses gene expression, but the functional role of gene-body DNA methylation in highly expressed genes has yet to be clarified. Here we show that, in mouse embryonic stem cells, Dnmt3b-dependent intragenic DNA methylation protects the gene body from spurious RNA polymerase II entry and cryptic transcription initiation. Using different genome-wide approaches, we demonstrate that this Dnmt3b function is dependent on its enzymatic activity and recruitment to the gene body by H3K36me3. Furthermore, the spurious transcripts can either be degraded by the RNA exosome complex or capped, polyadenylated, and delivered to the ribosome to produce aberrant proteins. Elongating RNA polymerase II therefore triggers an epigenetic crosstalk mechanism that involves SetD2, H3K36me3, Dnmt3b and DNA methylation to ensure the fidelity of gene transcription initiation, with implications for intragenic hypomethylation in cancer.
Date: 
April 5, 2017
Where: 
HSW 1057 at noon

Greg Kronmal, NGS Technical Sales Specialist and Sumathi Venkatapathy, Field Applications Scientist, Microarray Solutions from Thermo Fisher Scientific

Using New Clariom™ Solutions to Supplement RNA Profiling Workflows
We will show how the new Clariom microarrays fit with qPCR and NGS in a workflow that can help you determine gene expression for single genes or to explore the entire transcriptome to find actionable biomarkers.
Date: 
February 1, 2017
Where: 
HSW 1057 at noon

D'Juan Farmer

Exosomal MicroRNA Transport from Salivary Mesenchyme Regulates Epithelial Progenitor Expansion during Organogenesis
Hayashi T1, Lombaert IM1, Hauser BR1, Patel VN1, Hoffman MP2.
Dev Cell. 2017 Jan 9;40(1):95-103. doi: 10.1016/j.devcel.2016.12.001. Epub 2016 Dec 29.
January 9, 2017
1Matrix and Morphogenesis Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA. 2Matrix and Morphogenesis Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: [email protected]
Epithelial-mesenchymal interactions involve fundamental communication between tissues during organogenesis and are primarily regulated by growth factors and extracellular matrix. It is unclear whether RNA-containing exosomes are mobile genetic signals regulating epithelial-mesenchymal interactions. Here we identify that exosomes loaded with mesenchyme-specific mature microRNA contribute mobile genetic signals from mesenchyme to epithelium. The mature mesenchymal miR-133b-3p, loaded into exosomes, was transported from mesenchyme to the salivary epithelium, which did not express primary miR-133b-3p. Knockdown of miR-133b-3p in culture decreased endbud morphogenesis, reduced proliferation of epithelial KIT+ progenitors, and increased expression of a target gene, Disco-interacting protein 2 homolog B (Dip2b). DIP2B, which is involved in DNA methylation, was localized with 5-methylcytosine in the prophase nucleus of a subset of KIT+ progenitors during mitosis. In summary, exosomal transport of miR-133b-3p from mesenchyme to epithelium decreases DIP2B, which may function as an epigenetic regulator of genes responsible for KIT+ progenitor expansion during organogenesis.
Date: 
January 18, 2017
Where: 
HSW 1057 at noon

Eleonora De Klerk

Translation of poly(A) tails leads to precise mRNA cleavage
Guydosh NR1, Green R2
RNA. 2017 Feb 13. pii: rna.060418.116. doi: 10.1261/rna.060418.116. [Epub ahead of print]
February 13, 2017
National Institute of Diabetes and Digestive and Kidney Diseases. Johns Hopkins University School of Medicine [email protected]
Translation of poly(A) tails leads to mRNA cleavage but the mechanism and global pervasiveness of this "nonstop/no-go" decay process is not understood. Here we performed ribosome profiling (in a yeast strain lacking exosome function) of short 15-18 nt mRNA footprints to identify ribosomes stalled at 3' ends of mRNA decay intermediates. In this background, we found mRNA cleavage extending hundreds of nucleotides upstream of ribosome stalling in poly(A) and predominantly in one reading frame. These observations suggest that decay-triggering endonucleolytic cleavage is closely associated with the ribosome. Surprisingly, ribosomes appeared to accumulate (i.e. stall) in the transcriptome when as few as 3 consecutive ORF-internal lysine codons were positioned in the A, P, and E sites though significant mRNA degradation was not observed. Endonucleolytic cleavage was found, however, at sites of premature polyadenylation (encoding polylysine) and rescue of the ribosomes stalled at these sites was dependent on Dom34. These results suggest this process may be critical when changes in the polyadenylation site occur during development, tumorigenesis, or when translation termination/recycling is impaired.
Date: 
March 29, 2017
Where: 
HSW 1057 at noon

Theodore Roth

3D structures of individual mammalian genomes studied by single-cell Hi-C
Tim J. Stevens, David Lando, Srinjan Basu, Liam P. Atkinson, Yang Cao, Steven F. Lee, Martin Leeb, Kai J. Wohlfahrt, Wayne Boucher, Aoife O’Shaughnessy-Kirwan, Julie Cramard, Andre J. Faure, Meryem Ralser, Enrique Blanco, Lluis Morey, Miriam Sansó, Matthieu G. S. Palayret, Ben Lehner, Luciano Di Croce, Anton Wutz, Brian Hendrich, Dave Klenerman & Ernest D. Laue.
Nature. 2017 Mar 13. doi: 10.1038/nature21429. [Epub ahead of print]
March 13, 2017
1 Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK. 2 MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK. 3 Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK. 4 Wellcome Trust - MRC Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK. 5 EMBL-CRG Systems Biology Unit, Centre for Genomic Regulation (CRG), 08003 Barcelona, Spain. 6 Universitat Pompeu Fabra, 08003 Barcelona, Spain. 7 Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain.
The folding of genomic DNA from the beads-on-a-string-like structure of nucleosomes into higher-order assemblies is crucially linked to nuclear processes. Here we calculate 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. The technique enables genome folding to be examined at a scale of less than 100 kb, and chromosome structures to be validated. The structures of individual topological-associated domains and loops vary substantially from cell to cell. By contrast, A and B compartments, lamina-associated domains and active enhancers and promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. By studying genes regulated by pluripotency factor and nucleosome remodelling deacetylase (NuRD), we illustrate how the determination of single-cell genome structure provides a new approach for investigating biological processes.
Date: 
March 22, 2017
Where: 
HSW 1057 at noon

Not in session

N/A
Date: 
March 15, 2017
Where: 
HSW 1057 at noon