UCSF RNA Journal Club

A newsletter announcing the next presenter for RNA Journal Club

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

Ryan Wagner

Genome-wide mapping of autonomous promoter activity in human cells
van Arensbergen J1, FitzPatrick VD2,3, de Haas M1, Pagie L1, Sluimer J1, Bussemaker HJ2,3, van Steensel B1.
Nat Biotechnol. 2017 Feb;35(2):145-153. doi: 10.1038/nbt.3754. Epub 2016 Dec 26.
February 1, 2017
1Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, the Netherlands. 2Department of Biological Sciences, Columbia University, New York, New York, USA. 3Department of Systems Biology, Columbia University Medical Center, New York, New York, USA.
Previous methods to systematically characterize sequence-intrinsic activity of promoters have been limited by relatively low throughput and the length of the sequences that could be tested. Here we present 'survey of regulatory elements' (SuRE), a method that assays more than 108 DNA fragments, each 0.2-2 kb in size, for their ability to drive transcription autonomously. In SuRE, a plasmid library of random genomic fragments upstream of a 20-bp barcode is constructed, and decoded by paired-end sequencing. This library is used to transfect cells, and barcodes in transcribed RNA are quantified by high-throughput sequencing. When applied to the human genome, we achieve 55-fold genome coverage, allowing us to map autonomous promoter activity genome-wide in K562 cells. By computational modeling we delineate subregions within promoters that are relevant for their activity. We show that antisense promoter transcription is generally dependent on the sense core promoter sequences, and that most enhancers and several families of repetitive elements act as autonomous transcription initiation sites.
Date: 
March 8, 2017
Where: 
HSW 1057 at noon

Vanille Greiner

Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing
Amabile A1, Migliara A1, Capasso P2, Biffi M2, Cittaro D3, Naldini L4, Lombardo A5.
Cell. 2016 Sep 22;167(1):219-232.e14. doi: 10.1016/j.cell.2016.09.006.
September 22, 2016
1San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy; Vita-Salute San Raffaele University, Via Olgettina 58, 20132 Milan, Italy. 2San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy. 3Center for Translational Genomics and Bioinformatics, IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy. 4San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy; Vita-Salute San Raffaele University, Via Olgettina 58, 20132 Milan, Italy. Electronic address: [email protected] 5San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy; Vita-Salute San Raffaele University, Via Olgettina 58, 20132 Milan, Italy. Electronic address: [email protected] Abstract
Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine.
Date: 
March 1, 2017
Where: 
HSW 1057 at noon

Malin Akerblom

Synthetic recording and in situ readout of linage information in single cells
Frieda KL1, Linton JM1, Hormoz S1, Choi J2, Chow KK1, Singer ZS1, Budde MW1, Elowitz MB1,3, Cai L2.
Nature. 2017 Jan 5;541(7635):107-111. doi: 10.1038/nature20777. Epub 2016 Nov 21.
January 5, 2017
1Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA. 2Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA. 3Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA.
Reconstructing the lineage relationships and dynamic event histories of individual cells within their native spatial context is a long-standing challenge in biology. Many biological processes of interest occur in optically opaque or physically inaccessible contexts, necessitating approaches other than direct imaging. Here we describe a synthetic system that enables cells to record lineage information and event histories in the genome in a format that can be subsequently read out of single cells in situ. This system, termed memory by engineered mutagenesis with optical in situ readout (MEMOIR), is based on a set of barcoded recording elements termed scratchpads. The state of a given scratchpad can be irreversibly altered by CRISPR/Cas9-based targeted mutagenesis, and later read out in single cells through multiplexed single-molecule RNA fluorescence hybridization (smFISH). Using MEMOIR as a proof of principle, we engineered mouse embryonic stem cells to contain multiple scratchpads and other recording components. In these cells, scratchpads were altered in a progressive and stochastic fashion as the cells proliferated. Analysis of the final states of scratchpads in single cells in situ enabled reconstruction of lineage information from cell colonies. Combining analysis of endogenous gene expression with lineage reconstruction in the same cells further allowed inference of the dynamic rates at which embryonic stem cells switch between two gene expression states. Finally, using simulations, we show how parallel MEMOIR systems operating in the same cell could enable recording and readout of dynamic cellular event histories. MEMOIR thus provides a versatile platform for information recording and in situ, single-cell readout across diverse biological systems.
Date: 
February 15, 2017
Where: 
HSW 1057 at noon

John Gagnon

An Argonaute phosphorylation cycle promotes microRNA-mediated silencing
Golden RJ1,2, Chen B3,4, Li T1, Braun J1, Manjunath H1, Chen X1, Wu J5, Schmid V6, Chang TC1, Kopp F1, Ramirez-Martinez A1, Tagliabracci VS1, Chen ZJ1,7, Xie Y3,4,8, Mendell JT1,7,8,9.
Nature. 2017 Jan 23. doi: 10.1038/nature21025. [Epub ahead of print]
February 23, 2017
1Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 2Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 3Quantitative Biomedical Research Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 4Department of Clinical Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 5Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California 94143, USA. 6Eugene McDermott Center for Human Growth &Development, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 7Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 8Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 9Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). Here we use clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity in human cells to identify new regulators of the miRNA pathway. By using iterative rounds of screening, we reveal a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrate that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 reveals a pronounced expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per-target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.
Date: 
February 8, 2017
Where: 
HSW 1057 at noon