UCSF RNA Journal Club

A newsletter announcing the next presenter for RNA Journal Club

Hui Li

Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer
Kamerkar S, LeBleu VS, Sugimoto H, Yang S, Ruivo CF, Melo SA, Lee JJ, Kalluri R.
Nature. 2017 Jun 22;546(7659):498-503. doi: 10.1038/nature22341. Epub 2017 Jun 7.
Department of Cancer Biology, Metastasis Research Center, University of Texas MD Anderson Cancer Center, Houston, Texas 77005, USA. Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal (I3S), 4200 Porto, Portugal; Institute of Pathology and Molecular Immunology of the University of Porto (IPATIMUP), 4200 Porto, Portugal. Department of Biostatistics, University of Texas MD Anderson Cancer Center, Houston, Texas 77005, USA.
The mutant form of the GTPase KRAS is a key driver of pancreatic cancer but remains a challenging therapeutic target. Exosomes are extracellular vesicles generated by all cells, and are naturally present in the blood. Here we show that enhanced retention of exosomes, compared to liposomes, in the circulation of mice is likely due to CD47-mediated protection of exosomes from phagocytosis by monocytes and macrophages. Exosomes derived from normal fibroblast-like mesenchymal cells were engineered to carry short interfering RNA or short hairpin RNA specific to oncogenic KrasG12D, a common mutation in pancreatic cancer. Compared to liposomes, the engineered exosomes (known as iExosomes) target oncogenic KRAS with an enhanced efficacy that is dependent on CD47, and is facilitated by macropinocytosis. Treatment with iExosomes suppressed cancer in multiple mouse models of pancreatic cancer and significantly increased overall survival. Our results demonstrate an approach for direct and specific targeting of oncogenic KRAS in tumours using iExosomes.
Date: 
July 12, 2017
Where: 
HSW 1057 at noon

Fang Huang

Extensive RNA editing and splicing increase immune self-representation diversity in medullary thymic epithelial cells
Danan-Gotthold M, Guyon C, Giraud M, Levanon EY, Abramson J.
Genome Biol. 2016 Oct 24;17(1):219.
The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 52900, Israel. Department of Infection Immunity and Inflammation, Cochin Institute, Paris, France. The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 52900, Israel. [email protected] Department of Immunology, Weizmann Institute of Science, Rehovot, Israel. [email protected]
BACKGROUND: In order to become functionally competent but harmless mediators of the immune system, T cells undergo a strict educational program in the thymus, where they learn to discriminate between self and non-self. This educational program is, to a large extent, mediated by medullary thymic epithelial cells that have a unique capacity to express, and subsequently present, a large fraction of body antigens. While the scope of promiscuously expressed genes by medullary thymic epithelial cells is well-established, relatively little is known about the expression of variants that are generated by co-transcriptional and post-transcriptional processes. RESULTS: Our study reveals that in comparison to other cell types, medullary thymic epithelial cells display significantly higher levels of alternative splicing, as well as A-to-I and C-to-U RNA editing, which thereby further expand the diversity of their self-antigen repertoire. Interestingly, Aire, the key mediator of promiscuous gene expression in these cells, plays a limited role in the regulation of these transcriptional processes. CONCLUSIONS: Our results highlight RNA processing as another layer by which the immune system assures a comprehensive self-representation in the thymus which is required for the establishment of self-tolerance and prevention of autoimmunity. KEYWORDS: Alternative splicing; Medullary thymic epithelial cells (mTECs); RNA editing; RNA sequencing; Self-tolerance; Thymus
Date: 
July 5, 2017
Where: 
HSW 1057 at noon

Theodore Roth

Mapping the genomic landscape of CRISPR–Cas9 cleavage
Peter Cameron, Chris K Fuller, Paul D Donohoue, Brittnee N Jones, Matthew S Thompson, Matthew M Carter, Scott Gradia, Bastien Vidal, Elizabeth Garner, Euan M Slorach, Elaine Lau, Lynda M Banh, Alexandra M Lied, Leslie S Edwards, Alexander H Settle, Daniel Capurso, Victor Llaca, Stéphane Deschamps, Mark Cigan, Joshua K Young & Andrew P May.
Nature Methods
June 1, 2017
Caribou Biosciences, Berkeley, California, USA. DuPont Pioneer, Johnston, Iowa, USA. Present addresses: Omicia, Inc., Oakland, California, USA (B.N.J.); Genus Research, DeForest, Wisconsin, USA (M.C.) and Chan Zuckerberg Biohub, San Francisco, California, USA (A.P.M.). These authors contributed equally to this work. Correspondence should be addressed to J.K.Y. ([email protected]) or A.P.M. ([email protected]).
RNA-guided CRISPR–Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9–sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.
Date: 
June 28, 2017
Where: 
HSW 1057 at noon

John Gagnon

A maternal-effect selfish genetic element in Caenorhabditis elegans
Ben-David E, Burga A, Kruglyak L.
Science. 2017 Jun 9;356(6342):1051-1055. doi: 10.1126/science.aan0621. Epub 2017 May 11.
Department of Human Genetics, Department of Biological Chemistry, and Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095, USA. [email protected] [email protected] [email protected]
Selfish genetic elements spread in natural populations and have an important role in genome evolution. We discovered a selfish element causing embryonic lethality in crosses between wild strains of the nematode Caenorhabditis elegans The element is made up of sup-35, a maternal-effect toxin that kills developing embryos, and pha-1, its zygotically expressed antidote. pha-1 has long been considered essential for pharynx development on the basis of its mutant phenotype, but this phenotype arises from a loss of suppression of sup-35 toxicity. Inactive copies of the sup-35/pha-1 element show high sequence divergence from active copies, and phylogenetic reconstruction suggests that they represent ancestral stages in the evolution of the element. Our results suggest that other essential genes identified by genetic screens may turn out to be components of selfish elements.
Date: 
June 21, 2017
Where: 
HSW 1057 at noon

Ryan Wagner

Engineered Cpf1 variants with altered PAM specificities
Gao L, Cox DBT, Yan WX, Manteiga JC, Schneider MW, Yamano T, Nishimasu H, Nureki O, Crosetto N, Zhang F.
Nat Biotechnol. 2017 Jun 5. doi: 10.1038/nbt.3900. [Epub ahead of print]
Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. Department of Biological Engineering, Massachusetts Institute of Technology Cambridge, Massachusetts, USA. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. Harvard-MIT Division of Health Sciences and Technology, Harvard Medical School, Boston, Massachusetts, USA. Graduate Program in Biophysics, Harvard Medical School, Boston, Massachusetts, USA. Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan. JST, PRESTO, Tokyo, Japan. Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ∼11 bp.
Date: 
June 14, 2017
Where: 
HSW 1057 at noon

Kol Jia Yong (UPDATE: New Article)

High-resolution interrogation of functional elements in the noncoding genome
Neville E. Sanjana1,2,*,†,‡, Jason Wright1,2,†, Kaijie Zheng1,2, Ophir Shalem1,2, Pierre Fontanillas1, Julia Joung1,2, Christine Cheng1,3, Aviv Regev1,3, Feng
Science. 2016 Sep 30;353(6307):1545-1549.
September 30, 2016
1Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA. McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. [email protected] [email protected] 2Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA. McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 3Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA. 4Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA. Howard Hughes Medical Institute, David H. Koch Institute of Integrative Cancer Biology, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
The noncoding genome affects gene regulation and disease, yet we lack tools for rapid identification and manipulation of noncoding elements. We developed a CRISPR screen using ~18,000 single guide RNAs targeting >700 kilobases surrounding the genes NF1, NF2, and CUL3, which are involved in BRAF inhibitor resistance in melanoma. We find that noncoding locations that modulate drug resistance also harbor predictive hallmarks of noncoding function. With a subset of regions at the CUL3 locus, we demonstrate that engineered mutations alter transcription factor occupancy and long-range and local epigenetic environments, implicating these sites in gene regulation and chemotherapeutic resistance. Through our expansion of the potential of pooled CRISPR screens, we provide tools for genomic discovery and for elucidating biologically relevant mechanisms of gene regulation.
Date: 
February 22, 2017
Where: 
HSW 1057 at noon

Vanille Greiner

m6A-dependent maternal mRNA clearance facilitates zebrafish maternal-to-zygotic transition
Zhao BS, Wang X, Beadell AV, Lu Z, Shi H, Kuuspalu A, Ho RK, He C.
Nature. 2017 Feb 23;542(7642):475-478. doi: 10.1038/nature21355. Epub 2017 Feb 13.
Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA. Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA. Department of Organismal Biology and Anatomy, The University of Chicago, 1027 East 57th Street, Chicago, Illinois 60637, USA. Department of Biochemistry and Molecular Biology, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
The maternal-to-zygotic transition (MZT) is one of the most profound and tightly orchestrated processes during the early life of embryos, yet factors that shape the temporal pattern of vertebrate MZT are largely unknown. Here we show that over one-third of zebrafish maternal messenger RNAs (mRNAs) can be N6-methyladenosine (m6A) modified, and the clearance of these maternal mRNAs is facilitated by an m6A-binding protein, Ythdf2. Removal of Ythdf2 in zebrafish embryos decelerates the decay of m6A-modified maternal mRNAs and impedes zygotic genome activation. These embryos fail to initiate timely MZT, undergo cell-cycle pause, and remain developmentally delayed throughout larval life. Our study reveals m6A-dependent RNA decay as a previously unidentified maternally driven mechanism that regulates maternal mRNA clearance during zebrafish MZT, highlighting the critical role of m6A mRNA methylation in transcriptome switching and animal development.
Date: 
June 7, 2017
Where: 
HSW 1057 at noon

Malin Akerblom

Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis
Legnini I, Di Timoteo G, Rossi F, Morlando M, Briganti F, Sthandier O, Fatica A, Santini T, Andronache A, Wade M, Laneve P, Rajewsky N, Bozzoni I.
Mol Cell. 2017 Apr 6;66(1):22-37.e9. doi: 10.1016/j.molcel.2017.02.017. Epub 2017 Mar 23.
Department of Biology and Biotechnology Charles Darwin and IBPM, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy. Center for Life Nano [email protected], Istituto Italiano di Tecnologia, Viale Regina Elena 291, 00161 Rome, Italy. Center for Genomic Science of [email protected], Fondazione Istituto Italiano di Tecnologia (IIT), Via Adamello 16, 20139 Milan, Italy. Berlin Institute for Medical Systems Biology, Max-Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13125 Berlin, Germany. Department of Biology and Biotechnology Charles Darwin and IBPM, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy; Center for Life Nano [email protected], Istituto Italiano di Tecnologia, Viale Regina Elena 291, 00161 Rome, Italy; Institut Pasteur Italy, Fondazione Cenci-Bolognetti, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy. Electronic address: [email protected]
Circular RNAs (circRNAs) constitute a family of transcripts with unique structures and still largely unknown functions. Their biogenesis, which proceeds via a back-splicing reaction, is fairly well characterized, whereas their role in the modulation of physiologically relevant processes is still unclear. Here we performed expression profiling of circRNAs during in vitro differentiation of murine and human myoblasts, and we identified conserved species regulated in myogenesis and altered in Duchenne muscular dystrophy. A high-content functional genomic screen allowed the study of their functional role in muscle differentiation. One of them, circ-ZNF609, resulted in specifically controlling myoblast proliferation. Circ-ZNF609 contains an open reading frame spanning from the start codon, in common with the linear transcript, and terminating at an in-frame STOP codon, created upon circularization. Circ-ZNF609 is associated with heavy polysomes, and it is translated into a protein in a splicing-dependent and cap-independent manner, providing an example of a protein-coding circRNA in eukaryotes.
Date: 
May 24, 2017
Where: 
HSW 1057 at noon

Kol Jia Yong

Impact of cytosine methylation on DNA binding specificities of human transcription factors
Yin Y, Morgunova E, Jolma A, Kaasinen E, Sahu B, Khund-Sayeed S, Das PK, Kivioja T, Dave K, Zhong F, Nitta KR, Taipale M, Popov A, Ginno PA, Domcke S, Yan J, Schübeler D, Vinson C, Taipale J.
Science. 2017 May 5;356(6337). pii: eaaj2239. doi: 10.1126/science.aaj2239.
Division of Functional Genomics and Systems Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE 141 83 Stockholm, Sweden. Genome-Scale Biology Program, Post Office Box 63, FI-00014 University of Helsinki, Helsinki, Finland. Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Room 3128, Building 37, Bethesda, MD 20892, USA. European Synchrotron Radiation Facility, 38043 Grenoble, France. Friedrich-Miescher-Institute for Biomedical Research (FMI), Maulbeerstrasse 66, 4058 Basel, Switzerland. Faculty of Science, University of Basel, Petersplatz 1, 4003 Basel, Switzerland. Division of Functional Genomics and Systems Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE 141 83 Stockholm, Sweden. [email protected]
The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences.
Date: 
May 17, 2017
Where: 
HSW 1057 at noon

Maryia Barnett

CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome
Tyler S Klann, Joshua B Black, Malathi Chellappan, Alexias Safi, Lingyun Song, Isaac B Hilton, Gregory E Crawford, Timothy E Reddy & Charles A Gersbach
Nat Biotechnol. 2017 Apr 3. doi: 10.1038/nbt.3853. [Epub ahead of print]
Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA. Center for Genomic and Computational Biology, Duke University, Durham, North Carolina, USA. Department of Pediatrics, Division of Medical Genetics, Duke University Medical Center, Durham, North Carolina, USA. Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, North Carolina, USA. Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina, USA.
Large genome-mapping consortia and thousands of genome-wide association studies have identified non-protein-coding elements in the genome as having a central role in various biological processes. However, decoding the functions of the millions of putative regulatory elements discovered in these studies remains challenging. CRISPR-Cas9-based epigenome editing technologies have enabled precise perturbation of the activity of specific regulatory elements. Here we describe CRISPR-Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. Using dCas9KRAB repressor and dCas9p300 activator constructs and lentiviral single guide RNA libraries to target DNase I hypersensitive sites surrounding a gene of interest, we carried out both loss- and gain-of-function screens to identify regulatory elements for the β-globin and HER2 loci in human cells. CERES readily identified known and previously unidentified regulatory elements, some of which were dependent on cell type or direction of perturbation. This technology allows the high-throughput functional annotation of putative regulatory elements in their native chromosomal context.
Date: 
May 10, 2017
Where: 
HSW 1057 at noon