UCSF RNA Journal Club

A newsletter announcing the next presenter for RNA Journal Club

Koh Fujinaga

TT-seq maps the human transient transcriptome
Schwalb B1, Michel M1, Zacher B2, Frühauf K3, Demel C1, Tresch A4, Gagneur J5, Cramer P6.
Science. 2016 Jun 3;352(6290):1225-8. doi: 10.1126/science.aad9841.
June 3, 2016
1Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen, Germany. 2Gene Center Munich, Ludwig-Maximilians- Universität München, Feodor-Lynen-Straße 25, 81377 Munich, Germany. 3Department of Biosciences and Nutrition, Center for Innovative Medicine, and Science for Life Laboratory, Karolinska Institutet, Novum, Hälsovägen 7, 141 83 Huddinge, Sweden. 4Department of Biology, University of Cologne, Zülpicher Straße 47, 50647 Cologne, Germany. 5Max Planck Institute for Plant Breeding Research, Carl-von-Linné Weg 10, 50829 Cologne, Germany. *These authors contributed equally to this work. †Present address: Department of Informatics, Technische Universität München, Boltzmannstraße 3, 85748 Garching, Germany. ‡Corresponding author. Email: [email protected] (J.G.); [email protected] (P.C.)
Pervasive transcription of the genome produces both stable and transient RNAs. We developed transient transcriptome sequencing (TT-seq), a protocol that uniformly maps the entire range of RNA-producing units and estimates rates of RNA synthesis and degradation. Application of TT-seq to human K562 cells recovers stable messenger RNAs and long intergenic noncoding RNAs and additionally maps transient enhancer, antisense, and promoter-associated RNAs. TT-seq analysis shows that enhancer RNAs are short-lived and lack U1 motifs and secondary structure. TT-seq also maps transient RNA downstream of polyadenylation sites and uncovers sites of transcription termination; we found, on average, four transcription termination sites, distributed in a window with a median width of ~3300 base pairs. Termination sites coincide with a DNA motif associated with pausing of RNA polymerase before its release from the genome.
Date: 
August 3, 2016
Where: 
HSW 1057 at noon

Canceled

N/A
Date: 
July 27, 2016
Where: 
HSW 1057 at noon

Malin Akerblom

C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector
Abudayyeh OO1, Gootenberg JS2, Konermann S3, Joung J3, Slaymaker IM3, Cox DB4, Shmakov S5, Makarova KS6, Semenova E7, Minakhin L7, Severinov K8, Regev A9, Lander ES10, Koonin EV11, Zhang F12.
Science. 2016 Jun 2. pii: aaf5573. [Epub ahead of print]
June 2, 2016
1 Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. 2 Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA. 3 McGovern Institute for Brain Research at MIT, Cambridge, Massachusetts 02139, USA. 4 Departments of Brain and Cognitive Science and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 5 Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA. 6 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. 7 Skolkovo Institute of Science and Technology, Skolkovo, 143025, Russia. 8 National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA. 9 Waksman Institute for Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA. 10Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, 123182, Russia. *These authors contributed equally to this work.
The CRISPR-Cas adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the Class 2 type VI-A CRISPR-Cas effector C2c2 and demonstrate its RNA-guided RNase function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage. In vitro biochemical analysis show that C2c2 is guided by a single crRNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved HEPN domains, mutations in which generate catalytically inactive RNA-binding proteins. These results broaden our understanding of CRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools.
Date: 
July 20, 2016
Where: 
HSW 1057 at noon

John Gagnon

An Abundant Class of Non-coding DNA Can Prevent Stochastic Gene Silencing in the C. elegans Germline
Christian Frøkjær-Jensen,1,2,3 Nimit Jain,4 Loren Hansen,2 M. Wayne Davis,1 Yongbin Li,8 Di Zhao,8 Karine Rebora,6, Jonathan R.M. Millet,6 Xiao Liu,5,8 Stuart K. Kim,5,7 Denis Dupuy,6 Erik M. Jorgensen,1,* and Andrew Z. Fire2,7,*
Cell.
July 14, 2016
1Department of Biology, Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT 84112, USA 2Department of Pathology, Stanford University, Stanford, CA 94305, USA 3Department of Biomedical Sciences and Danish National Research Foundation Centre for Cardiac Arrhythmia, University of Copenhagen, 2200 Copenhagen N, Denmark 4Department of Bioengineering, Stanford University School of Medicine, Stanford, CA 94305, USA 5Department of Developmental Biology, Stanford University Medical Center, Stanford, CA 94305, USA 6IECB, University of Bordeaux, Laboratoire ARNA-INSERM, U869, 33600 Pessac, France 7Department of Genetics, Stanford University, Stanford, CA 94305, USA 8School of Life Sciences, Tsinghua University, Beijing 100084, China
Cells benefit from silencing foreign genetic elements but must simultaneously avoid inactivating endogenous genes. Although chromatin modifications and RNAs contribute to maintenance of silenced states, the establishment of silenced regions will inevitably reflect underlying DNA sequence and/or structure. Here, we demonstrate that a pervasive non-coding DNA feature in Caenorhabditis elegans, characterized by 10-base pair periodic An/Tn-clusters (PATCs), can license transgenes for germline expression within repressive chromatin domains. Transgenes containing natural or synthetic PATCs are resistant to position effect variegation and stochastic silencing in the germline. Among endogenous genes, intron length and PATC-character undergo dramatic changes as orthologs move from active to repressive chromatin over evolutionary time, indicating a dynamic character to the An/Tn periodicity. We propose that PATCs form the basis of a cellular immune system, identifying certain endogenous genes in heterochromatic contexts as privileged while foreign DNA can be suppressed with no requirement for a cellular memory of prior exposure.
Date: 
July 6, 2016
Where: 
HSW 1057 at noon

James Blau

Molecular recordings by directed CRISPR spacer acquisition
Shipman SL1, Nivala J2, Macklis JD3, Church GM4.
Science. 2016 Jun 9. pii: aaf1175. [Epub ahead of print]
June 9, 2016
Author information 1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. Department of Stem Cell and Regenerative Biology, Center for Brain Science, and Harvard Stem Cell Institute, Harvard University, Bauer Laboratory 103, Cambridge, MA 02138, USA. Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA 02138, USA. 2Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA 02138, USA. 3Department of Stem Cell and Regenerative Biology, Center for Brain Science, and Harvard Stem Cell Institute, Harvard University, Bauer Laboratory 103, Cambridge, MA 02138, USA. 4Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA 02138, USA. [email protected]
The ability to write a stable record of identified molecular events into a specific genomic locus would enable the examination of long cellular histories and have many applications, ranging from developmental biology to synthetic devices. We show that the type I-E CRISPR-Cas system of E. coli can mediate acquisition of defined pieces of synthetic DNA. We harnessed this feature to generate records of specific DNA sequences into a population of bacterial genomes. We then applied directed evolution to alter the recognition of a protospacer adjacent motif by the Cas1-Cas2 complex, which enabled recording in two modes simultaneously. We used this system to reveal aspects of spacer acquisition, fundamental to the CRISPR-Cas adaptation process. These results lay the foundations of a multimodal intracellular recording device.
Date: 
June 29, 2016
Where: 
HSW 1057 at noon

Brian Grone

The industrial melanism mutation in British peppered moths is a transposable element
Van't Hof AE1, Campagne P1, Rigden DJ1, Yung CJ1, Lingley J1, Quail MA2, Hall N1, Darby AC1, Saccheri IJ1.
Nature. 2016 Jun 1;534(7605):102-5. doi: 10.1038/nature17951.
June 1, 2016
1Institute of Integrative Biology, University of Liverpool, Biosciences Building, Crown Street, Liverpool L69 7ZB, UK. 2Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK. *These authors contributed equally to this work
Discovering the mutational events that fuel adaptation to environmental change remains an important challenge for evolutionary biology. The classroom example of a visible evolutionary response is industrial melanism in the peppered moth (Biston betularia): the replacement, during the Industrial Revolution, of the common pale typica form by a previously unknown black (carbonaria) form, driven by the interaction between bird predation and coal pollution. The carbonaria locus has been coarsely localized to a 200-kilobase region, but the specific identity and nature of the sequence difference controlling the carbonaria-typica polymorphism, and the gene it influences, are unknown. Here we show that the mutation event giving rise to industrial melanism in Britain was the insertion of a large, tandemly repeated, transposable element into the first intron of the gene cortex. Statistical inference based on the distribution of recombined carbonaria haplotypes indicates that this transposition event occurred around 1819, consistent with the historical record. We have begun to dissect the mode of action of the carbonaria transposable element by showing that it increases the abundance of a cortex transcript, the protein product of which plays an important role in cell-cycle regulation, during early wing disc development. Our findings fill a substantial knowledge gap in the iconic example of microevolutionary change, adding a further layer of insight into the mechanism of adaptation in response to natural selection. The discovery that the mutation itself is a transposable element will stimulate further debate about the importance of 'jumping genes' as a source of major phenotypic novelty.
Date: 
June 22, 2016
Where: 
HSW 1057 at noon

Roman Camarda

Long noncoding RNA LINP1 regulates repair of DNA double-strand breaks in triple-negative breast cancer
Zhang Y1, He Q1, Hu Z1, Feng Y1,2, Fan L1, Tang Z1, Yuan J1, Shan W1, Li C1,3, Hu X1,3, Tanyi JL3, Fan Y4, Huang Q5, Montone K6, Dang CV2,7,8, Zhang L1,3,8.
Nat Struct Mol Biol.
April 25, 2016
1Center for Research on Reproduction & Women’s Health, University of Pennsylvania, Philadelphia, Pennsylvania, USA. 2Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania, USA. 3Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, Pennsylvania, USA. 4Department of Radiation Oncology, University of Pennsylvania, Philadelphia, Pennsylvania, USA. 5Wistar Institute, Philadelphia, Pennsylvania, USA. 6Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. 7Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. 8Abramson Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania, USA. 9These authors
Long noncoding RNAs (lncRNAs) play critical roles during tumorigenesis by functioning as scaffolds that regulate protein-protein, protein-DNA or protein-RNA interactions. Using a clinically guided genetic screening approach, we identified lncRNA in nonhomologous end joining (NHEJ) pathway 1 (LINP1), which is overexpressed in human triple-negative breast cancer. We found that LINP1 enhances repair of DNA double-strand breaks by serving as a scaffold linking Ku80 and DNA-PKcs, thereby coordinating the NHEJ pathway. Importantly, blocking LINP1, which is regulated by p53 and epidermal growth factor receptor (EGFR) signaling, increases the sensitivity of the tumor-cell response to radiotherapy in breast cancer.
Date: 
June 15, 2016
Where: 
HSW 1057 at noon

Michael Boettcher

Whole organism lineage tracing by combinatorial and cumulative genome editing
McKenna A1, Findlay GM1, Gagnon JA2, Horwitz MS3, Schier AF4, Shendure J5.
Science.
May 26, 2016
1Department of Genome Sciences, University of Washington, Seattle, WA, USA. 2Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA. 3Department of Pathology, University of Washington, Seattle, WA, USA. 4Center for Brain Science, Harvard University, Cambridge, MA, USA. 5The Broad Institute of Harvard and MIT, Cambridge, MA, USA. 6FAS Center for Systems Biology, Harvard University, Cambridge, MA, USA. 7Howard Hughes Medical Institute, Seattle, WA, USA.
Multicellular systems develop from single cells through distinct lineages. However, current lineage tracing approaches scale poorly to whole, complex organisms. Here, we use genome editing to progressively introduce and accumulate diverse mutations in a DNA barcode over multiple rounds of cell division. The barcode, an array of CRISPR/Cas9 target sites, marks cells and enables the elucidation of lineage relationships via the patterns of mutations shared between cells. In cell culture and zebrafish, we show that rates and patterns of editing are tunable and that thousands of lineage-informative barcode alleles can be generated. By sampling hundreds of thousands of cells from individual zebrafish, we find that most cells in adult organs derive from relatively few embryonic progenitors. In future analyses, genome editing of synthetic target arrays for lineage tracing (GESTALT) can be used to generate large-scale maps of cell lineage in multicellular systems for normal development and disease.
Date: 
June 8, 2016
Where: 
HSW 1057 at noon

Ben Mansky

RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure
Lu Z1, Zhang QC2, Lee B1, Flynn RA1, Smith MA3, Robinson JT4, Davidovich C5, Gooding AR6, Goodrich KJ6, Mattick JS3, Mesirov JP4, Cech TR6, Chang HY7.
Cell
May 19, 2016
1Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA 94305, USA 2MOE Key Laboratory of Bioinformatics, Beijing Advanced Innovation Center for Structural Biology, Center for Synthetic and Systems Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China 3RNA Biology and Plasticity Group, Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia 4St Vincent’s Clinical School, UNSW Medicine, NSW 2052, Australia 5Department of Medicine and Moores Cancer Center, University of California San Diego, La Jolla, CA 92093, USA 6HHMI and Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80303, USA 7Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Victoria 3800, Australia 8EMBL Australia and the ARC Centre of Excellence in Advanced Molecular Imaging, Clayton, VIC 3800, Australia 9Co-first author *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cell.2016.04.028
RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Here, we develop PARIS, a method based on reversible psoralen crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells. PARIS analysis in three human and mouse cell types reveals frequent long-range structures, higher-order architectures, and RNA-RNA interactions in trans across the transcriptome. PARIS determines base-pairing interactions on an individual-molecule level, revealing pervasive alternative conformations. We used PARIS-determined helices to guide phylogenetic analysis of RNA structures and discovered conserved long-range and alternative structures. XIST, a long noncoding RNA (lncRNA) essential for X chromosome inactivation, folds into evolutionarily conserved RNA structural domains that span many kilobases. XIST A-repeat forms complex inter-repeat duplexes that nucleate higher-order assembly of the key epigenetic silencing protein SPEN. PARIS is a generally applicable and versatile method that provides novel insights into the RNA structurome and interactome. VIDEO ABSTRACT.
Date: 
June 1, 2016
Where: 
HSW 1057 at noon

Vanille Greiner

CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs
Mandegar MA1, Huebsch N2, Frolov EB3, Shin E3, Truong A3, Olvera MP3, Chan AH3, Miyaoka Y3, Holmes K3, Spencer CI3, Judge LM2, Gordon DE4, Eskildsen TV5, Villalta JE6, Horlbeck MA6, Gilbert LA6, Krogan NJ4, Sheikh SP5, Weissman JS6, Qi LS7, So PL3, Conklin BR8.
Cell Stem Cell.
April 7, 2016
1Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA 2Department of Pediatrics, University of California, San Francisco, San Francisco, CA 94158, USA 3Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA 4California Institute for Quantitative Biosciences, QB3, University of California, San Francisco, San Francisco, CA 94158, USA 5Gladstone Institute of Virology and Immunology, San Francisco, CA 94158, USA 6Department of Cardiovascular and Renal Research, University of Southern Denmark, 5000 Odense C, Denmark 7Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, 5000 Odense C, Denmark 8Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA 9Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA 10Department of Bioengineering, Stanford University, Stanford, CA 94305, USA 11Department of Medicine and Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA 12Present address: Regenerative Medicine Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, 156-8506, Japan *Correspondence: [email protected] (M.A.M.), [email protected] (B.R.C.) http://dx.doi.org/10.1016/j.stem.2016.01.022
Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease.
Date: 
May 11, 2016
Where: 
HSW 1057 at noon