UCSF RNA Journal Club

A newsletter announcing the next presenter for RNA Journal Club

Not in Session

Winter Break
Date: 
December 28, 2016
Where: 
HSW 1057 at noon

Not in Session

Winter Break
Date: 
December 21, 2016
Where: 
HSW 1057 at noon

Roman Camarda

LKB1 loss links serine metabolism to DNA methylation and tumorigenesis
Kottakis F1,2,3, Nicolay BN1,3, Roumane A1,2,3, Karnik R4,5,6, Gu H4,5,6, Nagle JM1,2,3, Boukhali M1,3, Hayward MC7, Li YY8,9, Chen T8,9,10, Liesa M11,12, Hammerman PS8,9,13, Wong KK8,9,10, Hayes DN7, Shirihai OS11,12, Dyson NJ1,3, Haas W1,3, Meissner A4,5,6, Bardeesy N1,2,3.
Nature. 2016 Oct 31;539(7629):390-395. doi: 10.1038/nature20132. [Epub ahead of print]
October 31, 2016
1Cancer Center, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, USA. 2Center for Regenerative Medicine, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, USA. 3Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114, USA. 4Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA. 5Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, USA. 6Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA. 7UNC, Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina 27599, USA. 8Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA. 9Department of Medical Oncology, Dana Farber Cancer Institute, Boston, Massachusetts 02215, USA. 10Belfer Institute for Applied Cancer Science, Dana Farber Cancer Institute, Boston, Massachusetts 02215, USA. 11Evans Center for Interdisciplinary Research, Department of Medicine, Mitochondria ARC, Boston University School of Medicine, Boston, Massachusetts 02118, USA. 12Department of Medicine, Division of Endocrinology, Diabetes and Hypertension, UCLA David Geffen School of Medicine, Los Angeles, California 90095, USA. 13Cancer Program, Broad Institute of Harvard and MIT, Cambridge, Massachusetts 02142, USA.
Intermediary metabolism generates substrates for chromatin modification, enabling the potential coupling of metabolic and epigenetic states. Here we identify a network linking metabolic and epigenetic alterations that is central to oncogenic transformation downstream of the liver kinase B1 (LKB1, also known as STK11) tumour suppressor, an integrator of nutrient availability, metabolism and growth. By developing genetically engineered mouse models and primary pancreatic epithelial cells, and employing transcriptional, proteomics, and metabolic analyses, we find that oncogenic cooperation between LKB1 loss and KRAS activation is fuelled by pronounced mTOR-dependent induction of the serine-glycine-one-carbon pathway coupled to S-adenosylmethionine generation. At the same time, DNA methyltransferases are upregulated, leading to elevation in DNA methylation with particular enrichment at retrotransposon elements associated with their transcriptional silencing. Correspondingly, LKB1 deficiency sensitizes cells and tumours to inhibition of serine biosynthesis and DNA methylation. Thus, we define a hypermetabolic state that incites changes in the epigenetic landscape to support tumorigenic growth of LKB1-mutant cells, while resulting in potential therapeutic vulnerabilities.
Date: 
December 14, 2016
Where: 
HSW 1057 at noon

Theodore Roth

In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration
Suzuki K1, Tsunekawa Y2, Hernandez-Benitez R1,3, Wu J1,4, Zhu J5,6, Kim EJ7, Hatanaka F1, Yamamoto M1, Araoka T1,4, Li Z8, Kurita M1, Hishida T1, Li M1, Aizawa E1, Guo S8, Chen S8, Goebl A1, Soligalla RD1, Qu J9,10, Jiang T6,11, Fu X5,6, Jafari M6, Esteban CR1, Berggren WT12, Lajara J4, Nuñez-Delicado E4, Guillen P4,13, Campistol JM14, Matsuzaki F2, Liu GH10,15,16,17, Magistretti P3, Zhang K8, Callaway EM7, Zhang K5,6,18,19, Belmonte JC1.
Nature. 2016 Dec 1;540(7631):144-149. doi: 10.1038/nature20565. Epub 2016 Nov 16.
December 1, 2016
1Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA. 2Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan. 34700 King Abdullah University of Science and Technology (KAUST) Thuwal 23955-6900, Saudi Arabia. 4Universidad Católica San Antonio de Murcia (UCAM) Campus de los Jerónimos, no. 135 Guadalupe 30107, Murcia, Spain. 5Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, China. 6Shiley Eye Institute, Institute for Genomic Medicine, Institute of Engineering in Medicine, University of California, San Diego, 9500 Gilman Drive #0946, La Jolla, California 92023, USA. 7Systems Neurobiology Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, California 92037, USA. 8Bioengineering, University of California, San Diego, 9500 Gilman Drive, MC0412, La Jolla, California 92093-0412, USA. 9State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China. 10University of Chinese Academy of Sciences, Beijing 100049, China. 11Guangzhou EliteHealth Biological Pharmaceutical Technology Company Ltd, Guangzhou 510005, China. 12Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA. 13Fundación Dr. Pedro Guillen, Investigación Biomedica de Clinica CEMTRO, Avenida Ventisquero de la Condesa, 42, 28035 Madrid, Spain. 14Hospital Clinic, University of Barcelona, IDIBAPS, 08036 Barcelona, Spain. 15National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. 16Key Laboratory of Regenerative Medicine of Ministry of Education, Institute of Aging and Regenerative Medicine, Jinan University, Guangzhou 510632, China. 17Beijing Institute for Brain Disorders, Beijing 100069, China. 18Molecular Medicine Research Center, West China Hospital, Sichuan University, Chengdu 610041, China. 19Veterans Administration Healthcare System, San Diego, California 92093, USA.
Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.
Date: 
December 7, 2016
Where: 
HSW 1057 at noon

Eleonora De Klerk

A novel translational control mechanism involving RNA structures within coding sequences
Jungfleisch J1, Nedialkova DD2, Dotu I3, Sloan KE4, Martinez-Bosch N3, Brüning L4, Raineri E5, Navarro P3, Bohnsack MT4, Leidel SA2, Diez J6.
Genome Res. 2016 Nov 7. pii: g
November 7, 2016
1University Pompeu Fabra. 2Max Planck Insitute for Molecular Biomedicine. 3Hospital del Mar Medical Research Institute. 4Goettingen University. 5Centro Nacional de Analisis Genomica. 6University Pompeu Fabra; [email protected]
The impact of RNA structures in coding sequences (CDS) within mRNAs is poorly understood. Here we identify a novel and highly conserved mechanism of translational control involving RNA structures within coding sequences and the DEAD-box helicase Dhh1. Using yeast genetics and genome-wide ribosome profiling analyses we show that this mechanism, initially derived from studies of the Brome Mosaic virus RNA genome, extends to yeast and human mRNAs highly enriched in membrane and secreted proteins. All Dhh1-dependent mRNAs, viral and cellular, share key common features. First, they contain long and highly structured CDSs, including a region located around nucleotide 70 after the translation initiation site, second, they are directly bound by Dhh1 with a specific binding distribution and third, complementary experimental approaches suggest that they are activated by Dhh1 at the translation initiation step. Our results show that ribosome translocation is not the only unwinding force of CDS and uncover a novel layer of translational control that involves RNA helicases and RNA folding within CDS providing novel opportunities for regulation of membrane and secretome proteins.
Date: 
November 30, 2016
Where: 
HSW 1057 at noon

Thanksgiving Holiday

N/A
Date: 
November 23, 2016
Where: 
HSW 1057 at noon

Michael Boettcher

CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells
Dever DP1, Bak RO1, Reinisch A2, Camarena J1, Washington G1, Nicolas CE1, Pavel-Dinu M1, Saxena N1, Wilkens AB1, Mantri S1, Uchida N3, Hendel A1, Narla A4, Majeti R2, Weinberg KI1, Porteus MH1.
Nature. 2016 Nov 7. doi: 10.1038/nature20134. [Epub ahead of print]
November 7, 2016
1Department of Pediatrics, Stanford University, Stanford, California 94305, USA. 2Department of Medicine, Division of Hematology, Cancer Institute, and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California 94305, USA. 3Stem Cells, Inc. 7707 Gateway Blvd., Suite 140, Newark, California 94560, USA. 4Division of Hematology/Oncology, Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94035, USA
The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for β-haemoglobinopathies.
Date: 
November 16, 2016
Where: 
HSW 1057 at noon

Kol Jia Yong

Editing DNA Methylation in the Mammalian Genome
Liu XS1, Wu H1, Ji X1, Stelzer Y1, Wu X1, Czauderna S2, Shu J1, Dadon D3, Young RA3, Jaenisch R4.
Cell
September 22, 2016
1Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA. 2Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, 31-007 Kraków, Poland. 3Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. 4Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address: [email protected]
Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation
Date: 
November 9, 2016
Where: 
HSW 1057 at noon

Vanille Greiner

Progressive Loss of Function in a Limb Enhancer during Snake Evolution
Kvon EZ1, Kamneva OK2, Melo US1, Barozzi I1, Osterwalder M1, Mannion BJ1, Tissières V3, Pickle CS1, Plajzer-Frick I1, Lee EA1, Kato M1, Garvin TH1, Akiyama JA1, Afzal V1, Lopez-Rios J3, Rubin EM4, Dickel DE1, Pennacchio LA5, Visel A6.
Cell. 2016 Oct 20;167(3):633-642.e11. doi: 10.1016/j.cell.2016.09.028.
October 20, 2016
1MS 84-171, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. 2Department of Biology, Stanford University, Stanford, CA 94305, USA. 3Department of Biomedicine, University of Basel, 4058 Basel, Switzerland. 4MS 84-171, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; U.S. Department of Energy Joint Genome Institute, Walnut Creek, CA 94598, USA. 5MS 84-171, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; U.S. Department of Energy Joint Genome Institute, Walnut Creek, CA 94598, USA. Electronic address: [email protected] 6MS 84-171, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; U.S. Department of Energy Joint Genome Institute, Walnut Creek, CA 94598, USA; School of Natural Sciences, University of California, Merced, CA 95343, USA. Electronic address: [email protected]
The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. We identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates, including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologs caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution. PAPERCLIP.
Date: 
November 2, 2016
Where: 
HSW 1057 at noon

Kol Jia Young

Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage
Komor AC1,2, Kim YB1,2, Packer MS1,2, Zuris JA1,2, Liu DR1,2.
Nature
December 20, 2015
1Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA. 2Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 02138, USA.
Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.
Date: 
May 18, 2016
Where: 
HSW 1057 at noon