UCSF RNA Journal Club

A newsletter announcing the next presenter for RNA Journal Club

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

Eleonora De Klerk

Redefining the Translational Status of 80S Monosomes
Heyer EE, Moore MJ.
Cell.;164(4):757-69. doi: 10.1016/j.cell.2016.01.003.
February 11, 2016
1Howard Hughes Medical Institute, RNA Therapeutics Institute and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cell.2016.01.003
Fully assembled ribosomes exist in two populations: polysomes and monosomes. While the former has been studied extensively, to what extent translation occurs on monosomes and its importance for overall translational output remain controversial. Here, we used ribosome profiling to examine the translational status of 80S monosomes in Saccharomyces cerevisiae. We found that the vast majority of 80S monosomes are elongating, not initiating. Further, most mRNAs exhibit some degree of monosome occupancy, with monosomes predominating on nonsense-mediated decay (NMD) targets, upstream open reading frames (uORFs), canonical ORFs shorter than ∼590 nt, and ORFs for which the total time required to complete elongation is substantially shorter than that required for initiation. Importantly, mRNAs encoding low-abundance regulatory proteins tend to be enriched in the monosome fraction. Our data highlight the importance of monosomes for the translation of highly regulated mRNAs.
Date: 
May 25, 2016
Where: 
HSW 1057 at noon

Eleonora De Klerk

Redefining the Translational Status of 80S Monosomes
Where: 
HSW 1057 at noon

Daniele Cary

Degradation of Stop Codon Read-through Mutant Proteins via the Ubiquitin-Proteasome System Causes Hereditary Disorders
Shibata N1, Ohoka N1, Sugaki Y2, Onodera C2, Inoue M3, Sakuraba Y4, Takakura D5, Hashii N5, Kawasaki N5, Gondo Y4, Naito M6.
J Biol Chem. 2015 Nov 20;290(47):28428-37. doi: 10.1074/jbc.M115.670901. Epub 2015 Oct 6.
November 20, 2015
1From the Division of Molecular Target and Gene Therapy Products and. 2the Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8561, Japan. 3the Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan. 4the Mutagenesis and Genomics Team, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan. 5the Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan. 6From the Division of Molecular Target and Gene Therapy Products and [email protected]
During translation, stop codon read-through occasionally happens when the stop codon is misread, skipped, or mutated, resulting in the production of aberrant proteins with C-terminal extension. These extended proteins are potentially deleterious, but their regulation is poorly understood. Here we show in vitro and in vivo evidence that mouse cFLIP-L with a 46-amino acid extension encoded by a read-through mutant gene is rapidly degraded by the ubiquitin-proteasome system, causing hepatocyte apoptosis during embryogenesis. The extended peptide interacts with an E3 ubiquitin ligase, TRIM21, to induce ubiquitylation of the mutant protein. In humans, 20 read-through mutations are related to hereditary disorders, and extended peptides found in human PNPO and HSD3B2 similarly destabilize these proteins, involving TRIM21 for PNPO degradation. Our findings indicate that degradation of aberrant proteins with C-terminal extension encoded by read-through mutant genes is a mechanism for loss of function resulting in hereditary disorders.
Date: 
October 26, 2016
Where: 
HSW 1057 at noon

Gabriel Eades

Systematic mapping of functional enhancer-promoter connections with CRISPR interference
Charles P. Fulco1,2, Mathias Munschauer1, Rockwell Anyoha1, Glen Munson1, Sharon R. Grossman1,3,4, Elizabeth M. Perez1, Michael Kane1, Brian Cleary1,5, Eric S. Lander1,2,4,*,†, Jesse M. Engreitz1,*,†
Science. 2016 Sep 29. pii: aag2445. [Epub ahead of print]
1Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Department of Systems Biology, Harvard Medical School, Boston, MA 02114, USA. 2Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. 3Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Division of Health Sciences and Technology, MIT, Cambridge, MA 02139, USA. Department of Biology, MIT, Cambridge, MA 02139, USA. 4Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Computational and Systems Biology Program, MIT, Cambridge, MA 02139, USA. 5Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Department of Systems Biology, Harvard Medical School, Boston, MA 02114, USA. Department of Biology, MIT, Cambridge, MA 02139, USA. [email protected] [email protected] 6Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. [email protected] [email protected]
Gene expression in mammals is regulated by noncoding elements that can impact physiology and disease, yet the functions and target genes of most noncoding elements remain unknown. We present a high-throughput approach that uses CRISPR interference (CRISPRi) to discover regulatory elements and identify their target genes. We assess >1 megabase (Mb) of sequence in the vicinity of 2 essential transcription factors, MYC and GATA1, and identify 9 distal enhancers that control gene expression and cellular proliferation. Quantitative features of chromatin state and chromosome conformation distinguish the 7 enhancers that regulate MYC from other elements that do not, suggesting a strategy for predicting enhancer-promoter connectivity. This CRISPRi-based approach can be applied to dissect transcriptional networks and interpret the contributions of noncoding genetic variation to human disease
Date: 
October 19, 2016
Where: 
HSW 1057 at noon