UCSF RNA Journal Club

A newsletter announcing the next presenter for RNA Journal Club

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

Malin Akerblom

Continuous genetic recording with self-targeting CRISPR-Cas in human cells
Perli SD1, Cui CH2, Lu TK3.
Science. 2016 Sep 9;353(6304). pii: aag0511. doi: 10.1126/science.aag0511. Epub 2016 Aug 18.
December 9, 2015
Synthetic Biology Group, MIT Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Harvard -MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
The ability to record molecular events in vivo would enable monitoring of signaling dynamics within cellular niches and critical factors that orchestrate cellular behavior. We present a self-contained analog memory device for longitudinal recording of molecular stimuli into DNA mutations in human cells. This device consists of a self-targeting guide RNA (stgRNA) that repeatedly directs Streptococcus pyogenes Cas9 nuclease activity toward the DNA that encodes the stgRNA, enabling localized, continuous DNA mutagenesis as a function of stgRNA expression. We demonstrate programmable and multiplexed memory storage in human cells triggered by exogenous inducers or inflammation, both in vitro and in vivo. This tool, Mammalian Synthetic Cellular Recorder Integrating Biological Events (mSCRIBE), provides a distinct strategy for investigating cell biology in vivo and enables continuous evolution of targeted DNA sequences.
Date: 
October 12, 2016
Where: 
HSW 1057 at noon

Roman Camarda

Different promoter affinities account for specificity in MYC-dependent gene regulation
Lorenzin F1, Benary U2, Baluapuri A1, Walz S3,4, Jung LA1,5, von Eyss B1, Kisker C5, Wolf J2, Eilers M1,4, Wolf E1.
Elife. 2016 Jul 27;5. pii: e15161. doi: 10.7554/eLife.15161.
July 27, 2016
1Department of Biochemistry and Molecular Biology, Biocenter, University of Wu¨ rzburg, Wu¨ rzburg, Germany; 2Group Mathematical Modeling of Cellular Processes, Max-Delbru¨ ck-Center for Molecular Medicine, Berlin, Germany; 3Core Unit Bioinformatics, Biocenter, University of Wu¨ rzburg, Wu¨ rzburg, Germany; 4Comprehensive Cancer Center Mainfranken, University of Wu¨ rzburg, Wu¨ rzburg, Germany; 5Rudolf-Virchow-Center for Experimental Biomedicine, University of Wu¨ rzburg, Wu¨ rzburg, Germanyv
Enhanced expression of the MYC transcription factor is observed in the majority of tumors. Two seemingly conflicting models have been proposed for its function: one proposes that MYC enhances expression of all genes, while the other model suggests gene-specific regulation. Here, we have explored the hypothesis that specific gene expression profiles arise since promoters differ in affinity for MYC and high-affinity promoters are fully occupied by physiological levels of MYC. We determined cellular MYC levels and used RNA- and ChIP-sequencing to correlate promoter occupancy with gene expression at different concentrations of MYC. Mathematical modeling showed that binding affinities for interactions of MYC with DNA and with core promoter-bound factors, such as WDR5, are sufficient to explain promoter occupancies observed in vivo. Importantly, promoter affinity stratifies different biological processes that are regulated by MYC, explaining why tumor-specific MYC levels induce specific gene expression programs and alter defined biological properties of cells.
Date: 
October 5, 2016
Where: 
HSW 1057 at noon

John Gagnon

The DEAD-Box Protein Dhh1p Couples mRNA Decay and Translation by Monitoring Codon Optimality
Radhakrishnan A1, Chen YH2, Martin S2, Alhusaini N2, Green R3, Coller J4.
Cell. 2016 Sep 22;167(1):122-132.e9. doi: 10.1016/j.cell.2016.08.053. Epub 2016 Sep 15.
September 15, 2016
1Program in Molecular Biophysics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA 2Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA 3Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106, USA 4Co-first author 5Lead Contact *Correspondence: [email protected] (R.G.), [email protected] (J.C.) http://dx.doi.org/10.1016/j.cell.2016.08.053
A major determinant of mRNA half-life is the codon-dependent rate of translational elongation. How the processes of translational elongation and mRNA decay communicate is unclear. Here, we establish that the DEAD-box protein Dhh1p is a sensor of codon optimality that targets an mRNA for decay. First, we find mRNAs whose translation elongation rate is slowed by inclusion of non-optimal codons are specifically degraded in a Dhh1p-dependent manner. Biochemical experiments show Dhh1p is preferentially associated with mRNAs with suboptimal codon choice. We find these effects on mRNA decay are sensitive to the number of slow-moving ribosomes on an mRNA. Moreover, we find Dhh1p overexpression leads to the accumulation of ribosomes specifically on mRNAs (and even codons) of low codon optimality. Lastly, Dhh1p physically interacts with ribosomes in vivo. Together, these data argue that Dhh1p is a sensor for ribosome speed, targeting an mRNA for repression and subsequent decay.
Date: 
September 28, 2016
Where: 
HSW 1057 at noon

Michael Boettcher

A multifunctional AAV–CRISPR–Cas9 and its host response
Chew WL1,2, Tabebordbar M2,3, Cheng JK3, Mali P1, Wu EY3, Ng AH1,4, Zhu K3,5, Wagers AJ3, Church GM1,6.
Nat Methods. 2016 Sep 5. doi: 10.1038/nmeth.3993. [Epub ahead of print]
September 5, 2016
1Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. 2Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts, USA. 3Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA. 4Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA. 5Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA. 6Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA. 7Present addresses: Department of Bioengineering, University of California San Diego, La Jolla, California, USA (P.M.) and RaNA Therapeutics, Cambridge, Massachusetts, USA (E.Y.W.). 8These authors contributed equally to this work. Correspondence should be addressed to G.M.C. ([email protected]) or A.J.W. ([email protected]).
CRISPR-Cas9 delivery by adeno-associated virus (AAV) holds promise for gene therapy but faces critical barriers on account of its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multifunctional platform customizable for genome editing, transcriptional regulation, and other previously impracticable applications of AAV-CRISPR-Cas9. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV-CRISPR-Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce extensive cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics.
Date: 
September 21, 2016
Where: 
HSW 1057 at noon

James Blau

m6A RNA methylation promotes XIST-mediated transcriptional repression
Patil DP1, Chen CK2, Pickering BF1, Chow A2, Jackson C2, Guttman M2, Jaffrey SR1
Nature. 2016 Sep 7. doi: 10.1038/nature19342. [Epub ahead of print]
September 7, 2016
1Department of Pharmacology, Weill-Cornell Medical College, Cornell University, New York, New York 10065, USA. 2Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA.
The long non-coding RNA X-inactive specific transcript (XIST) mediates the transcriptional silencing of genes on the X chromosome. Here we show that, in human cells, XIST is highly methylated with at least 78 N6-methyladenosine (m6A) residues-a reversible base modification of unknown function in long non-coding RNAs. We show that m6A formation in XIST, as well as in cellular mRNAs, is mediated by RNA-binding motif protein 15 (RBM15) and its paralogue RBM15B, which bind the m6A-methylation complex and recruit it to specific sites in RNA. This results in the methylation of adenosine nucleotides in adjacent m6A consensus motifs. Furthermore, we show that knockdown of RBM15 and RBM15B, or knockdown of methyltransferase like 3 (METTL3), an m6A methyltransferase, impairs XIST-mediated gene silencing. A systematic comparison of m6A-binding proteins shows that YTH domain containing 1 (YTHDC1) preferentially recognizes m6A residues on XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of m6A. These data reveal a pathway of m6A formation and recognition required for XIST-mediated transcriptional repression.
Date: 
September 14, 2016
Where: 
HSW 1057 at noon

Eleonora De Klerk

Targeted Epigenetic Remodeling of Endogenous Loci by CRISPR/Cas9-Based Transcriptional Activators Directly Converts Fibroblasts to Neuronal Cells
Joshua B. Black,1 Andrew F. Adler,1,9 Hong-Gang Wang,6,8,10 Anthony M. D’Ippolito,2,3 Hunter A. Hutchinson,1Timothy E. Reddy,2,4 Geoffrey S. Pitt,6,7,8 Kam W. Leong,1,11 and Charles A. Gersbach1,2,5,*
Cell Stem Cell. 2016 Aug 9. pii: S1934-5909(16)30196-5. doi: 10.1016/j.stem.2016.07.001. [Epub ahead of print]
August 9, 2016
1Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA 2Center for Genomic and Computational Biology, Duke University, Durham, NC 27708, USA 3University Program in Genetics and Genomics, Duke University Medical Center, Durham, NC 27710, USA 4Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, NC 27710, USA 5Department of Orthopaedic Surgery, Duke University Medical Center, Durham, NC 27710, USA 6Ion Channel Research Unit, Duke University Medical Center, Durham, NC 27710, USA 7Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, USA 8Division of Cardiology, Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA 9Present address: Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093, USA 10Present address: Cardiovascular Research Institute, Weill Cornell Medicine, New York, NY 10021, USA 11Present address: Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2016.07.001
Overexpression of exogenous fate-specifying transcription factors can directly reprogram differentiated somatic cells to target cell types. Here, we show that similar reprogramming can also be achieved through the direct activation of endogenous genes using engineered CRISPR/Cas9-based transcriptional activators. We use this approach to induce activation of the endogenous Brn2, Ascl1, and Myt1l genes (BAM factors) to convert mouse embryonic fibroblasts to induced neuronal cells. This direct activation of endogenous genes rapidly remodeled the epigenetic state of the target loci and induced sustained endogenous gene expression during reprogramming. Thus, transcriptional activation and epigenetic remodeling of endogenous master transcription factors are sufficient for conversion between cell types. The rapid and sustained activation of endogenous genes in their native chromatin context by this approach may facilitate reprogramming with transient methods that avoid genomic integration and provides a new strategy for overcoming epigenetic barriers to cell fate specification.
Date: 
September 7, 2016
Where: 
HSW 1057 at noon