UCSF RNA Journal Club

A newsletter announcing the next presenter for RNA Journal Club

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.
October 12, 2016
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.
October 5, 2016
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.
September 28, 2016
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.
September 21, 2016
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.
September 14, 2016
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.
September 7, 2016
HSW 1057 at noon

D'Juan Farmer

Dynamic Axonal Translation in Developing and Mature Visual Circuits
Shigeoka T1, Jung H2, Jung J3, Turner-Bridger B1, Ohk J3, Lin JQ1, Amieux PS4, Holt CE5.
Cell. 2016 Jun 30;166(1):181-92. doi: 10.1016/j.cell.2016.05.029. Epub 2016 Jun 16.
June 30, 2016
1Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK 2Department of Anatomy, Brain Research Institute, and Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 03722, Republic of Korea 3Bastyr University Research Institute, Bastyr University, Kenmore, WA 98028, USA 4Co-first author 5Co-senior author *Correspondence: [email protected] (H.J.), [email protected] (C.E.H.) http://dx.doi.org/10.1016/j.cell.2016.05.029
Local mRNA translation mediates the adaptive responses of axons to extrinsic signals, but direct evidence that it occurs in mammalian CNS axons in vivo is scant. We developed an axon-TRAP-RiboTag approach in mouse that allows deep-sequencing analysis of ribosome-bound mRNAs in the retinal ganglion cell axons of the developing and adult retinotectal projection in vivo. The embryonic-to-postnatal axonal translatome comprises an evolving subset of enriched genes with axon-specific roles, suggesting distinct steps in axon wiring, such as elongation, pruning, and synaptogenesis. Adult axons, remarkably, have a complex translatome with strong links to axon survival, neurotransmission, and neurodegenerative disease. Translationally coregulated mRNA subsets share common upstream regulators, and sequence elements generated by alternative splicing promote axonal mRNA translation. Our results indicate that intricate regulation of compartment-specific mRNA translation in mammalian CNS axons supports the formation and maintenance of neural circuits in vivo.
August 31, 2016
HSW 1057 at noon

Kol Jia Yong

A comprehensive analysis of 3′ end sequencing data sets reveals novel polyadenylation signals and the repressive role of heterogeneous ribonucleoprotein C on cleavage and polyadenylation
Gruber AJ1, Schmidt R1, Gruber AR1, Martin G1, Ghosh S1, Belmadani M1, Keller W1, Zavolan M1.
Genome Res. 2016 Aug;26(8):1145-59. doi: 10.1101/gr.202432.115. Epub 2016 Jul 5.
August 1, 2016
Present address: University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada Corresponding author: [email protected] Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.202432.115. Freely available online through the Genome Research Open Access option. © 2016 Gruber et al. This article, published in Genome Research, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
Alternative polyadenylation (APA) is a general mechanism of transcript diversification in mammals, which has been recently linked to proliferative states and cancer. Different 3' untranslated region (3' UTR) isoforms interact with different RNA-binding proteins (RBPs), which modify the stability, translation, and subcellular localization of the corresponding transcripts. Although the heterogeneity of pre-mRNA 3' end processing has been established with high-throughput approaches, the mechanisms that underlie systematic changes in 3' UTR lengths remain to be characterized. Through a uniform analysis of a large number of 3' end sequencing data sets, we have uncovered 18 signals, six of which are novel, whose positioning with respect to pre-mRNA cleavage sites indicates a role in pre-mRNA 3' end processing in both mouse and human. With 3' end sequencing we have demonstrated that the heterogeneous ribonucleoprotein C (HNRNPC), which binds the poly(U) motif whose frequency also peaks in the vicinity of polyadenylation (poly(A)) sites, has a genome-wide effect on poly(A) site usage. HNRNPC-regulated 3' UTRs are enriched in ELAV-like RBP 1 (ELAVL1) binding sites and include those of the CD47 gene, which participate in the recently discovered mechanism of 3' UTR-dependent protein localization (UDPL). Our study thus establishes an up-to-date, high-confidence catalog of 3' end processing sites and poly(A) signals, and it uncovers an important role of HNRNPC in regulating 3' end processing. It further suggests that U-rich elements mediate interactions with multiple RBPs that regulate different stages in a transcript's life cycle.
August 24, 2016
HSW 1057 at noon

Gabriel Eades

Translation readthrough mitigation
Joshua A. Arribere, Elif S. Cenik, Nimit Jain, Gaelen T. Hess, Cameron H. Lee, Michael C. Bassik & Andrew Z. Fire
Nature. 2016 Jun 30;534(7609):719-23.
June 1, 2016
Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, USA. Department of Bioengineering, Stanford University, Stanford, California 94305, USA. Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA.
A fraction of ribosomes engaged in translation will fail to terminate when reaching a stop codon, yielding nascent proteins inappropriately extended on their C termini. Although such extended proteins can interfere with normal cellular processes, known mechanisms of translational surveillance are insufficient to protect cells from potential dominant consequences. Here, through a combination of transgenics and CRISPR–Cas9 gene editing in Caenorhabditis elegans, we demonstrate a consistent ability of cells to block accumulation of C-terminal-extended proteins that result from failure to terminate at stop codons. Sequences encoded by the 3′ untranslated region (UTR) were sufficient to lower protein levels. Measurements of mRNA levels and translation suggested a co- or post-translational mechanism of action for these sequences in C. elegans. Similar mechanisms evidently operate in human cells, in which we observed a comparable tendency for translated human 3′ UTR sequences to reduce mature protein expression in tissue culture assays, including 3′ UTR sequences from the hypomorphic ‘Constant Spring’ haemoglobin stop codon variant. We suggest that 3′ UTRs may encode peptide sequences that destabilize the attached protein, providing mitigation of unwelcome and varied translation errors.
August 17, 2016
HSW 1057 at noon

Vanille Greiner

Versatile in vivo regulation of tumor phenotypes by dCas9-mediated transcriptional perturbation
Braun CJ1, Bruno PM1, Horlbeck MA2, Gilbert LA2, Weissman JS2, Hemann MT3.
Proc Natl Acad Sci U S A. 2016 Jul 5;113(27):E3892-900. doi: 10.1073/pnas.1600582113. Epub 2016 Jun 20.
July 5, 2016
The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA 94158; Howard Hughes Medical Institute, University of California, San Francisco, CA 94158; and eCenter for RNA Systems Biology, University of California, San Francisco, CA 94158
Targeted transcriptional regulation is a powerful tool to study genetic mediators of cellular behavior. Here, we show that catalytically dead Cas9 (dCas9) targeted to genomic regions upstream or downstream of the transcription start site allows for specific and sustainable gene-expression level alterations in tumor cells in vitro and in syngeneic immune-competent mouse models. We used this approach for a high-coverage pooled gene-activation screen in vivo and discovered previously unidentified modulators of tumor growth and therapeutic response. Moreover, by using dCas9 linked to an activation domain, we can either enhance or suppress target gene expression simply by changing the genetic location of dCas9 binding relative to the transcription start site. We demonstrate that these directed changes in gene-transcription levels occur with minimal off-target effects. Our findings highlight the use of dCas9-mediated transcriptional regulation as a versatile tool to reproducibly interrogate tumor phenotypes in vivo.
August 10, 2016
HSW 1057 at noon