UCSF RNA Journal Club

A newsletter announcing the next presenter for RNA Journal Club

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

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.
August 3, 2016
HSW 1057 at noon


July 27, 2016
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.
July 20, 2016
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,*
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.
July 6, 2016
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.
June 29, 2016
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.
June 22, 2016
HSW 1057 at noon