Sebastiaan van Heesch, Franziska Witte, Valentin Schneider-Lunitz, Jana F. Schulz,Eleonora Adami, Allison B. Faber, Marieluise Kirchner, Henrike Maatz, Susanne Blachut, Clara-Louisa Sandmann, Masatoshi Kanda,1Catherine L. Worth,1 Sebastian Schafer,2,4 Lorenzo Calviello,5,6,33 Rhys Merriott,1 Giannino Patone,1 Oliver Hummel,1Emanuel Wyler,5 Benedikt Obermayer,5,7 Michael B. Mu¨ cke,1 Eric L. Lindberg,1 Franziska Trnka,1 Sebastian Memczak,5,36
Marcel Schilling,5 Leanne E. Felkin,8,9 Paul J.R. Barton,8,9 Nicholas M. Quaife,8,9,32 Konstantinos Vanezis,9,32, Sebastian Diecke,10,11,12 Masaya Mukai,13 Nancy Mah,14 Su-Jun Oh,14 Andreas Kurtz,14 Christoph Schramm,15,16, Dorothee Schwinge,16 Marcial Sebode,16 Magdalena Harakalova,17,18 Folkert W. Asselbergs,17,19,20 Aryan Vink,18, Roel A. de Weger,18 Sivakumar Viswanathan,2 Anissa A. Widjaja,2 Anna Ga¨rtner-Rommel,21 Hendrik Milting,21, Cris dos Remedios,22 Christoph Knosalla,11,23,24 Philipp Mertins,3 Markus Landthaler,5,25 Martin Vingron,26, Wolfgang A. Linke,27,28 Jonathan G. Seidman,29 Christine E. Seidman,29,30,31 Nikolaus Rajewsky,5 Uwe Ohler,5,6, Stuart A. Cook,2,4,9,32 and Norbert Hubner1,11,12,24,35,37,*
Cell 178, 1–19, June 27, 2019 ª 2019 Elsevier Inc.
Elizabeth Crouch
Time
12:00pm
Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing proteintruncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.
Harleen Saini, Alicia A. Bicknell, Sean R. Eddy, Melissa J. Moore
bioRxiv preprint first posted online Apr. 30, 2019; doi: http://dx.doi.org/10.1101/623447. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The polarized structure of axons and dendrites in neuronal cells depends in part on RNA localization. Previous studies have looked at which polyadenylated RNAs are enriched in neuronal projections or at synapses, but less is known about the distribution of non-adenylated RNAs. By physically dissecting projections from cell bodies of primary rat hippocampal neurons and sequencing total RNA, we found an unexpected set of free circular introns with a non-canonical branchpoint enriched in neuronal projections. These introns appear to be tailless lariats that escape debranching. They lack ribosome occupancy, sequence conservation, and known localization signals, and their function, if any, is not known. Nonetheless, their enrichment in projections has important implications for our understanding of the mechanisms by which RNAs reach distal compartments of asymmetric cells.
RNA aptamers and RNA aptamer-based devices can be genetically encoded and expressed in cells to probe and manipulate cellular function. However, their usefulness in the mammalian cell is limited by low expression and rapid degradation. Here we describe the Tornado (Twister-optimized RNA for durable overexpression) expression system for achieving rapid RNA circularization, resulting in RNA aptamers with high stability and expression levels. Tornado-expressed transcripts contain an RNA of interest flanked by Twister ribozymes. The ribozymes rapidly undergo autocatalytic cleavage, leaving termini that are ligated by the ubiquitous endogenous RNA ligase RtcB. Using this approach, protein-binding aptamers that otherwise have minimal effects in cells become potent inhibitors of cellular signaling. Additionally, an RNA-based fluorescent metabolite biosensor for S-adenosyl methionine (SAM) that is expressed at low levels when expressed as a linear RNA achieves levels sufficient for detection of intracellular SAM dynamics when expressed as a circularRNA. The Tornado expression system thus markedly enhances the utility of RNA-based approaches in the mammalian cell.
bioRxiv preprint first posted online Nov. 18, 2018; doi: http://dx.doi.org/10.1101/473207. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.All rights reserved. No reuse allowed without permission. All rights reserved. No reuse allowed without permission.
Circular RNAs (circRNAs) are an abundant and conserved class of RNAs with ambiguous functions. Some circRNAs have been shown to be translated through IRES driven cap-independent translation, yet the scope of circRNA translation is unclear because endogenous IRESs are rare. To determine the prevalence and importance of circRNA translation, we screened a random 10-nt library using a cell-based reporter system and identified a large number of AU-rich motifs that are capable of initiating circRNA translation. These IRES-like elements are significantly enriched in circRNAs vs. linear mRNA and are sufficient to drive robust translation of circRNAs containing solely the coding sequences. Importantly, these elements are predicted to present in thousands of circRNAs with 67% of which encode a truncated isoform of host genes, and are found to bind trans-acting factors that promote circRNA translation. Our results indicate that extensive circRNA translation can be driven by short IRES-like elements, suggesting a general function of circRNAs in cytoplasm through translation.
We characterized the epigenetic landscape of genes encoding long noncoding RNAs (lncRNAs) across 6,475 tumors and 455 cancer cell lines. In stark contrast to the CpG island hypermethylation phenotype in cancer, we observed a recurrent hypomethylation of 1,006 lncRNAgenes in cancer, including EPIC1 (epigenetically-induced lncRNA1). Overexpression of EPIC1 is associated with poor prognosis in luminal B breast cancer patients and enhances tumor growth in vitro and in vivo. Mechanistically, EPIC1 promotes cell-cycle progression by interacting with MYC through EPIC1's 129-283 nt region. EPIC1 knockdown reduces the occupancy of MYC to its target genes (e.g., CDKN1A, CCNA2, CDC20, and CDC45). MYC depletion abolishes EPIC1's regulation of MYC target and luminal breast cancer tumorigenesis in vitro and in vivo.
Mihir Metkar, Hakan Ozadam, Bryan R. Lajoie, Maxim Imakaev, Leonid A. Mirny, Job Dekker, Melissa J. Moore
bioRxiv preprint first posted online Mar. 8, 2018; doi: http://dx.doi.org/10.1101/278747. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
Stephen Floor
Time
12:00pm
Compared to noncoding RNAs (ncRNAs) such as rRNAs and ribozymes, for which high resolution structures abound, little is known about the tertiary structures of mRNAs. In eukaryotic cells, newly made mRNAs are packaged with proteins in highly compacted mRNPs, but the manner of this mRNA compaction is unknown. Here we developed and implemented RIPPLiT (RNA ImmunoPrecipitation and Proximity Ligation in Tandem), a transcriptome-wide method for probing the 3D conformations of RNAs stably-associated with defined proteins, in this case exon junction complex (EJC) core factors. EJCs multimerize with other mRNP components to form megadalton sized complexes that protect large swaths of newly synthesized mRNAs from endonuclease digestion. Unlike ncRNAs, mRNAs behave more like flexible polymers without strong locus-specific interactions. Polymer analysis of proximity ligation data for hundreds of mRNA species demonstrates that pre-translational mammalian mRNPs fold as linear rod-like structures with no strong propensity for 5' and 3' end interaction.
We recently described a new class of long noncoding RNAs (lncRNAs) that are distinguished by especially tight chromatin association and whose presence is strongly correlated to expression of nearby genes. Here, we examine the cis-enhancer mechanism of this class of chromatin-enriched RNA (cheRNA) across multiple human cell lines. cheRNAs are largely cell type specific and provide the most reliable chromatin signature to predict cis-gene transcription in every human cell type examined. Targeted depletion of three cheRNAs decreases expression of their neighboring genes, indicating potential co-activator function, and single-molecule fluorescence in situ hybridization (smFISH) of one cheRNA-distal target gene pair suggests a spatial overlap consistent with a role in chromosome looping. Additionally, the cheRNA HIDALGO stimulates the fetal hemoglobin subunit gamma 1 (HBG1) gene during erythroid differentiation by promoting contacts to a downstream enhancer. Our results suggest that multiple cheRNAs activate proximal lineage-specific gene transcription.
Piwecka M, Glažar P, Hernandez-Miranda LR, Memczak S, Wolf SA, Rybak-Wolf A, Filipchyk A, Klironomos F, Cerda Jara CA, Fenske P, Trimbuch T, Zywitza V, Plass M, Schreyer L, Ayoub S, Kocks C, Kühn R, Rosenmund C, Birchmeier C, Rajewsky N.
Science. 2017 Aug 10. pii: eaam8526. doi: 10.1126/science.aam8526. [Epub ahead of print]
Malin Akerblom
Time
12:00pm
Hundreds of circular RNAs (circRNAs) are highly abundant in mammalian brain, with oftentimes conserved expression. Here, we show that the circRNA Cdr1as is massively bound by miR-7 and miR-671 in the human and mouse brain. When the Cdr1as locus was removed from the mouse genome, knockout animals displayed impaired sensorimotor gating, a deficit in the ability to filter out unnecessary information associated with neuropsychiatric disorders. Electrophysiological recordings revealed dysfunctional synaptic transmission. Expression of microRNAs miR-7 and miR-671 was specifically and post-transcriptionally misregulated in all brain regions analyzed. Expression of immediate early genes such as Fos, a direct miR-7 target, was enhanced in Cdr1as-deficient brains, providing a possible molecular link to the behavioral phenotype. Our data indicate an in vivo loss-of-function circRNA phenotype and suggest that interactions between circRNAs and miRNAs are important for normal brain function.
Circular RNAs (circRNAs) constitute a family of transcripts with unique structures and still largely unknown functions. Their biogenesis, which proceeds via a back-splicing reaction, is fairly well characterized, whereas their role in the modulation of physiologically relevant processes is still unclear. Here we performed expression profiling of circRNAs during in vitro differentiation of murine and human myoblasts, and we identified conserved species regulated in myogenesis and altered in Duchenne muscular dystrophy. A high-content functional genomic screen allowed the study of their functional role in muscle differentiation. One of them, circ-ZNF609, resulted in specifically controlling myoblast proliferation. Circ-ZNF609 contains an open reading frame spanning from the start codon, in common with the linear transcript, and terminating at an in-frame STOP codon, created upon circularization. Circ-ZNF609 is associated with heavy polysomes, and it is translated into a protein in a splicing-dependent and cap-independent manner, providing an example of a protein-coding circRNA in eukaryotes.
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