Hyeshik Chang

Citrate can act as a partial magnesium chelator to facilitate the synthesis of RNA within a protocell-like vesicle. Authors: Katarzyna Adamala, Jack W. Szostak

Author: Jing-Woei Li

Authors: Burcu Bolukbasi, Nicholas Berente, Joel Cutcher-Gershenfeld, Leslie Dechurch, Courtney Flint, Michael Haberman, John Leslie King, Eric Knight, Barbara Lawrence, Ethan Masella, Charles McElroy, Barbara Mittleman, Mark Nolan, Melanie Radik, Namchul Shin, Cheryl A. Thompson, Susan Winter, Ilya Zaslavsky, M. Lee Allison, David Arctur, Jennifer Arrigo, Anthony K. Aufdenkampe, Jay Bass, Jim Crowell, Mike Daniels, Stephen Diggs, Christopher Duffy, Yolanda Gil, Basil Gomez, Sara Graves, Robert Hazen, Leslie Hsu, Danie Kinkade, Kerstin Lehnert, Chris Marone, Don Middleton, Anders Noren, Genevieve Pearthree, Mohan Ramamurthy, Erin Robinson, George Percivall, Stephen Richard, Celina Suarez, Doug Walker

The DIY dilemma

Nature 503, 7477 (2013). doi:10.1038/503437b

Misconceptions about do-it-yourself biology mean that opportunities are being missed.

Nature Methods 10, 1165 (2013). doi:10.1038/nmeth.2735

Author: Ian Korf

RNA-seq is a recent and immensely popular technology for cataloging and comparing gene expression. Two papers from the international RGASP consortium report on large-scale competitions to identify the best algorithms for RNA-seq analysis, with surprising variability in the results.

Nature Methods 10, 1144 (2013). doi:10.1038/nmeth.2750

Author: Tal Nawy

Long-read sequencing uncovers transcript features missed by short-read methods.

Nature Methods 10, 1135 (2013). doi:10.1038/nmeth.2760

The dangers of phototoxicity in fluorescence microscopy experiments are too often ignored.

Article

Whether microRNA processing mediated by Dicer is regulated in a cell-cycle-dependent manner is unknown. Here, Chen et al. show that Cyclin D1, which is important in the control of the cell cycle, regulates the expression of Dicer, and that Cyclin D1 and Dicer expression levels correlate in breast cancer.

Nature Communications doi: 10.1038/ncomms3812

Authors: Zuoren Yu, Liping Wang, Chenguang Wang, Xiaoming Ju, Min Wang, Ke Chen, Emanuele Loro, Zhiping Li, Yuzhen Zhang, Kongming Wu, Mathew C. Casimiro, Michael Gormley, Adam Ertel, Paolo Fortina, Yihan Chen, Aydin Tozeren, Zhongmin Liu, Richard G. Pestell

Molecular Systems Biology 9, (2013). doi:10.1038/msb.2013.68

Authors: Jean Hausser, Afzal Pasha Syed, Nathalie Selevsek, Erik van Nimwegen, Lukasz Jaskiewicz, Ruedi Aebersold & Mihaela Zavolan

Nature advance online publication 27 November 2013. doi:10.1038/nature12861

Authors: Ashley Acevedo, Leonid Brodsky & Raul Andino

RNA viruses exist as genetically diverse populations. It is thought that diversity and genetic structure of viral populations determine the rapid adaptation observed in RNA viruses and hence their pathogenesis. However, our understanding of the mechanisms underlying virus evolution has been limited by the inability to accurately describe the genetic structure of virus populations. Next-generation sequencing technologies generate data of sufficient depth to characterize virus populations, but are limited in their utility because most variants are present at very low frequencies and are thus indistinguishable from next-generation sequencing errors. Here we present an approach that reduces next-generation sequencing errors and allows the description of virus populations with unprecedented accuracy. Using this approach, we define the mutation rates of poliovirus and uncover the mutation landscape of the population. Furthermore, by monitoring changes in variant frequencies on serially passaged populations, we determined fitness values for thousands of mutations across the viral genome. Mapping of these fitness values onto three-dimensional structures of viral proteins offers a powerful approach for exploring structure–function relationships and potentially uncovering new functions. To our knowledge, our study provides the first single-nucleotide fitness landscape of an evolving RNA virus and establishes a general experimental platform for studying the genetic changes underlying the evolution of virus populations.

Nature advance online publication 27 November 2013. doi:10.1038/nature12730

Authors: Xiao Wang, Zhike Lu, Adrian Gomez, Gary C. Hon, Yanan Yue, Dali Han, Ye Fu, Marc Parisien, Qing Dai, Guifang Jia, Bing Ren, Tao Pan & Chuan He

N6-methyladenosine (m6A) is the most prevalent internal (non-cap) modification present in the messenger RNA of all higher eukaryotes. Although essential to cell viability and development, the exact role of m6A modification remains to be determined. The recent discovery of two m6A demethylases in mammalian cells highlighted the importance of m6A in basic biological functions and disease. Here we show that m6A is selectively recognized by the human YTH domain family 2 (YTHDF2) ‘reader’ protein to regulate mRNA degradation. We identified over 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs, but which also include non-coding RNAs, with a conserved core motif of G(m6A)C. We further establish the role of YTHDF2 in RNA metabolism, showing that binding of YTHDF2 results in the localization of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies. The carboxy-terminal domain of YTHDF2 selectively binds to m6A-containing mRNA, whereas the amino-terminal domain is responsible for the localization of the YTHDF2–mRNA complex to cellular RNA decay sites. Our results indicate that the dynamic m6A modification is recognized by selectively binding proteins to affect the translation status and lifetime of mRNA.

Modulation of epigenetic regulators and cell fate decisions by miRNAs.

Epigenomics. 2013 Dec;5(6):671-83

Authors: Gruber AJ, Zavolan M

Abstract
Mammalian gene expression is controlled at multiple levels by a variety of regulators, including chromatin modifiers, transcription factors and miRNAs. The latter are small, ncRNAs that inhibit the expression of target mRNAs by reducing both their stability and translation rate. In this review, we summarize the recent work towards characterizing miRNA targets that are themselves involved in the regulation of gene expression at the epigenetic level. Epigenetic regulators are strongly enriched among the predicted targets of miRNAs, which may contribute to the documented importance of miRNAs for pluripotency, organism development and somatic cell reprogramming.

PMID: 24283881 [PubMed - in process]

by Daniele Fanelli

Direct sequencing of RNA by primer extension is a fast and accurate method that is useful in a variety of situations. It is a valuable technique for determining the faithfulness of in vitro processing reactions, such as splicing or RNA editing. It is often used as an alternative to reverse transcription PCR (RT–PCR) followed by cloning and DNA sequencing. In the primer extension reaction, a radiolabeled probe (almost always a 5'-end-labeled DNA oligonucleotide) is annealed to the target RNA of interest. After hybridization, cDNA synthesis by reverse transcription proceeds from the 3' end of the primer in the presence of chain-terminating dideoxynucleotide triphosphates. The cDNA products are fractionated on denaturing polyacrylamide gels and analyzed by phosphorimaging or autoradiography.

Here we present two simple methods for preparing radiolabeled size markers for gel electrophoresis. The first procedure describes the generation of an RNA marker ladder by the alkaline hydrolysis of ³²P 5'- or 3'-end-labeled RNA. The second procedure describes the labeling of DNA fragments produced by digestion of pBR322 with the restriction enzyme MspI.

RNAi combined with next-generation sequencing has proven to be a powerful and cost-effective genetic screening platform in mammalian cells. Still, this technology has its limitations and is incompatible with in situ mutagenesis screens on a genome-wide scale. Using p53 as a proof-of-principle target, we readapted the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated 9) genome-editing system to demonstrate the feasibility of this methodology for targeted gene disruption positive selection assays. By using novel "all-in-one" lentiviral and retroviral delivery vectors heterologously expressing both a codon-optimized Cas9 and its synthetic guide RNA (sgRNA), we show robust selection for the CRISPR-modified Trp53 locus following drug treatment. Furthermore, by linking Cas9 expression to GFP fluorescence, we use an "all-in-one" system to track disrupted Trp53 in chemoresistant lymphomas in the Eμ-myc mouse model. Deep sequencing analysis of the tumor-derived endogenous Cas9-modified Trp53 locus revealed a wide spectrum of mutants that were enriched with seemingly limited off-target effects. Taken together, these results establish Cas9 genome editing as a powerful and practical approach for positive in situ genetic screens.

Phillip A. Dumesic, Hiten D. Madhani.

Lin Yang, John E. Froberg, Jeannie T. Lee.

Valeria Cherkasov, Sarah Hofmann, Silke Druffel-Augustin, Axel Mogk, Jens Tyedmers, Georg Stoecklin, Bernd Bukau. BackgroundExposure of cells to severe heat stress causes not only misfolding and aggregation of proteins but also inhibition of translation and storage of mRNA in cytosolic heat stress granules (h....

It has recently been established that synthesis of double-stranded cDNA can be done from a single cell for use in DNA sequencing. Global gene expression can be quantified from the number of reads mapping to each gene, and mutations and mRNA splicing variants determined from the sequence reads. Here we...

A major limitation of high-throughput DNA sequencing is the high rate of erroneous base calls produced. For instance, Illumina sequencing machines produce errors at a rate of ∼0.1–1 × 10−2 per base sequenced. These technologies typically produce billions of base calls per experiment, translating to millions of errors. We have...

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Lower and upper stem-single-stranded RNA junctions together determine the Drosha cleavage site.

Proc Natl Acad Sci U S A. 2013 Dec 2;

Authors: Ma H, Wu Y, Choi JG, Wu H

Abstract
Microprocessor [Drosha-DGCR8 (DiGeorge syndrome critical region gene 8) complex] processing of primary microRNA (pri-miRNA) is the critical first step in miRNA biogenesis, but how the Drosha cleavage site is determined has been unclear. Previous models proposed that the Drosha-DGCR8 complex measures either ∼22 nt from the upper stem-single-stranded RNA (ssRNA, terminal loop) junction or ∼11 nt from the lower stem-ssRNA junction to determine the cleavage site. Here, using miRNA-offset RNAs to determine the Drosha cleavage site, we show that the Microprocessor measures the distances from both the lower and upper stem-ssRNA junctions to determine the cleavage site in human cells, and optimal distances from both structures are critical to the precision of Drosha processing. If the distances are not optimal, Drosha tends to cleave at multiple sites, which can, in turn, generate multiple 5' isomiRs. Thus, our results also reveal a mechanism of 5' isomiR generation.

PMID: 24297910 [PubMed - as supplied by publisher]

Nature Structural & Molecular Biology 20, 1434 (2013). doi:10.1038/nsmb.2699

Authors: Michael T Lovci, Dana Ghanem, Henry Marr, Justin Arnold, Sherry Gee, Marilyn Parra, Tiffany Y Liang, Thomas J Stark, Lauren T Gehman, Shawn Hoon, Katlin B Massirer, Gabriel A Pratt, Douglas L Black, Joe W Gray, John G Conboy & Gene W Yeo

Nature Structural & Molecular Biology 20, 1458 (2013). doi:10.1038/nsmb.2701

Authors: Young-Kook Kim, Gabbine Wee, Joha Park, Jongkyu Kim, Daehyun Baek, Jin-Soo Kim & V Narry Kim

Nature Structural & Molecular Biology 20, 1367 (2013). doi:10.1038/nsmb.2703

Authors: Peter Refsing Andersen, Michal Domanski, Maiken S Kristiansen, Helena Storvall, Evgenia Ntini, Celine Verheggen, Aleks Schein, Jakob Bunkenborg, Ina Poser, Marie Hallais, Rickard Sandberg, Anthony Hyman, John LaCava, Michael P Rout, Jens S Andersen, Edouard Bertrand & Torben Heick Jensen

Nature Structural & Molecular Biology 20, 1358 (2013). doi:10.1038/nsmb.2720

Authors: Marie Hallais, Frédéric Pontvianne, Peter Refsing Andersen, Marcello Clerici, Daniela Lener, Nour El Houda Benbahouche, Thierry Gostan, Franck Vandermoere, Marie-Cécile Robert, Stephen Cusack, Céline Verheggen, Torben Heick Jensen & Edouard Bertrand

Nature Structural & Molecular Biology 20, 1344 (2013). doi:10.1038/nsmb.2728

Authors: Loyal A Goff & John L Rinn

An unresolved question in mammalian epigenetic regulation is how ubiquitously expressed chromatin-modifying complexes such as Polycomb group complex 2 (PRC2) find their specific target sites across an intricate choreography of localization events in time and space. Two recent studies now provide critical new insights into an intriguing genome-wide role for RNA in PRC2 regulation.

Nature Structural & Molecular Biology 20, 1341 (2013). doi:10.1038/nsmb.2725

Authors: Kalyan Das, Stefan G Sarafianos & Eddy Arnold

Nature advance online publication 04 December 2013. doi:10.1038/nature12786

Authors: In-Uck Park, Mike W. Peacey & Marcus R. Munafò

The objective of science is to advance knowledge, primarily in two interlinked ways: circulating ideas, and defending or criticizing the ideas of others. Peer review acts as the gatekeeper to these mechanisms. Given the increasing concern surrounding the reproducibility of much published research, it is critical to understand whether peer review is intrinsically susceptible to failure, or whether other extrinsic factors are responsible that distort scientists’ decisions. Here we show that even when scientists are motivated to promote the truth, their behaviour may be influenced, and even dominated, by information gleaned from their peers’ behaviour, rather than by their personal dispositions. This phenomenon, known as herding, subjects the scientific community to an inherent risk of converging on an incorrect answer and raises the possibility that, under certain conditions, science may not be self-correcting. We further demonstrate that exercising some subjectivity in reviewer decisions, which serves to curb the herding process, can be beneficial for the scientific community in processing available information to estimate truth more accurately. By examining the impact of different models of reviewer decisions on the dynamic process of publication, and thereby on eventual aggregation of knowledge, we provide a new perspective on the ongoing discussion of how the peer-review process may be improved.

Nature advance online publication 04 December 2013. doi:10.1038/nature12785

Authors: Madhu S. Kumar, Elena Armenteros-Monterroso, Philip East, Probir Chakravorty, Nik Matthews, Monte M. Winslow & Julian Downward

Non-small-cell lung cancer (NSCLC) is the most prevalent histological cancer subtype worldwide. As the majority of patients present with invasive, metastatic disease, it is vital to understand the basis for lung cancer progression. Hmga2 is highly expressed in metastatic lung adenocarcinoma, in which it contributes to cancer progression and metastasis. Here we show that Hmga2 promotes lung cancer progression in mouse and human cells by operating as a competing endogenous RNA (ceRNA) for the let-7 microRNA (miRNA) family. Hmga2 can promote the transformation of lung cancer cells independent of protein-coding function but dependent upon the presence of let-7 sites; this occurs without changes in the levels of let-7 isoforms, suggesting that Hmga2 affects let-7 activity by altering miRNA targeting. These effects are also observed in vivo, where Hmga2 ceRNA activity drives lung cancer growth, invasion and dissemination. Integrated analysis of miRNA target prediction algorithms and metastatic lung cancer gene expression data reveals the TGF-β co-receptor Tgfbr3 (ref. 12) as a putative target of Hmga2 ceRNA function. Tgfbr3 expression is regulated by the Hmga2 ceRNA through differential recruitment to Argonaute 2 (Ago2), and TGF-β signalling driven by Tgfbr3 is important for Hmga2 to promote lung cancer progression. Finally, analysis of NSCLC-patient gene-expression data reveals that HMGA2 and TGFBR3 are coordinately regulated in NSCLC-patient material, a vital corollary to ceRNA function. Taken together, these results suggest that Hmga2 promotes lung carcinogenesis both as a protein-coding gene and as a non-coding RNA; such dual-function regulation of gene-expression networks reflects a novel means by which oncogenes promote disease progression.