Hyeshik Chang

Nature Genetics 45, 877 (2013). doi:10.1038/ng.2672

Authors: Gregoriy A Dokshin, Andrew E Baltus, John J Eppig & David C Page

A freely available microscopic model of human brain architecture with a spatial resolution of 20 micrometers is presented.

Authors: Katrin Amunts, Claude Lepage, Louis Borgeat, Hartmut Mohlberg, Timo Dickscheid, Marc-Étienne Rousseau, Sebastian Bludau, Pierre-Louis Bazin, Lindsay B. Lewis, Ana-Maria Oros-Peusquens, Nadim J. Shah, Thomas Lippert, Karl Zilles, Alan C. Evans

by Matthew J. Hangauer, Ian W. Vaughn, Michael T. McManus

Known protein coding gene exons compose less than 3% of the human genome. The remaining 97% is largely uncharted territory, with only a small fraction characterized. The recent observation of transcription in this intergenic territory has stimulated debate about the extent of intergenic transcription and whether these intergenic RNAs are functional. Here we directly observed with a large set of RNA-seq data covering a wide array of human tissue types that the majority of the genome is indeed transcribed, corroborating recent observations by the ENCODE project. Furthermore, using de novo transcriptome assembly of this RNA-seq data, we found that intergenic regions encode far more long intergenic noncoding RNAs (lincRNAs) than previously described, helping to resolve the discrepancy between the vast amount of observed intergenic transcription and the limited number of previously known lincRNAs. In total, we identified tens of thousands of putative lincRNAs expressed at a minimum of one copy per cell, significantly expanding upon prior lincRNA annotation sets. These lincRNAs are specifically regulated and conserved rather than being the product of transcriptional noise. In addition, lincRNAs are strongly enriched for trait-associated SNPs suggesting a new mechanism by which intergenic trait-associated regions may function. These findings will enable the discovery and interrogation of novel intergenic functional elements.

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Genome-wide profiling of human cap-independent translation-enhancing elements.

Nat Methods. 2013 Jun 16;

Authors: Wellensiek BP, Larsen AC, Stephens B, Kukurba K, Waern K, Briones N, Liu L, Snyder M, Jacobs BL, Kumar S, Chaput JC

Abstract
We report an in vitro selection strategy to identify RNA sequences that mediate cap-independent initiation of translation. This method entails mRNA display of trillions of genomic fragments, selection for initiation of translation and high-throughput deep sequencing. We identified >12,000 translation-enhancing elements (TEEs) in the human genome, generated a high-resolution map of human TEE-bearing regions (TBRs), and validated the function of a subset of sequences in vitro and in cultured cells.

PMID: 23770754 [PubMed - as supplied by publisher]

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Identification of LIN28B-bound mRNAs reveals features of target recognition and regulation.

RNA Biol. 2013 May 29;10(7)

Authors: Graf R, Munschauer M, Mastrobuoni G, Mayr F, Heinemann U, Kempa S, Rajewsky N, Landthaler M

Abstract
The conserved human LIN28 RNA-binding proteins function in development, maintenance of pluripotency and oncogenesis. We used PAR-CLIP and a newly developed variant of this method, iDo-PAR-CLIP, to identify LIN28B targets as well as sites bound by the individual RNA-binding domains of LIN28B in the human transcriptome at nucleotide resolution. The position of target binding sites reflected the known structural relative orientation of individual LIN28B-binding domains, validating iDo-PAR-CLIP. Our data suggest that LIN28B directly interacts with most expressed mRNAs and members of the let-7 microRNA family. The Lin28-binding motif detected in pre-let-7 was enriched in mRNA sequences bound by LIN28B. Upon LIN28B knockdown, cell proliferation and the cell cycle were strongly impaired. Quantitative shotgun proteomics of LIN28B depleted cells revealed significant reduction of protein synthesis from its RNA targets that function in translation, mRNA splicing and cell cycle control. Computational analyses provided evidence that the strength of protein synthesis reduction correlated with the location of LIN28B binding sites within target transcripts.

PMID: 23770886 [PubMed - as supplied by publisher]

Clusters of microRNAs emerge by new hairpins in existing transcripts.

Nucleic Acids Res. 2013 Jun 17;

Authors: Marco A, Ninova M, Ronshaugen M, Griffiths-Jones S

Abstract
Genetic linkage may result in the expression of multiple products from a polycistronic transcript, under the control of a single promoter. In animals, protein-coding polycistronic transcripts are rare. However, microRNAs are frequently clustered in the genomes of animals, and these clusters are often transcribed as a single unit. The evolution of microRNA clusters has been the subject of much speculation, and a selective advantage of clusters of functionally related microRNAs is often proposed. However, the origin of microRNA clusters has not been so far explored. Here, we study the evolution of microRNA clusters in Drosophila melanogaster. We observed that the majority of microRNA clusters arose by the de novo formation of new microRNA-like hairpins in existing microRNA transcripts. Some clusters also emerged by tandem duplication of a single microRNA. Comparative genomics show that these clusters are unlikely to split or undergo rearrangements. We did not find any instances of clusters appearing by rearrangement of pre-existing microRNA genes. We propose a model for microRNA cluster evolution in which selection over one of the microRNAs in the cluster interferes with the evolution of the other linked microRNAs. Our analysis suggests that the study of microRNAs and small RNAs must consider linkage associations.

PMID: 23775791 [PubMed - as supplied by publisher]

Micro RNAs regulate hormone synthesis in the pituitary and the production of oocytes.

Authors: Hidetoshi Hasuwa, Jun Ueda, Masahito Ikawa, Masaru Okabe

The EMBO Journal. doi:10.1038/emboj.2013.135

Authors: Michal Lubas, Christian K Damgaard, Rafal Tomecki, Dominik Cysewski, Torben Heick Jensen & Andrzej Dziembowski

The EMBO Journal. doi:10.1038/emboj.2013.134

Authors: Johanna C Scheuermann & Laurie A Boyer

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Alternative Splicing Regulates Biogenesis of miRNAs Located across Exon-Intron Junctions.

Mol Cell. 2013 Jun 4;

Authors: Melamed Z, Levy A, Ashwal-Fluss R, Lev-Maor G, Mekahel K, Atias N, Gilad S, Sharan R, Levy C, Kadener S, Ast G

Abstract
The initial step in microRNA (miRNA) biogenesis requires processing of the precursor miRNA (pre-miRNA) from a longer primary transcript. Many pre-miRNAs originate from introns, and both a mature miRNA and a spliced RNA can be generated from the same transcription unit. We have identified a mechanism in which RNA splicing negatively regulates the processing of pre-miRNAs that overlap exon-intron junctions. Computational analysis identified dozens of such pre-miRNAs, and experimental validation demonstrated competitive interaction between the Microprocessor complex and the splicing machinery. Tissue-specific alternative splicing regulates maturation of one such miRNA, miR-412, resulting in effects on its targets that code a protein network involved in neuronal cell death processes. This mode of regulation specifically controls maturation of splice-site-overlapping pre-miRNAs but not pre-miRNAs located completely within introns or exons of the same transcript. Our data present a biological role of alternative splicing in regulation of miRNA biogenesis.

PMID: 23747012 [PubMed - as supplied by publisher]

Nature Reviews Genetics. doi:10.1038/nrg3455

Authors: David B. Goldstein, Andrew Allen, Jonathan Keebler, Elliott H. Margulies, Steven Petrou, Slavé Petrovski & Shamil Sunyaev

Recently, researchers have uncovered the presence of many long noncoding RNAs (lncRNAs) in embryonic stem cells and believe they are important regulators of the differentiation process. However, there are only a few examples explicitly linking lncRNA activity to transcriptional regulation. Here, we used transcript counting and spatial localization to characterize a lncRNA (dubbed linc-HOXA1) located ~50 kb from the Hoxa gene cluster in mouse embryonic stem cells. Single-cell transcript counting revealed that linc-HOXA1 and Hoxa1 RNA are highly variable at the single-cell level and that whenever linc-HOXA1 RNA abundance was high, Hoxa1 mRNA abundance was low and vice versa. Knockdown analysis revealed that depletion of linc-HOXA1 RNA at its site of transcription increased transcription of the Hoxa1 gene cis to the chromosome and that exposure of cells to retinoic acid can disrupt this interaction. We further showed that linc-HOXA1 RNA represses Hoxa1 by recruiting the protein PURB as a transcriptional cofactor. Our results highlight the power of transcript visualization to characterize lncRNA function and also suggest that PURB can facilitate lncRNA-mediated transcriptional regulation.

Guanine-rich RNA sequences can fold into non-canonical, four stranded helical structures called G-quadruplexes that have been shown to be widely distributed within the mammalian transcriptome, as well as being key regulatory elements in various biological mechanisms. That said, their role within the 3'-untranslated region (UTR) of mRNA remains to be elucidated and appreciated. A bioinformatic analysis of the 3'-UTRs of mRNAs revealed enrichment in G-quadruplexes. To shed light on the role(s) of these structures, those found in the LRP5 and FXR1 genes were characterized both in vitro and in cellulo. The 3'-UTR G-quadruplexes were found to increase the efficiencies of alternative polyadenylation sites, leading to the expression of shorter transcripts and to possess the ability to interfere with the miRNA regulatory network of a specific mRNA. Clearly, G-quadruplexes located in the 3'-UTRs of mRNAs are cis-regulatory elements that have a significant impact on gene expression.

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Integrated platform for genome-wide screening and construction of high-density genetic interaction maps in mammalian cells.

Proc Natl Acad Sci U S A. 2013 Jun 18;110(25):E2317-26

Authors: Kampmann M, Bassik MC, Weissman JS

Abstract
A major challenge of the postgenomic era is to understand how human genes function together in normal and disease states. In microorganisms, high-density genetic interaction (GI) maps are a powerful tool to elucidate gene functions and pathways. We have developed an integrated methodology based on pooled shRNA screening in mammalian cells for genome-wide identification of genes with relevant phenotypes and systematic mapping of all GIs among them. We recently demonstrated the potential of this approach in an application to pathways controlling the susceptibility of human cells to the toxin ricin. Here we present the complete quantitative framework underlying our strategy, including experimental design, derivation of quantitative phenotypes from pooled screens, robust identification of hit genes using ultra-complex shRNA libraries, parallel measurement of tens of thousands of GIs from a single double-shRNA experiment, and construction of GI maps. We describe the general applicability of our strategy. Our pooled approach enables rapid screening of the same shRNA library in different cell lines and under different conditions to determine a range of different phenotypes. We illustrate this strategy here for single- and double-shRNA libraries. We compare the roles of genes for susceptibility to ricin and Shiga toxin in different human cell lines and reveal both toxin-specific and cell line-specific pathways. We also present GI maps based on growth and ricin-resistance phenotypes, and we demonstrate how such a comparative GI mapping strategy enables functional dissection of physical complexes and context-dependent pathways.

PMID: 23739767 [PubMed - indexed for MEDLINE]

Science is now accepting submissions for the best dance interpretations of doctoral work

Susanta K. Sarkar, Ambika Bumb, Maria Mills, Keir C. Neuman.

Dilek Colak, Sheng-Jian Ji, Bo T. Porse, Samie R. Jaffrey. Growth cones enable axons to navigate toward their targets by responding to extracellular signaling molecules. Growth-cone responses are mediated in part by the local translation of axonal messeng....

Nature Reviews Genetics. doi:10.1038/nrg3462

Author: Gunter Meister

Suzanne R. Lee, Jens Lykke-Andersen.

Motivation: Ribosome profiling is a new technique that allows monitoring locations of translating ribosomes on mRNA at a whole transcriptome level. A recent ribosome profiling study demonstrated that internal Shine–Dalgarno (SD) sequences have a major global effect on translation rates in bacteria: ribosomes pause at SD sites in mRNA. Therefore, it is important to understand how SD sites effect mRNA movement through the ribosome and generation of ribosome footprints.

Results: Here, we provide evidence that in addition to pausing effect, internal SD sequences induce a caterpillar-like movement of mRNA through the ribosome cavity. Once an SD site binds to the ribosome, it remains attached to it while the ribosome decodes a few subsequent codons. This leads to asymmetric progressive elongation of ribosome footprints at the 3'-end. It is likely that internal SD sequences induce a pause not on a single, but on several adjacent codons. This finding is important for our understanding of mRNA movement through the ribosome and also should facilitate interpretation of ribosome profiling data.

Contact: brave.oval.pan@gmail.com

A Rapid Ribosome Profiling Method Elucidates Chloroplast Ribosome Behavior in Vivo.

Plant Cell. 2013 Jun 4;

Authors: Zoschke R, Watkins KP, Barkan A

Abstract
The profiling of ribosome footprints by deep sequencing has revolutionized the analysis of translation by mapping ribosomes with high resolution on a genome-wide scale. We present a variation on this approach that offers a rapid and cost-effective alternative for the genome-wide profiling of chloroplast ribosomes. Ribosome footprints from leaf tissue are hybridized to oligonucleotide tiling microarrays of the plastid ORFeome and report the abundance and translational status of every chloroplast mRNA. Each assay replaces several time-consuming traditional methods while also providing information that was previously inaccessible. To illustrate the utility of the approach, we show that it detects known defects in chloroplast gene expression in several nuclear mutants of maize (Zea mays) and that it reveals previously unsuspected defects. Furthermore, it provided firm answers to several lingering questions in chloroplast gene expression: (1) the overlapping atpB/atpE open reading frames, whose translation had been proposed to be coupled, are translated independently in vivo; (2) splicing is not a prerequisite for translation initiation on an intron-containing chloroplast RNA; and (3) a feedback control mechanism that links the synthesis of ATP synthase subunits in Chlamydomonas reinhardtii does not exist in maize. An analogous approach is likely to be useful for studies of mitochondrial gene expression.

PMID: 23735295 [PubMed - as supplied by publisher]

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Dicer-processed small RNAs: rules and exceptions.

J Exp Zool B Mol Dev Evol. 2013 Jan;320(1):35-46

Authors: Langenberger D, Çakir MV, Hoffmann S, Stadler PF

Abstract
Canonical microRNAs are excised from their hairpin-shaped precursors by Dicer. In order to find possible exceptions to this rule and to identify additional substrates for Dicer processing we re-evaluate the small RNA sequencing data of the Dicer knockdown experiment in MCF-7 cells orignally published by Friedländer et al. [Friedländer et al., 2012, Nucleic Acids Res 40:37-52]. While the well-known non-Dicer mir-451 is not sufficiently expressed in these experiments, there are several additional Dicer-independent microRNAs, among them the important tumor supressor mir-663a. We recover previously described examples of non-miRNA Dicer substrates such as tRNA-Gln and several snoRNAs. Interestingly, sdRNAs derived from box C/D snoRNAs are Dicer-independent, while those derived from box H/ACA snoRNAs are often Dicer dependent. Several pol-III transcripts, in particular the vault RNAs and the great ape specific snaRs are processed by Dicer, while the small RNAs originating from Y RNAs seem to be Dicer independent.

PMID: 23165937 [PubMed - in process]

An alternative translation start site produces an elongated PTEN that can enter tumor cells and kill them.

Authors: Benjamin D. Hopkins, Barry Fine, Nicole Steinbach, Meaghan Dendy, Zachary Rapp, Jacquelyn Shaw, Kyrie Pappas, Jennifer S. Yu, Cindy Hodakoski, Sarah Mense, Joshua Klein, Sarah Pegno, Maria-Luisa Sulis, Hannah Goldstein, Benjamin Amendolara, Liang Lei, Matthew Maurer, Jeffrey Bruce, Peter Canoll, Hanina Hibshoosh, Ramon Parsons

Nature advance online publication 02 June 2013. doi:10.1038/nature12210

Authors: Wenbo Li, Dimple Notani, Qi Ma, Bogdan Tanasa, Esperanza Nunez, Aaron Yun Chen, Daria Merkurjev, Jie Zhang, Kenneth Ohgi, Xiaoyuan Song, Soohwan Oh, Hong-Sook Kim, Christopher K. Glass & Michael G. Rosenfeld

The functional importance of gene enhancers in regulated gene expression is well established. In addition to widespread transcription of long non-coding RNAs (lncRNAs) in mammalian cells, bidirectional ncRNAs are transcribed on enhancers, and are thus referred to as enhancer RNAs (eRNAs). However, it has remained unclear whether these eRNAs are functional or merely a reflection of enhancer activation. Here we report that in human breast cancer cells 17β-oestradiol (E2)-bound oestrogen receptor α (ER-α) causes a global increase in eRNA transcription on enhancers adjacent to E2-upregulated coding genes. These induced eRNAs, as functional transcripts, seem to exert important roles for the observed ligand-dependent induction of target coding genes, increasing the strength of specific enhancer–promoter looping initiated by ER-α binding. Cohesin, present on many ER-α-regulated enhancers even before ligand treatment, apparently contributes to E2-dependent gene activation, at least in part by stabilizing E2/ER-α/eRNA-induced enhancer–promoter looping. Our data indicate that eRNAs are likely to have important functions in many regulated programs of gene transcription.

Nature advance online publication 02 June 2013. doi:10.1038/nature12243

Authors: James O’Malley, Stavroula Skylaki, Kumiko A. Iwabuchi, Eleni Chantzoura, Tyson Ruetz, Anna Johnsson, Simon R. Tomlinson, Sten Linnarsson & Keisuke Kaji

The generation of induced pluripotent stem (iPS) cells presents a challenge to normal developmental processes. The low efficiency and heterogeneity of most methods have hindered understanding of the precise molecular mechanisms promoting, and roadblocks preventing, efficient reprogramming. Although several intermediate populations have been described, it has proved difficult to characterize the rare, asynchronous transition from these intermediate stages to iPS cells. The rapid expansion of minor reprogrammed cells in the heterogeneous population can also obscure investigation of relevant transition processes. Understanding the biological mechanisms essential for successful iPS cell generation requires both accurate capture of cells undergoing the reprogramming process and identification of the associated global gene expression changes. Here we demonstrate that in mouse embryonic fibroblasts, reprogramming follows an orderly sequence of stage transitions, marked by changes in the cell-surface markers CD44 and ICAM1, and a Nanog–enhanced green fluorescent protein (Nanog–eGFP) reporter. RNA-sequencing analysis of these populations demonstrates two waves of pluripotency gene upregulation, and unexpectedly, transient upregulation of several epidermis-related genes, demonstrating that reprogramming is not simply the reversal of the normal developmental processes. This novel high-resolution analysis enables the construction of a detailed reprogramming route map, and the improved understanding of the reprogramming process will lead to new reprogramming strategies.

Cys₂-His₂ zinc finger proteins (ZFPs) are the largest group of transcription factors in higher metazoans. A complete characterization of these ZFPs and their associated target sequences is pivotal to fully annotate transcriptional regulatory networks in metazoan genomes. As a first step in this process, we have characterized the DNA-binding specificities of 129 zinc finger sets from Drosophila using a bacterial one-hybrid system. This data set contains the DNA-binding specificities for at least one encoded ZFP from 70 unique genes and 23 alternate splice isoforms representing the largest set of characterized ZFPs from any organism described to date. These recognition motifs can be used to predict genomic binding sites for these factors within the fruit fly genome. Subsets of fingers from these ZFPs were characterized to define their orientation and register on their recognition sequences, thereby allowing us to define the recognition diversity within this finger set. We find that the characterized fingers can specify 47 of the 64 possible DNA triplets. To confirm the utility of our finger recognition models, we employed subsets of Drosophila fingers in combination with an existing archive of artificial zinc finger modules to create ZFPs with novel DNA-binding specificity. These hybrids of natural and artificial fingers can be used to create functional zinc finger nucleases for editing vertebrate genomes.

A Review of Computational Tools in microRNA Discovery.

Front Genet. 2013;4:81

Authors: Gomes CP, Cho JH, Hood L, Franco OL, Pereira RW, Wang K

Abstract
Since microRNAs (miRNAs) were discovered, their impact on regulating various biological activities has been a surprising and exciting field. Knowing the entire repertoire of these small molecules is the first step to gain a better understanding of their function. High throughput discovery tools such as next-generation sequencing significantly increased the number of known miRNAs in different organisms in recent years. However, the process of being able to accurately identify miRNAs is still a complex and difficult task, requiring the integration of experimental approaches with computational methods. A number of prediction algorithms based on characteristics of miRNA molecules have been developed to identify new miRNA species. Different approaches have certain strengths and weaknesses and in this review, we aim to summarize several commonly used tools in metazoan miRNA discovery.

PMID: 23720668 [PubMed - as supplied by publisher]

Erik Gutierrez, Byung-Sik Shin, Christopher J. Woolstenhulme, Joo-Ran Kim, Preeti Saini, Allen R. Buskirk, Thomas E. Dever. Translation factor eIF5A, containing the unique amino acid hypusine, was originally shown to stimulate Met-puromycin synthesis, a model assay for peptide bond formation. More recently, eIF5A was s....

Nature Protocols 8, 1216 (2013). doi:10.1038/nprot.2013.071

Authors: Maciej Antkowiak, Maria L Torres-Mapa, David J Stevenson, Kishan Dholakia & Frank J Gunn-Moore

Laser-mediated gene transfection into mammalian cells has recently emerged as a powerful alternative to more traditional transfection techniques. In particular, the use of a femtosecond-pulsed laser operating in the near-infrared (NIR) region has been proven to provide single-cell selectivity, localized delivery, low toxicity and consistent

Nature Methods 10, 550 (2013). doi:10.1038/nmeth.2473

Authors: Kristina Woodruff, Luis M Fidalgo, Samy Gobaa, Matthias P Lutolf & Sebastian J Maerkl

High-content assays have the potential to drastically increase throughput in cell biology and drug discovery, but handling and culturing large libraries of cells such as primary tumor or cancer cell lines requires expensive, dedicated robotic equipment. We developed a simple yet powerful method that uses contact spotting to generate high-density nanowell arrays of live mammalian cells for the culture and interrogation of cell libraries.