Hyeshik Chang

Amrita Basu, Nicole E. Bodycombe, Jaime H. Cheah, Edmund V. Price, Ke Liu, Giannina I. Schaefer, Richard Y. Ebright, Michelle L. Stewart, Daisuke Ito, Stephanie Wang, Abigail L. Bracha, Ted Liefeld, Mathias Wawer, Joshua C. Gilbert, Andrew J. Wilson, Nicolas Stransky, Gregory V. Kryukov, Vlado Dancik, Jordi Barretina, Levi A. Garraway, C. Suk-Yee Hon, Benito Munoz, Joshua A. Bittker, Brent R. Stockwell, Dineo Khabele, Andrew M. Stern, Paul A. Clemons, Alykhan F. Shamji, Stuart L. Schreiber. The high rate of clinical response to protein-kinase-targeting drugs matched to cancer patients with specific genomic alterations has prompted efforts to use cancer cell line (CCL) profiling to id....

Intrinsic Noise of microRNA-Regulated Genes and the ceRNA Hypothesis.

PLoS One. 2013;8(8):e72676

Authors: Noorbakhsh J, Lang AH, Mehta P

Abstract
MicroRNAs are small noncoding RNAs that regulate genes post-transciptionally by binding and degrading target eukaryotic mRNAs. We use a quantitative model to study gene regulation by inhibitory microRNAs and compare it to gene regulation by prokaryotic small non-coding RNAs (sRNAs). Our model uses a combination of analytic techniques as well as computational simulations to calculate the mean-expression and noise profiles of genes regulated by both microRNAs and sRNAs. We find that despite very different molecular machinery and modes of action (catalytic vs stoichiometric), the mean expression levels and noise profiles of microRNA-regulated genes are almost identical to genes regulated by prokaryotic sRNAs. This behavior is extremely robust and persists across a wide range of biologically relevant parameters. We extend our model to study crosstalk between multiple mRNAs that are regulated by a single microRNA and show that noise is a sensitive measure of microRNA-mediated interaction between mRNAs. We conclude by discussing possible experimental strategies for uncovering the microRNA-mRNA interactions and testing the competing endogenous RNA (ceRNA) hypothesis.

PMID: 23991139 [PubMed - in process]

Kimiko Saka, Satoru Ide, Austen R.D. Ganley, Takehiko Kobayashi. Genomic instability is a conserved factor in lifespan reduction, although the molecular mechanism is not known [1]. Studies in the yeast Saccharomyces cerevisiae over the past 20 years have....

Alex Charles Tuck, David Tollervey. Eukaryotic genomes generate a heterogeneous ensemble of mRNAs and long noncoding RNAs (lncRNAs). LncRNAs and mRNAs are both transcribed by Pol II and acquire 5′ caps and poly(A) tails, but only mR....

F. Ann Ran, Patrick D. Hsu, Chie-Yu Lin, Jonathan S. Gootenberg, Silvana Konermann, Alexandro E. Trevino, David A. Scott, Azusa Inoue, Shogo Matoba, Yi Zhang, Feng Zhang. Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by....

Nature Methods 10, (2013). doi:10.1038/nmeth.f.357

Author: Scott Kuersten

Nature Methods 10, 843 (2013). doi:10.1038/nmeth.2564

Authors: James A Anderson, Marleen Eijkholt & Judy Illes

Optimism about biomedicine is challenged by the increasingly complex ethical, legal and social issues it raises. Reporting of scientific methods is no longer sufficient to address the complex relationship between science and society. To promote 'ethical reproducibility', we call for transparent reporting of research ethics methods used in biomedical research.

Activating silent argonautes.

Nat Struct Mol Biol. 2013 Jul;20(7):769-71

Authors: Kidwell MA, Doudna JA

Abstract
Multiple Argonaute proteins are implicated in gene silencing by RNA interference (RNAi), but only one is known to be an endonuclease that can cleave target mRNAs. Chimeric Argonaute proteins now reveal an unexpected mechanism by which mutations distal to the catalytic center can unmask intrinsic catalytic activity, results hinting at structurally mediated regulation.

PMID: 23984440 [PubMed - in process]

Authors: David R. Corey, Stefanie Dimmeler, Jan-Wilhelm Kornfeld

by The PLOS Medicine Editors

Background: A composite biological structure, such as an insect head or abdomen, contains many internal structures with distinct functions. Composite structures are often used in RNA-seq studies, though it is unclear how expression of the same gene in different tissues and structures within the same structure affects the measurement (or even utility) of the resulting patterns of gene expression. Here we determine how complex composite tissue structure affects measures of gene expression using RNA-seq. Results: We focus on two structures in the honey bee (the sting gland and digestive tract) both contained within one larger structure, the whole abdomen. For each of the three structures, we used RNA-seq to identify differentially expressed genes between two developmental stages, nurse bees and foragers. Based on RNA-seq for each structure-specific extraction, we found that RNA-seq with composite structures leads to many false negatives (genes strongly differentially expressed in particular structures which are not found to be differentially expressed within the composite structure). We also found a significant number of genes with one pattern of differential expression in the tissue-specific extraction, and the opposite in the composite extraction, suggesting multiple signals from such genes within the composite structure. We found these patterns for different classes of genes including transcription factors. Conclusions: Many RNA-seq studies currently use composite extractions, and even whole insect extractions, when tissue and structure specific extractions are possible. This is due to the logistical difficultly of micro-dissection and unawareness of the potential errors associated with composite extractions. The present study suggests that RNA-seq studies of composite structures are prone to false negatives and difficult to interpret positive signals for genes with variable patterns of local expression. In general, our results suggest that RNA-seq on large composite structures should be avoided unless it is possible to demonstrate that the effects shown here do not exist for the genes of interest.

Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system.

Cell Res. 2013 Aug 27;

Authors: Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan S, Shivalila CS, Dadon DB, Jaenisch R

Abstract
Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore, we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3-4 sgRNAs binding to the proximal promoters, suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system.Cell Research advance online publication 27 Aug 2013; doi:10.1038/cr.2013.122.

PMID: 23979020 [PubMed - as supplied by publisher]

Current microRNA target predictions are based on sequence information and empirically derived rules but do not make use of the expression of microRNAs and their targets. This study aimed to improve microRNA target predictions in a given biological context, using in silico predictions, microRNA and mRNA expression. We used target prediction tools to produce lists of predicted targets and used a gene set test designed to detect consistent effects of microRNAs on the joint expression of multiple targets. In a single test, association between microRNA expression and target gene set expression as well as the contribution of the individual target genes on the association are determined. The strongest negatively associated mRNAs as measured by the test were prioritized. We applied our integration method to a well-defined muscle differentiation model. Validation of our predictions in C2C12 cells confirmed predicted targets of known as well as novel muscle-related microRNAs. We further studied associations between microRNA–mRNA pairs in human prostate cancer, finding some pairs that have been recently experimentally validated by others. Using the same study, we showed the advantages of the global test over Pearson correlation and lasso. We conclude that our integrated approach successfully identifies regulated microRNAs and their targets.

MicroRNAs (miRNAs) are short RNA species derived from hairpin-forming miRNA precursors (pre-miRNA) and acting as key posttranscriptional regulators. Most computational tools labeled as miRNA predictors are in fact pre-miRNA predictors and provide no information about the putative miRNA location within the pre-miRNA. Sequence and structural features that determine the location of the miRNA, and the extent to which these properties vary from species to species, are poorly understood. We have developed miRdup, a computational predictor for the identification of the most likely miRNA location within a given pre-miRNA or the validation of a candidate miRNA. MiRdup is based on a random forest classifier trained with experimentally validated miRNAs from miRbase, with features that characterize the miRNA–miRNA* duplex. Because we observed that miRNAs have sequence and structural properties that differ between species, mostly in terms of duplex stability, we trained various clade-specific miRdup models and obtained increased accuracy. MiRdup self-trains on the most recent version of miRbase and is easy to use. Combined with existing pre-miRNA predictors, it will be valuable for both de novo mapping of miRNAs and filtering of large sets of candidate miRNAs obtained from transcriptome sequencing projects. MiRdup is open source under the GPLv3 and available at http://www.cs.mcgill.ca/~blanchem/mirdup/.

Disruption of microRNA Biogenesis Confers Resistance to ER Stress-Induced Cell Death Upstream of the Mitochondrion.

PLoS One. 2013;8(8):e73870

Authors: Cawley K, Logue SE, Gorman AM, Zeng Q, Patterson J, Gupta S, Samali A

Abstract
Global downregulation of microRNAs (miRNAs) is a common feature of human tumors and has been shown to enhance cancer progression. Several components of the miRNA biogenesis machinery (XPO5, DICER and TRBP) have been shown to act as haploinsufficient tumor suppressors. How the deregulation of miRNA biogenesis promotes tumor development is not clearly understood. Here we show that loss of miRNA biogenesis increased resistance to endoplasmic reticulum (ER) stress-induced cell death. We observed that HCT116 cells with a DICER hypomorphic mutation (Exn5/Exn5) or where DICER or DROSHA were knocked down were resistant to ER stress-induced cell death. Extensive analysis revealed little difference in the unfolded protein response (UPR) of WT compared to Exn5/Exn5 HCT116 cells upon ER stress treatment. However, analysis of the intrinsic apoptotic pathway showed that resistance occurred upstream of the mitochondria. In particular, BAX activation and dissipation of mitochondrial membrane potential was attenuated, and there was altered expression of BCL-2 family proteins. These observations demonstrate a key role for miRNAs as critical modulators of the ER stress response. In our model, downregulation of miRNA biogenesis delays ER stress-induced apoptosis. This suggests that disrupted miRNA biogenesis may contribute to cancer progression by inhibiting ER stress-induced cell death.

PMID: 23977393 [PubMed - in process]

Use of Bru-Seq and BruChase-Seq for genome-wide assessment of the synthesis and stability of RNA.

Methods. 2013 Aug 21;

Authors: Paulsen MT, Veloso A, Prasad J, Bedi K, Ljungman EA, Magnuson B, Wilson TE, Ljungman M

Abstract
Gene expression studies commonly examine total cellular RNA, which only provides information about its steady-state pool of RNA. It remains unclear whether differences in this steady-state difference reflects variable rates of transcription or RNA degradation. To specifically monitor RNA synthesis and degradation genome-wide, we developed Bru-Seq and BruChase-Seq. These assays are based on metabolic pulse-chase labeling of RNA using bromouridine (Bru). In Bru-Seq, recently labeled RNAs are sequenced to reveal spans of nascent transcription in the genome. In BruChase-Seq, cells are chased in uridine for different periods of time following Bru-labeling, allowing for the isolation of RNA populations of specific ages. Here we describe these methodologies in detail and highlight their usefulness in assessing RNA synthesis and stability as well as splicing kinetics with examples of specific genes from different human cell lines.

PMID: 23973811 [PubMed - as supplied by publisher]

We report an approach for generating immobilized monoclonal templates for next- generation sequencing applications. Our isothermal amplification method is based on a template walking mechanism using a pair of low-melting temperature (Tm) solid-surface homopolymer primers and a low-Tm solution phase primer. The method can generate more than one billion submicrometer-sized...

Lionel Tafforeau, Christiane Zorbas, Jean-Louis Langhendries, Sahra-Taylor Mullineux, Vassiliki Stamatopoulou, Romain Mullier, Ludivine Wacheul, Denis L.J. Lafontaine. Mature ribosomal RNAs (rRNAs) are produced from polycistronic precursors following complex processing. Precursor (pre)-rRNA processing has been extensively characterized in yeast and was assumed t....

Benjamin Guglielmi, Natalie La Rochelle, Robert Tjian. To bridge the gap between in vivo and in vitro molecular mechanisms, we dissected the transcriptional control of the endogenous histone gene cluster (His-C) by single-cell imaging. A combination o....

A rare, small RNA turns a gene-splicing machine into a switch that controls the expression of hundreds of human genes. Howard Hughes Medical Institute Investigator and professor of Biochemistry Gideon Dreyfuss, PhD, and his team from the Perelman School of Medicine at the University of Pennsylvania, discovered an entirely new aspect of the gene-splicing process that produces messenger RNA (mRNA).

The investigators found that a scarce, small RNA, called U6atac, controls the expression of hundreds of genes that have critical functions in cell growth, cell-cycle control, and global control of physiology. Their results were published in the journal eLife.

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Nature Protocols 8, 1765 (2013). doi:10.1038/nprot.2013.099

Authors: Simon Anders, Davis J McCarthy, Yunshun Chen, Michal Okoniewski, Gordon K Smyth, Wolfgang Huber & Mark D Robinson

RNA sequencing (RNA-seq) has been rapidly adopted for the profiling of transcriptomes in many areas of biology, including studies into gene regulation, development and disease. Of particular interest is the discovery of differentially expressed genes across different conditions (e.g., tissues, perturbations) while optionally adjusting for

The EMBO Journal. doi:10.1038/emboj.2013.188

Authors: Kiranmai Gumireddy, Anping Li, Jinchun Yan, Tetsuro Setoyama, Gregg J Johannes, Ulf A Ørom, Julia Tchou, Qin Liu, Lin Zhang, David W Speicher, George A Calin & Qihong Huang

This paper describes the identification of an exonuclease in a kinetoplastid that is related to the XRN family of exonucleases present in higher eukaryotes. Functional studies demonstrate that knockdown of this exonuclease is not lethal but results in defects in rRNA processing.

This paper focuses on the C. elegans RNA-binding protein, GLD-1. GLD-1 is a translational repressor whose targets have been difficult to predict. The authors develop a simple biophysical model, which, by taking into account target accessibility, significantly improves target prediction. They find that CDS-located binding sites contribute most to translational repression.

Related Articles

Decreased dicer expression enhances SRP-mediated protein targeting.

PLoS One. 2013;8(2):e56950

Authors: Ren YF, Li G, Xue YF, Zhang XJ, Song YJ, Lv L, Wu J, Fang YX, Wang YQ, Shi KQ, Chen YP, Tang KF

Abstract
We have shown that Dicer processes 7SL RNA into different fragments ranging from ∼20 to more than 200 nucleotides. Here we addressed the molecular functions of these 7SL RNA fragments and found that some of them functioned as dominant-negative regulators of the full-length 7SL RNA, interfering with signal recognition particle (SRP) complex formation. Transfection of these 7SL RNA fragments inhibited the expression of cell surface glycoproteins, the targeting of a reporter protein to the endoplasmic reticulum, and the secretion of secreted alkaline phosphatase. These results suggest that some Dicer-processed 7SL RNA fragments interfered with SRP-mediated protein targeting. Moreover, we showed that Dicer knockdown enhanced SRP-mediated protein targeting and that transfection of a mixture of the 7SL RNA fragments partially restored this effect. Our data indicate that Dicer can fine-tune the efficiency of SRP-mediated protein targeting via processing a proportion of 7SL RNA into fragments of different lengths.

PMID: 23468895 [PubMed - indexed for MEDLINE]

Proteomic identification of target proteins following Drosha knockdown in cervical cancer.

Oncol Rep. 2013 Aug 20;

Authors: Zhou J, Cai J, Huang Z, Ding H, Wang J, Jia J, Zhao Y, Huang D, Wang Z

Abstract
The nuclear microRNA (miRNA) processing enzyme Drosha is upregulated in cervical cancer, and its overexpression is related to an invasive tumour phenotype. However, the mechanisms that underlie this effect remain poorly understood. The aim of this study was to identify the potential targets of Drosha in cervical cancer. Here, we demonstrated that Drosha knockdown (Drosha-KD) inhibited proliferation, colony formation and the migration of cervical cancer cells in vitro. A global upregulation of proteins in Drosha-KD cells was revealed by two-dimensional gel electrophoresis (2-DE). Eighteen proteins were identified by liquid chromatography and tandem mass spectrometry technology (LC-MS/MS) from 21 selected protein spots that exhibited significant alterations in Drosha-KD cells. The majority of the identified proteins have been previously associated with tumour formation. The downregulation of tubulin 5β in Drosha-KD cervical cancer cells was further confirmed by western blotting. Our results suggest that Drosha affects the biological activity of cervical cancer cells by regulating the expression of numerous tumour-associated proteins.

PMID: 23969986 [PubMed - as supplied by publisher]

WaRSwap is a randomization algorithm that for the first time provides a practical network motif discovery method for large multi-layer networks, for example those that include transcription factors, microRNAs, and non-regulatory protein coding genes. The algorithm is applicable to systems with tens of thousands of genes, while accounting for critical aspects of biological networks, including self-loops, large hubs, and target rearrangements. We validate WaRSwap on a newly inferred regulatory network from Arabidopsis thaliana, and compare outcomes on published Drosophila and human networks. Specifically, sustained input switches are among the few over-represented circuits across this diverse set of eukaryotes.

A large-scale RNA in vitro selection study systematically identified RNA recognition elements for 205 RNA-binding proteins belonging to families conserved in most eukaryotes.

Optical control of mammalian endogenous transcription and epigenetic states

Nature 500, 7463 (2013). doi:10.1038/nature12466

Authors: Silvana Konermann, Mark D. Brigham, Alexandro E. Trevino, Patrick D. Hsu, Matthias Heidenreich, Le Cong, Randall J. Platt, David A. Scott, George M. Church & Feng Zhang

The dynamic nature of gene expression enables cellular programming, homeostasis and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high-precision spatiotemporal control of many cellular functions. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here we describe the development of light-inducible transcriptional effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical cofactors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of freely behaving mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states.

Related Articles

Exceptional stories of microRNAs.

Exp Biol Med (Maywood). 2013 Apr;238(4):339-43

Authors: Lee HJ

Abstract
MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs that regulate the expression of target genes in eukaryotic cells and have been extensively studied in the past decade. However, recent evidence suggests that miRNAs have additional, important roles and functions other than post-transcriptional regulation through binding at the 3' untranslated regions of their target genes. This review describes newly discovered information about the biogenesis and functions of miRNAs as well as presents different points of view about the miRNA system. Our increasing knowledge of the exceptional stories of miRNAs will offer new insights into these powerful gene regulators from virus and bacteria to animals.

PMID: 23759998 [PubMed - indexed for MEDLINE]