Hyeshik Chang

Related Articles

Dicer-dependent and -independent Argonaute2 protein interaction networks in mammalian cells.

Mol Cell Proteomics. 2012 Nov;11(11):1442-56

Authors: Frohn A, Eberl HC, Stöhr J, Glasmacher E, Rüdel S, Heissmeyer V, Mann M, Meister G

Abstract
Argonaute (Ago) proteins interact with small regulatory RNAs such as microRNAs (miRNAs) and facilitate gene-silencing processes. miRNAs guide Ago proteins to specific mRNAs leading to translational silencing or mRNA decay. In order to understand the mechanistic details of miRNA function, it is important to characterize Ago protein interactors. Although several proteomic studies have been performed, it is not clear how the Ago interactome changes on miRNA or mRNA binding. Here, we report the analysis of Ago protein interactions in miRNA-containing and miRNA-depleted cells. Using stable isotope labeling in cell culture in conjunction with Dicer knock out mouse embryonic fibroblasts, we identify proteins that interact with Ago2 in the presence or the absence of Dicer. In contrast to our current view, we find that Ago-mRNA interactions can also take place in the absence of miRNAs. Our proteomics approach provides a rich resource for further functional studies on the cellular roles of Ago proteins.

PMID: 22918229 [PubMed - indexed for MEDLINE]

Je-Hyun Yoon, Kotb Abdelmohsen, Subramanya Srikantan, Xiaoling Yang, Jennifer L. Martindale, Supriyo De, Maite Huarte, Ming Zhan, Kevin G. Becker, Myriam Gorospe.

A class of circular RNAs that regulates microRNAs is abundant in mammalian cells.

Authors: Jeremy E. Wilusz, Phillip A. Sharp

Luke A. Yates, Chris J. Norbury, Robert J.C. Gilbert. MicroRNAs (miRNAs) are versatile regulators of gene expression in higher eukaryotes. In order to silence many different mRNAs in a precise manner, miRNA stability and efficacy is controlled by hig....

Aleksandra Helwak, Grzegorz Kudla, Tatiana Dudnakova, David Tollervey. MicroRNAs (miRNAs) play key roles in gene regulation, but reliable bioinformatic or experimental identification of their targets remains difficult. To provide an unbiased view of human miRNA targe....

Shengben Li, Lin Liu, Xiaohong Zhuang, Yu Yu, Xigang Liu, Xia Cui, Lijuan Ji, Zhiqiang Pan, Xiaofeng Cao, Beixin Mo, Fuchun Zhang, Natasha Raikhel, Liwen Jiang, Xuemei Chen. Translation inhibition is a major but poorly understood mode of action of microRNAs (miRNAs) in plants and animals. In particular, the subcellular location where this process takes place is unknow....

Taiowa A. Montgomery, Gary Ruvkun. Little is known about where microRNAs (miRNAs) regulate their targets within the cell. In this issue, Li et al. identify a new player in the plant miRNA pathway that implicates the endoplasmic ret....

Hiromitsu Ota, Masayuki Sakurai, Ravi Gupta, Louis Valente, Bjorn-Erik Wulff, Kentaro Ariyoshi, Hisashi Iizasa, Ramana V. Davuluri, Kazuko Nishikura. Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosine residues to inosine specifically in double-stranded RNAs. In this study, we investigated the interact....

ShortStack is a new stand-alone and single-command tool for discovery of genes/loci that are precursors of small interfering and microRNAs using deeply sequenced small RNA libraries. The analysis can be performed for distant species and allows the quantification of the inferred small RNA genes.

Nature Reviews Molecular Cell Biology 14, 266 (2013). doi:10.1038/nrm3573

Author: Rachel David

microRNAs (miRNAs) regulate gene expression, but whether this involves inhibition of translation or altered mRNA decay is debated. Here, the authors observed that mRNA destabilization only occurred for mRNAs that had been translationally repressed (through removal of translation initiation factors), suggesting that translation inhibition preceeds

MicroRNAs (miRNAs) can group together along the human genome to form stable secondary structures made of several hairpins hosting miRNAs in their stems. The few known examples of such structures are all involved in cancer development. A large scale computational analysis of human chromosomes crossing sequence analysis and deep sequencing data revealed the presence of >400 structural clusters of miRNAs in the human genome. An a posteriori analysis validates predictions as bona fide miRNAs. A functional analysis of structural clusters position along the chromosomes co-localizes them with genes involved in several key cellular processes like immune systems, sensory systems, signal transduction and development. Immune systems diseases, infectious diseases and neurodegenerative diseases are characterized by genes that are especially well organized around structural clusters of miRNAs. Target genes functional analysis strongly supports a regulatory role of most predicted miRNAs and, notably, a strong involvement of predicted miRNAs in the regulation of cancer pathways. This analysis provides new fundamental insights on the genomic organization of miRNAs in human chromosomes.

Stress response mechanisms that modulate the dynamics of tRNA degradation and accumulation from the cytoplasm to the nucleus have been studied in yeast, the rat hepatoma and human cells. In the current study, we investigated tRNA degradation and accumulation in HeLa cells under various forms of stress. We found that initiator tRNA^Met (tRNA(iMet)) was specifically degraded under heat stress. Two exonucleases, Xrn1 and Xrn2, are involved in the degradation of tRNA(iMet) in the cytoplasm and the nucleus, respectively. In addition to degradation, we observed accumulation of tRNA(iMet) in the nucleus. We also found that the mammalian target of rapamycin (mTOR), which regulates tRNA trafficking in yeast, is partially phosphorylated at Ser2448 in the presence of rapamycin and/or during heat stress. Our results suggest phosphorylation of mTOR may correlate with accumulation of tRNA(iMet) in heat-treated HeLa cells.

Scott A. Lujan, Jessica S. Williams, Anders R. Clausen, Alan B. Clark, Thomas A. Kunkel. To maintain genome stability, mismatch repair of nuclear DNA replication errors must be directed to the nascent strand, likely by DNA ends and PCNA. Here we show that the efficiency of mismatch re....

Protein- and RNA-based transcriptional regulation governs the removal of mitochondria during red blood cell differentiation.

Authors: Isabelle Barde, Benjamin Rauwel, Ray Marcel Marin-Florez, Andrea Corsinotti, Elisa Laurenti, Sonia Verp, Sandra Offner, Julien Marquis, Adamandia Kapopoulou, Jiri Vanicek, Didier Trono

Related Articles

Roles of R2D2, a cytoplasmic D2 body component, in the endogenous siRNA pathway in Drosophila.

Mol Cell. 2013 Feb 21;49(4):680-91

Authors: Nishida KM, Miyoshi K, Ogino A, Miyoshi T, Siomi H, Siomi MC

Abstract
Endogenous small interfering RNAs (endo-siRNAs) in Drosophila are processed by Dicer-2 (Dcr-2) and loaded onto Ago2 by the Dcr-2/R2D2 heterodimer. In r2d2 mutants, the level of endo-siRNAs is unchanged, but endo-siRNAs are misloaded onto Ago1. However, the mechanism underlying the control of endo-siRNA sorting by R2D2 remains unknown. Here, we show that R2D2 controls endo-siRNA sorting by localizing Dcr-2, and presumably endo-siRNA duplexes, to cytoplasmic foci, D2 bodies. Ago2, but not Ago1, localized to D2 bodies. dsRNA-binding-deficient mutant, but not wild-type, R2D2 failed to localize D2 bodies and caused endo-siRNA misdirection to Ago1 in R2D2-depleted cells. However, R2D2 was dispensable for sorting miRNAs and exogenous siRNAs onto Ago1 and Ago2, respectively, in vivo. Endo- and exo-siRNA guide selection also occurred R2D2 independently. The functions of R2D2 are required to avoid endo-siRNA misdirection to Ago1, because Ago1 is capable of loading incompletely complementary miRNA duplexes and endo-siRNA duplexes.

PMID: 23375501 [PubMed - in process]

Yonatan Savir, Tsvi Tlusty. The ribosome is a complex molecular machine that, in order to synthesize proteins, has to decode mRNAs by pairing their codons with matching tRNAs. Decoding is a major determinant of fitness and r....

Kai Papenfort, Yan Sun, Masatoshi Miyakoshi, Carin K. Vanderpool, Jörg Vogel. Glucose homeostasis is strictly controlled in all domains of life. Bacteria that are unable to balance intracellular sugar levels and deal with potentially toxic phosphosugars cease growth and ris....

Nature advance online publication 17 April 2013. doi:10.1038/nature12119

Authors: Hao-Ming Chang, Robinson Triboulet, James E. Thornton & Richard I. Gregory

The pluripotency factor Lin28 blocks the expression of let-7 microRNAs in undifferentiated cells during development, and functions as an oncogene in a subset of cancers. Lin28 binds to let-7 precursor (pre-let-7) RNAs and recruits 3′ terminal uridylyl transferases to selectively inhibit let-7 biogenesis. Uridylated pre-let-7 is refractory to processing by Dicer, and is rapidly degraded by an unknown RNase. Here we identify Dis3l2 as the 3′–5′ exonuclease responsible for the decay of uridylated pre-let-7 in mouse embryonic stem cells. Biochemical reconstitution assays show that 3′ oligouridylation stimulates Dis3l2 activity in vitro, and knockdown of Dis3l2 in mouse embryonic stem cells leads to the stabilization of pre-let-7. Our study establishes 3′ oligouridylation as an RNA decay signal for Dis3l2, and identifies the first physiological RNA substrate of this new exonuclease, which is mutated in the Perlman syndrome of fetal overgrowth and causes a predisposition to Wilms’ tumour development.

by Nancy Maizels, Lucas T. Gray

Recent experiments provide fascinating examples of how G4 DNA and G4 RNA structures—aka quadruplexes—may contribute to normal biology and to genomic pathologies. Quadruplexes are transient and therefore difficult to identify directly in living cells, which initially caused skepticism regarding not only their biological relevance but even their existence. There is now compelling evidence for functions of some G4 motifs and the corresponding quadruplexes in essential processes, including initiation of DNA replication, telomere maintenance, regulated recombination in immune evasion and the immune response, control of gene expression, and genetic and epigenetic instability. Recognition and resolution of quadruplex structures is therefore an essential component of genome biology. We propose that G4 motifs and structures that participate in key processes compose the G4 genome, analogous to the transcriptome, proteome, or metabolome. This is a new view of the genome, which sees DNA as not only a simple alphabet but also a more complex geography. The challenge for the future is to systematically identify the G4 motifs that form quadruplexes in living cells and the features that confer on specific G4 motifs the ability to function as structural elements.

Integration of Genome-wide Approaches Identifies lncRNAs of Adult Neural Stem Cells and Their Progeny In Vivo.

Cell Stem Cell. 2013 Apr 10;

Authors: Ramos AD, Diaz A, Nellore A, Delgado RN, Park KY, Gonzales-Roybal G, Oldham MC, Song JS, Lim DA

Abstract
Long noncoding RNAs (lncRNAs) have been described in cell lines and various whole tissues, but lncRNA analysis of development in vivo is limited. Here, we comprehensively analyze lncRNA expression for the adult mouse subventricular zone neural stem cell lineage. We utilize complementary genome-wide techniques including RNA-seq, RNA CaptureSeq, and ChIP-seq to associate specific lncRNAs with neural cell types, developmental processes, and human disease states. By integrating data from chromatin state maps, custom microarrays, and FACS purification of the subventricular zone lineage, we stringently identify lncRNAs with potential roles in adult neurogenesis. shRNA-mediated knockdown of two such lncRNAs, Six3os and Dlx1as, indicate roles for lncRNAs in the glial-neuronal lineage specification of multipotent adult stem cells. Our data and workflow thus provide a uniquely coherent in vivo lncRNA analysis and form the foundation of a user-friendly online resource for the study of lncRNAs in development and disease.

PMID: 23583100 [PubMed - as supplied by publisher]

CLIPing the brain: Studies of protein-RNA interactions important for neurodegenerative disorders.

Mol Cell Neurosci. 2013 Apr 10;

Authors: Modic M, Ule J, Sibley CR

Abstract
The fate of an mRNA is largely determined by its interactions with RNA binding proteins (RBPs). Post-transcriptional processing, RNA stability, localisation and translation are some of the events regulated by the plethora of RBPs present within cells. Mutations in various RBPs cause several diseases of the central nervous system, including frontotemporal lobar degeneration, amyotrophic lateral sclerosis and fragile X syndrome. Here we review the studies that integrated UV-induced cross-linked immunoprecipitation (CLIP) with other genome-wide methods to comprehensively characterise the function of diverse RBPs in the brain. We discuss the technical challenges of these studies, and review the strategies that can be used to reliably identify the RNAs bound and regulated by an RBP. We conclude by highlighting how CLIP and related techniques have been instrumental in addressing the role of RBPs in neurologic diseases.

PMID: 23583633 [PubMed - as supplied by publisher]

Retinoic acid inducible gene I (RIG-I) senses viral RNAs and triggers innate antiviral responses through induction of type I IFNs and inflammatory cytokines. However, whether RIG-I interacts with host cellular RNA remains undetermined. Here we report that Rig-I interacts with multiple cellular mRNAs, especially Nf-κb1. Rig-I is required for NF-κB...

Transcription steps are marked by different modifications of the C-terminal domain of RNA polymerase II (RNAPII). Phosphorylation of Ser5 and Ser7 by cyclin-dependent kinase 7 (CDK7) as part of TFIIH marks initiation, whereas phosphorylation of Ser2 by CDK9 marks elongation. These processes are thought to take place in localized transcription foci in the nucleus, known as "transcription factories," but it has been argued that the observed clusters/foci are mere fixation or labeling artifacts. We show that transcription factories exist in living cells as distinct foci by live-imaging fluorescently labeled CDK9, a kinase known to associate with active RNAPII. These foci were observed in different cell types derived from CDK9-mCherry knock-in mice. We show that these foci are very stable while highly dynamic in exchanging CDK9. Chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) data show that the genome-wide binding sites of CDK9 and initiating RNAPII overlap on transcribed genes. Immunostaining shows that CDK9-mCherry foci colocalize with RNAPII-Ser5P, much less with RNAPII-Ser2P, and not with CDK12 (a kinase reported to be involved in the Ser2 phosphorylation) or with splicing factor SC35. In conclusion, transcription factories exist in living cells, and initiation and elongation of transcripts takes place in different nuclear compartments.

Nature Structural & Molecular Biology 20, 440 (2013). doi:10.1038/nsmb.2516

Authors: Per Johnsson, Amanda Ackley, Linda Vidarsdottir, Weng-Onn Lui, Martin Corcoran, Dan Grandér & Kevin V Morris

Increasing attention has focused on the significance of RNA in sperm, in light of its contribution to the birth and long-term health of a child, role in sperm function and diagnostic potential. As the composition of sperm RNA is in flux, assigning specific roles to individual RNAs presents a significant challenge. For the first time RNA-seq was used to characterize the population of coding and non-coding transcripts in human sperm. Examining RNA representation as a function of multiple methods of library preparation revealed unique features indicative of very specific and stage-dependent maturation and regulation of sperm RNA, illuminating their various transitional roles. Correlation of sperm transcript abundance with epigenetic marks suggested roles for these elements in the pre- and post-fertilization genome. Several classes of non-coding RNAs including lncRNAs, CARs, pri-miRNAs, novel elements and mRNAs have been identified which, based on factors including relative abundance, integrity in sperm, available knockout data of embryonic effect and presence or absence in the unfertilized human oocyte, are likely to be essential male factors critical to early post-fertilization development. The diverse and unique attributes of sperm transcripts that were revealed provides the first detailed analysis of the biology and anticipated clinical significance of spermatozoal RNAs.

Loop-miRs: active microRNAs generated from single-stranded loop regions.

Nucleic Acids Res. 2013 Apr 10;

Authors: Winter J, Link S, Witzigmann D, Hildenbrand C, Previti C, Diederichs S

Abstract
MicroRNAs (miRNAs) are key mediators of post-transcriptional gene regulation. The miRNA precursors are processed by the endonucleases Drosha and Dicer into a duplex, bound to an Argonaute protein and unwound into two single-stranded miRNAs. Although alternative ways to generate miRNAs have been discovered, e.g. pre-miRNA cleavage by Ago2 or cleavage products of snoRNAs or tRNAs, all known pathways converge on a double-stranded RNA duplex. Exogenous single-stranded siRNAs (ss-siRNAs) can elicit an effective RNA interference reaction; recent studies have identified chemical modifications increasing their stability and activity. Here, we provide first evidence that endogenous, unmodified, single-stranded RNA sequences are generated from single-stranded loop regions of human pre-miRNA hairpins, the so called loop-miRs. Luciferase assays and immunoprecipitation validate loop-miR activity and incorporation into RNA-induced silencing complexes. This study identifies endogenous miRNAs that are generated from single-stranded regions; hence, it provides evidence that precursor-miRNAs can give rise to three distinct endogenous miRNAs: the guide strand, the passenger strand and the loop-miR.

PMID: 23580554 [PubMed - as supplied by publisher]

RNA chaperones promote assembly of complex RNA structures by binding to and “neutralizing” guanosine bases.

Authors: Jacob K. Grohman, Robert J. Gorelick, Colin R. Lickwar, Jason D. Lieb, Brian D. Bower, Brent M. Znosko, Kevin M. Weeks

Background: Perfectly formed duplex elements in RNA occur within folding units, often as a part of hairpin motifs which can be reliably predicted by various RNA folding algorithms. Double helices with consecutive Watson-Crick base-pairing may also be formed between distant RNA segments thereby facilitating long-range interactions of long-chain RNA that may be biologically functional. Here we addressed the potential formation of RNA duplex motifs by long-range RNA-RNA interactions of distantly located matching sequence elements of a single long-chain RNA. Results: We generated a Python-based software tool that identifies consecutive RNA duplex elements at any given length and nucleotide content formed by distant sequences. The software tool, dubbed RNAslider, is built on the theoretical RNA structure prediction algorithm Mfold. Source code and sample data sets are available on demand. We found that a small ratio of human genes including the Argonaute (Ago)-like gene family encode mRNAs containing highly GC-rich non-hairpin duplex elements (GC-helix) of equal to or more than 8 base pairs in length and we provide experimental evidence for their biological significance. Conclusion: GC-helices are observed preferentially within the 5′-region of mRNAs in an evolutionarily conserved fashion indicating their potential biological role. This view is supported experimentally by post-transcriptional regulation of gene expression of a fusion transcript containing 5′-sequences of human mRNAAgo2 harbouring GC-helices and down-stream coding sequences of Renilla luciferase.