Hyeshik Chang

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Poly(A) RNA-binding proteins and polyadenosine RNA: new members and novel functions.

Wiley Interdiscip Rev RNA. 2014 Apr 30;

Authors: Wigington CP, Williams KR, Meers MP, Bassell GJ, Corbett AH

Abstract
Poly(A) RNA-binding proteins (Pabs) bind with high affinity and specificity to polyadenosine RNA. Textbook models show a nuclear Pab, PABPN1, and a cytoplasmic Pab, PABPC, where the nuclear PABPN1 modulates poly(A) tail length and the cytoplasmic PABPC stabilizes poly(A) RNA in the cytoplasm and also enhances translation. While these conventional roles are critically important, the Pab family has expanded recently both in number and in function. A number of novel roles have emerged for both PAPBPN1 and PABPC that contribute to the fine-tuning of gene expression. Furthermore, as the characterization of the nucleic acid binding properties of RNA-binding proteins advances, additional proteins that show high affinity and specificity for polyadenosine RNA are being discovered. With this expansion of the Pab family comes a concomitant increase in the potential for Pabs to modulate gene expression. Further complication comes from an expansion of the potential binding sites for Pab proteins as revealed by an analysis of templated polyadenosine stretches present within the transcriptome. Thus, Pabs could influence mRNA fate and function not only by binding to the nontemplated poly(A) tail but also to internal stretches of adenosine. Understanding the diverse functions of Pab proteins is not only critical to understand how gene expression is regulated but also to understand the molecular basis for tissue-specific diseases that occur when Pab proteins are altered. Here we describe both conventional and recently emerged functions for PABPN1 and PABPC and then introduce and discuss three new Pab family members, ZC3H14, hnRNP-Q1, and LARP4. For further resources related to this article, please visit the WIREs website. Conflict of interest: The authors have declared no conflicts of interest for this article.

PMID: 24789627 [PubMed - as supplied by publisher]

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Mechanisms of genome instability induced by RNA-processing defects.

Trends Genet. 2014 Jun;30(6):245-53

Authors: Chan YA, Hieter P, Stirling PC

Abstract
The role of normal transcription and RNA processing in maintaining genome integrity is becoming increasingly appreciated in organisms ranging from bacteria to humans. Several mutations in RNA biogenesis factors have been implicated in human cancers, but the mechanisms and potential connections to tumor genome instability are not clear. Here, we discuss how RNA-processing defects could destabilize genomes through mutagenic R-loop structures and by altering expression of genes required for genome stability. A compelling body of evidence now suggests that researchers should be directly testing these mechanisms in models of human cancer.

PMID: 24794811 [PubMed - indexed for MEDLINE]

Nature Protocols 9, 1282 (2014). doi:10.1038/nprot.2014.085

Authors: Myriam Heiman, Ruth Kulicke, Robert J Fenster, Paul Greengard & Nathaniel Heintz

Cellular diversity and architectural complexity create barriers to understanding the function of the mammalian CNS at a molecular level. To address this problem, we have recently developed a methodology that provides the ability to profile the entire translated mRNA complement of any genetically defined cell

Nature Biotechnology 32, 453 (2014). doi:10.1038/nbt.2890

Authors: William R Jeck & Norman E Sharpless

Publication date: July 2014
Source:Trends in Cell Biology, Volume 24, Issue 7
Author(s): Xiuju Li , Qinglian Wang , Yongsheng Liu

Publication date: 8 May 2014
Source:Cell, Volume 157, Issue 4

As part of Cell’s 40th anniversary celebration, we are spotlighting 40 principal investigators under the age of 40. Among the questions we posed to this group was, “What is the biggest challenge facing young scientists?” A sampling of their responses appears below. But to see the full profiles of all 40 scientists, including their responses to this and other questions, please visit http://www.cell.com/40/under40.

It is widely assumed, if not definitively proved, that miRNAs exert their biological functions only when associated with an Argonaute protein. Many investigators have used 3'-biotin-tagged miRNAs to determine target specificity via affinity pulldowns. This short paper shows that a specific 3'-biotin-tagged miRNA does not efficiently associate with Argonaute proteins, a result indicating that 3'-biotin-tagging may not be a reliable way of determining target specificity.

Utilization of antiviral small interfering RNAs is thought to be largely restricted to plants, nematodes, and arthropods. In an effort to determine whether a physiological interplay exists between the host small RNA machinery and the cellular response to virus infection in mammals, we evaluated antiviral activity in the presence and...

MicroRNAs (miRNAs) are small evolutionarily conserved regulatory RNAs that modulate mRNA stability and translation in a wide range of cell types. MiRNAs are involved in a broad array of biological processes, including cellular proliferation, differentiation, and apoptosis. To identify previously unidentified regulators of miRNA, we initiated a systematic discovery-type proteomic...

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Retrotransposon silencing during embryogenesis: dicer cuts in LINE.

PLoS Genet. 2013 Nov;9(11):e1003944

Authors: Faulkner GJ

PMID: 24244199 [PubMed - indexed for MEDLINE]

Drosha protein levels are translationally regulated during Xenopus oocyte maturation.

Mol Biol Cell. 2014 May 14;

Authors: Muggenhumer D, Vesely C, Nimpf S, Tian N, Yongfeng J, Jantsch MF

Abstract
MicroRNAs (miRNAs) are ∼21nt nucleotide long, single-stranded noncoding RNAs that regulate gene expression. Biogenesis of miRNAs is mediated by the two RNase III-like enzymes Drosha and Dicer. Here we study miRNA biogenesis during maturation of Xenopus oocytes to eggs using microinjection of pri-miRNAs. We show that processing of exogenous and endogenous pri-miRNAs is strongly enhanced upon maturation of oocytes to eggs. Overexpression of cloned Xenopus Drosha in oocytes, however, boosts pri-miRNA processing dramatically, indicating that Drosha is a rate-limiting factor in Xenopus oocytes. This developmental regulation of Drosha is controlled by poly-A length addition to the Drosha mRNA that boosts translation upon transition from oocytes to eggs. Processing of pri-miRNAs by Drosha and Dicer has been shown to be affected by adenosine to inosine deamination type RNA-editing. Using activated Xenopus eggs for microinjection experiments we demonstrate that RNA-editing can reduce pri-miRNA processing in vivo. This processing block is determined by the structural but not sequence changes introduced by RNA-editing.

PMID: 24829383 [PubMed - as supplied by publisher]

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Global and Local Competition between Exogenously Introduced microRNAs and Endogenously Expressed microRNAs.

Mol Cells. 2014 May 13;

Authors: Kim D, Kim J, Baek D

Abstract
It has been reported that exogenously introduced micro-RNA (exo-miRNA) competes with endogenously expressed miRNAs (endo-miRNAs) in human cells, resulting in a detectable upregulation of mRNAs with endo-miRNA target sites (TSs). However, the detailed mechanisms of the competition between exo- and endo-miRNAs remain uninvestigated. In this study, using 74 microarrays that monitored the whole-transcriptome response after introducing miRNAs or siRNAs into HeLa cells, we systematically examined the derepression of mRNAs with exoand/ or endo-miRNA TSs. We quantitatively assessed the effect of the number of endo-miRNA TSs on the degree of mRNA derepression. As a result, we observed that the number of endo-miRNA TSs was significantly associated with the degree of derepression, supporting that the derepression resulted from the competition between exo- and endo-miRNAs. However, when we examined whether the site proficiency of exomiRNA TSs could also influence mRNA derepression, to our surprise, we discovered a strong positive correlation. Our analysis indicates that site proficiencies of both exoand endo-miRNA TSs are important determinants for the degree of mRNA derepression, implying that the derepression of mRNAs in response to exo-miRNA is more complex than that currently perceived. Our observations may lead to a more complete understanding of the detailed mechanisms of the competition between exo- and endo-miRNAs and to a more accurate prediction of miRNA targets. Our analysis also suggests an interesting hypothesis that long 3'-UTRs may function as molecular buffer against gene expression regulation by individual miRNAs.

PMID: 24823356 [PubMed - as supplied by publisher]

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RNAbrowse: RNA-Seq De Novo Assembly Results Browser.

PLoS One. 2014;9(5):e96821

Authors: Mariette J, Noirot C, Nabihoudine I, Bardou P, Hoede C, Djari A, Cabau C, Klopp C

Abstract
Transcriptome analysis based on a de novo assembly of next generation RNA sequences is now performed routinely in many laboratories. The generated results, including contig sequences, quantification figures, functional annotations and variation discovery outputs are usually bulky and quite diverse. This article presents a user oriented storage and visualisation environment permitting to explore the data in a top-down manner, going from general graphical views to all possible details. The software package is based on biomart, easy to install and populate with local data. The software package is available under the GNU General Public License (GPL) at http://bioinfo.genotoul.fr/RNAbrowse.

PMID: 24823498 [PubMed - in process]

Although transcriptional elongation by RNA polymerase II is coupled with many RNA-related processes, genomewide elongation rates remain unknown. We describe a method, called 4sUDRB-seq, based on reversible inhibition of transcription elongation coupled with tagging newly transcribed RNA with 4-thiouridine and high throughput sequencing to measure simultaneously with high confidence genome-wide transcription elongation rates in cells. We find that most genes are transcribed at about 3.5 kb/min, with elongation rates varying between 2 kb/min - 6 kb/min. 4sUDRB-seq can facilitate genomewide exploration of the involvement of specific elongation factors in transcription and the contribution of deregulated transcription elongation to various pathologies.

Unlike short interfering RNAs (siRNAs), which are commonly designed to repress a single messenger RNA (mRNA) target through perfect base pairing, microRNAs (miRNAs) are endogenous small RNAs that have evolved to concurrently repress multiple mRNA targets through imperfect complementarity. MicroRNA target recognition is primarily determined by pairing of the miRNA seed sequence (nucleotides 2–8) to complementary match sites in each mRNA target. Whereas siRNA technology is well established for single target knockdown, the design of artificial miRNAs for multi-target repression is largely unexplored. We designed and functionally analysed over 200 artificial miRNAs for simultaneous repression of pyruvate carboxylase and glutaminase by selecting all seed matches shared by their 3' untranslated regions. Although we identified multiple miRNAs that repressed endogenous protein expression of both genes, seed-based artificial miRNA design was highly inefficient, as the majority of miRNAs with even perfect seed matches did not repress either target. Moreover, commonly used target prediction programs did not substantially discriminate effective artificial miRNAs from ineffective ones, indicating that current algorithms do not fully capture the features important for artificial miRNA targeting and are not yet sufficient for designing artificial miRNAs. Our analysis suggests that additional factors are strong determinants of the efficacy of miRNA-mediated target repression and remain to be discovered.

Non-coding (nc)RNAs are important structural and regulatory molecules. Accurate determination of the primary sequence and secondary structure of ncRNAs is important for understanding their functions. During cDNA synthesis, RNA 3' end stem-loops can self-prime reverse transcription, creating RNA–cDNA chimeras. We found that chimeric RNA–cDNA fragments can also be detected at 5' end stem-loops, although at much lower frequency. Using the Gubler–Hoffman method, both types of chimeric fragments can be converted to cDNA during library construction, and they are readily detectable in high-throughput RNA sequencing (RNA-seq) experiments. Here, we show that these chimeric reads contain valuable information about the boundaries of ncRNAs. We developed a bioinformatic method, called Vicinal, to precisely map the ends of numerous fruitfly, mouse and human ncRNAs. Using this method, we analyzed chimeric reads from over 100 RNA-seq datasets, the results of which we make available for users to find RNAs of interest. In summary, we show that Vicinal is a useful tool for determination of the precise boundaries of uncharacterized ncRNAs, facilitating further structure/function studies.

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3'UTRs take a long shot in the brain.

Bioessays. 2014 Jan;36(1):39-45

Authors: Wang L, Yi R

Abstract
The fast advancing RNA-seq technology has unveiled an unexpected diversity and expression specificity of 3' untranslated regions (3'UTRs) of mRNAs. In particular, neural mRNAs seem to express significantly longer 3'UTRs, some of which are over 10 kb in length. The extensive elongation of 3'UTRs in neural tissues provides intriguing possibilities for cell type-specific regulations that are governed by miRNAs, RNA-binding proteins and ribonucleoprotein aggregates. In this article, we review recent progress in the characterization of mRNA 3'UTRs and discuss their implications in the understanding of 3'UTR-mediated gene regulation.

PMID: 24115048 [PubMed - indexed for MEDLINE]

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What do you mean by transcription rate?: the conceptual difference between nascent transcription rate and mRNA synthesis rate is essential for the proper understanding of transcriptomic analyses.

Bioessays. 2013 Dec;35(12):1056-62

Authors: Pérez-Ortín JE, Medina DA, Chávez S, Moreno J

Abstract
mRNA synthesis in all organisms is performed by RNA polymerases, which work as nanomachines on DNA templates. The rate at which their product is made is an important parameter in gene expression. Transcription rate encompasses two related, yet different, concepts: the nascent transcription rate, which measures the in situ mRNA production by RNA polymerase, and the rate of synthesis of mature mRNA, which measures the contribution of transcription to the mRNA concentration. Both parameters are useful for molecular biologists, but they are not interchangeable and they are expressed in different units. It is important to distinguish when and where each one should be used. We propose that for functional genomics the use of nascent transcription rates should be restricted to the evaluation of the transcriptional process itself, whereas mature mRNA synthesis rates should be employed to address the transcriptional input to mRNA concentration balance leading to variation of gene expression.

PMID: 24105897 [PubMed - indexed for MEDLINE]

Background: Adipogenesis is a complex process, in which immature pre-adipocytes change morphology, micro-anatomy and physiology to become mature adipocytes. These store and accumulate fat and release diverse hormones. Massive changes in protein content and protein composition of the transforming cell take place within a short time-frame.In a previous study we analyzed changes in the abundance of free and polysomal, i.e. ribosome bound, RNAs in the first hours of adipogenesis in the murine cell line 3 T3-L1. Here we analyze changes of mRNA levels and their potential contribution to the changing protein pool by determination of mRNA levels and ribosome binding to mRNAs in 3 T3-L1 cells stimulated for adipogenesis. We grouped mRNA species into categories with respect to up- or down-regulated transcription and translation and analyzed the groups regarding specific functionalities based on Gene Ontology (GO). Results: A shift towards up-regulation of gene expression in early adipogenesis was detected. Genes up-regulated at the transcriptional (TC:up) and translational (TL:up) level (TC:up/TL:up) are very likely involved in control and logistics of translation. Many of them are known to contain a TOP motif. In the TC:up/TL:unchanged group we detected most of the metal binding proteins and metal transporters. In the TC:unchanged/TL:up group several factors of the olfactory receptor family were identified, while in TC:unchanged/TL:down methylation and repair genes are represented. In the TC:down/TL:up group we detected many signaling factors. The TC:down/TL:unchanged group mainly consists of regulatory factors. Conclusions: Within the first hours of adipogenesis, changes in transcriptional and translational regulation take place. Notably, genes with a specific biological or molecular function tend to cluster in groups according to their transcriptional and translational regulation.

Article

High-resolution structures of large RNAs and protein–RNA complexes are difficult to solve due to inherent structural flexibility and a high risk of crystallization artefacts. Here, Duss et al . present a novel EPR-based approach to aid structure determination of large RNAs and protein–RNA complexes in solution.

Nature Communications doi: 10.1038/ncomms4669

Authors: Olivier Duss, Maxim Yulikov, Gunnar Jeschke, Frédéric H.-T. Allain

MicroRNAs delicately regulate the balance of angiogenesis. Here we show that depletion of all microRNAs suppresses tumor angiogenesis. We generated microRNA-deficient tumors by knocking out Dicer1. These tumors are highly hypoxic but poorly vascularized, suggestive of deficient angiogenesis signaling. Expression profiling revealed that angiogenesis genes were significantly down-regulated as a result of the microRNA deficiency. Factor inhibiting hypoxia-inducible factor 1 (HIF-1), FIH1, is derepressed under these conditions and suppresses HIF transcription. Knocking out FIH1 using CRISPR/Cas9-mediated genome engineering reversed the phenotypes of microRNA-deficient cells in HIF transcriptional activity, VEGF production, tumor hypoxia, and tumor angiogenesis. Using multiplexed CRISPR/Cas9, we deleted regions in FIH1 3' untranslated regions (UTRs) that contain microRNA-binding sites, which derepresses FIH1 protein and represses hypoxia response. These data suggest that microRNAs promote tumor responses to hypoxia and angiogenesis by repressing FIH1.

Uncovering how an RNA-protein molecular scalpel targets DNA will advance our ability to engineer genomes. Author: Rodolphe Barrangou

Publication date: 5 June 2014
Source:Molecular Cell, Volume 54, Issue 5
Author(s): Nicole Lambert , Alex Robertson , Mohini Jangi , Sean McGeary , Phillip A. Sharp , Christopher B. Burge
Specific protein-RNA interactions guide posttranscriptional gene regulation. Here, we describe RNA Bind-n-Seq (RBNS), a method that comprehensively characterizes sequence and structural specificity of RNA binding proteins (RBPs), and its application to the developmental alternative splicing factors RBFOX2, CELF1/CUGBP1, and MBNL1. For each factor, we recovered both canonical motifs and additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in motifs containing UGC and/or GCU. Dissociation constants calculated from RBNS data using a novel algorithm correlated highly with values measured by surface plasmon resonance. Motifs identified by RBNS were conserved, were bound and active in vivo, and distinguished the subset of motifs enriched by CLIP-Seq that had regulatory activity. Together, our data demonstrate that RBNS complements crosslinking-based methods and show that in vivo binding and activity of these splicing factors is driven largely by intrinsic RNA affinity.

Graphical abstract

Teaser

Lambert et al. develop a sequencing-based method to determine the sequence and structural specificity of RNA binding proteins (RBPs). They identify new motifs for three RBPs, detect effects of RNA structure on binding, and distinguish the subset of motifs identified by UV crosslinking that have regulatory activity.

Nature advance online publication 14 May 2014. doi:10.1038/nature13271

Authors: Olivier Duss, Erich Michel, Maxim Yulikov, Mario Schubert, Gunnar Jeschke & Frédéric H.-T. Allain

Nature advance online publication 14 May 2014. doi:10.1038/nature13317

Authors: Vera C. Martins, Katrin Busch, Dilafruz Juraeva, Carmen Blum, Carolin Ludwig, Volker Rasche, Felix Lasitschka, Sergey E. Mastitsky, Benedikt Brors, Thomas Hielscher, Hans Joerg Fehling & Hans-Reimer Rodewald

Nature advance online publication 14 May 2014. doi:10.1038/nature13315

Authors: Takashi Kiuchi, Hikaru Koga, Munetaka Kawamoto, Keisuke Shoji, Hiroki Sakai, Yuji Arai, Genki Ishihara, Shinpei Kawaoka, Sumio Sugano, Toru Shimada, Yutaka Suzuki, Masataka G. Suzuki & Susumu Katsuma

The silkworm Bombyx mori uses a WZ sex determination system that is analogous to the one found in birds and some reptiles. In this system, males have two Z sex chromosomes, whereas females have Z and W sex chromosomes. The silkworm W chromosome has a dominant role in female determination, suggesting the existence of a dominant feminizing gene in this chromosome. However, the W chromosome is almost fully occupied by transposable element sequences, and no functional protein-coding gene has been identified so far. Female-enriched PIWI-interacting RNAs (piRNAs) are the only known transcripts that are produced from the sex-determining region of the W chromosome, but the function(s) of these piRNAs are unknown. Here we show that a W-chromosome-derived, female-specific piRNA is the feminizing factor of B. mori. This piRNA is produced from a piRNA precursor which we named Fem. Fem sequences were arranged in tandem in the sex-determining region of the W chromosome. Inhibition of Fem-derived piRNA-mediated signalling in female embryos led to the production of the male-specific splice variants of B. mori doublesex (Bmdsx), a gene which acts at the downstream end of the sex differentiation cascade. A target gene of Fem-derived piRNA was identified on the Z chromosome of B. mori. This gene, which we named Masc, encoded a CCCH-type zinc finger protein. We show that the silencing of Masc messenger RNA by Fem piRNA is required for the production of female-specific isoforms of Bmdsx in female embryos, and that Masc protein controls both dosage compensation and masculinization in male embryos. Our study characterizes a single small RNA that is responsible for primary sex determination in the WZ sex determination system.

Nature advance online publication 14 May 2014. doi:10.1038/nature13336

Author: František Marec

Sex determination in the silkworm Bombyx mori has been found to depend on the presence or absence of a small RNA. This is thought to be the first example of a molecule other than a protein mediating this process.

Nature advance online publication 07 May 2014. doi:10.1038/nature13318

Authors: Deepankar Pratap Singh, Baptiste Saudemont, Gérard Guglielmi, Olivier Arnaiz, Jean-François Goût, Malgorzata Prajer, Alexey Potekhin, Ewa Przybòs, Anne Aubusson-Fleury, Simran Bhullar, Khaled Bouhouche, Maoussi Lhuillier-Akakpo, Véronique Tanty, Corinne Blugeon, Adriana Alberti, Karine Labadie, Jean-Marc Aury, Linda Sperling, Sandra Duharcourt & Eric Meyer

Motivation: MicroRNAs (miRNAs) are small non-coding RNAs that are extensively involved in gene expression regulation. One major roadblock in functional miRNA studies is the reliable prediction of genes targeted by miRNAs, as rules defining miRNA target recognition have not been well-established to date. Availability of high-throughput experimental data from a recent CLASH (cross linking, ligation and sequencing of hybrids) study has presented an unprecedented opportunity to characterize miRNA target recognition patterns, which may provide guidance for improved miRNA target prediction.

Results: The CLASH data were analysed to identify distinctive sequence features that characterize canonical and non-canonical miRNA target types. Most miRNA targets were of non-canonical type, i.e. without involving perfect pairing to canonical miRNA seed region. Different miRNAs have distinct targeting patterns, and this miRNA-to-miRNA variability was associated with seed sequence composition. Specifically, seed-based canonical target recognition was dependent on the GC content of the miRNA seed. For miRNAs with low GC content of the seed region, non-canonical targeting was the dominant mechanism for target recognition. In contrast to canonical targeting, non-canonical targeting did not lead to significant target downregulation at either the RNA or protein level.

Contact: xwang@radonc.wustl.edu

The human RNA-editing enzyme adenosine deaminase acting on RNA (ADAR1) carries a unique nuclear localization signal (NLS) that overlaps one of its double-stranded RNA-binding domains (dsRBDs). This dsRBD-NLS is recognized by the nuclear import receptor transportin 1 (Trn1; also called karyopherin-β2) in an RNA-sensitive manner. Most Trn1 cargos bear a...