Hyeshik Chang

Nature Methods 10, 1139 (2013). doi:10.1038/nmeth.2738

Authors: Martin Krzywinski & Naomi Altman

The ability to detect experimental effects is undermined in studies that lack power.

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In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features.

Nature. 2013 Nov 24;

Authors: Ding Y, Tang Y, Kwok CK, Zhang Y, Bevilacqua PC, Assmann SM

Abstract
RNA structure has critical roles in processes ranging from ligand sensing to the regulation of translation, polyadenylation and splicing. However, a lack of genome-wide in vivo RNA structural data has limited our understanding of how RNA structure regulates gene expression in living cells. Here we present a high-throughput, genome-wide in vivo RNA structure probing method, structure-seq, in which dimethyl sulphate methylation of unprotected adenines and cytosines is identified by next-generation sequencing. Application of this method to Arabidopsis thaliana seedlings yielded the first in vivo genome-wide RNA structure map at nucleotide resolution for any organism, with quantitative structural information across more than 10,000 transcripts. Our analysis reveals a three-nucleotide periodic repeat pattern in the structure of coding regions, as well as a less-structured region immediately upstream of the start codon, and shows that these features are strongly correlated with translation efficiency. We also find patterns of strong and weak secondary structure at sites of alternative polyadenylation, as well as strong secondary structure at 5' splice sites that correlates with unspliced events. Notably, in vivo structures of messenger RNAs annotated for stress responses are poorly predicted in silico, whereas mRNA structures of genes related to cell function maintenance are well predicted. Global comparison of several structural features between these two categories shows that the mRNAs associated with stress responses tend to have more single-strandedness, longer maximal loop length and higher free energy per nucleotide, features that may allow these RNAs to undergo conformational changes in response to environmental conditions. Structure-seq allows the RNA structurome and its biological roles to be interrogated on a genome-wide scale and should be applicable to any organism.

PMID: 24270811 [PubMed - as supplied by publisher]

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Computational Methods for MicroRNA Target Prediction.

Methods Mol Biol. 2014;1107:207-21

Authors: Hamzeiy H, Allmer J, Yousef M

Abstract
MicroRNAs (miRNAs) are important players in gene regulation. The final and maybe the most important step in their regulatory pathway is the targeting. Targeting is the binding of the miRNA to the mature RNA via the RNA-induced silencing complex. Expression patterns of miRNAs are highly specific in respect to external stimuli, developmental stage, or tissue. This is used to diagnose diseases such as cancer in which the expression levels of miRNAs are known to change considerably. Newly identified miRNAs are increasing in number with every new release of miRBase which is the main online database providing miRNA sequences and annotation. Many of these newly identified miRNAs do not yet have identified targets. This is especially the case in animals where the miRNA does not bind to its target as perfectly as it does in plants. Valid targets need to be identified for miRNAs in order to properly understand their role in cellular pathways. Experimental methods for target validations are difficult, expensive, and time consuming. Having considered all these facts it is of crucial importance to have accurate computational miRNA target predictions. There are many proposed methods and algorithms available for predicting targets for miRNAs, but only a few have been developed to become available as independent tools and software. There are also databases which collect and store information regarding predicted miRNA targets. Current approaches to miRNA target prediction produce a huge amount of false positive and an unknown amount of false negative results, and thus the need for better approaches is evermore evident. This chapter aims to give some detail about the current tools and approaches used for miRNA target prediction, provides some grounds for their comparison, and outlines a possible future.

PMID: 24272439 [PubMed - in process]

Hiro-oki Iwakawa, Yukihide Tomari. microRNAs (miRNAs) bind Argonaute proteins in order to form RNA-induced silencing complexes (RISCs) that can silence the expression of complementary mRNAs. Plant miRNAs can mediate the cleavage of....

Schraga Schwartz, Sudeep D. Agarwala, Maxwell R. Mumbach, Marko Jovanovic, Philipp Mertins, Alexander Shishkin, Yuval Tabach, Tarjei S. Mikkelsen, Rahul Satija, Gary Ruvkun, Steven A. Carr, Eric S. Lander, Gerald R. Fink, Aviv Regev. N⁶-methyladenosine (m⁶A) is the most ubiquitous mRNA base modification, but little is known about its precise location, temporal dynamics, and regulation. Here, we gen....

Martin S. Taylor, John LaCava, Paolo Mita, Kelly R. Molloy, Cheng Ran Lisa Huang, Donghui Li, Emily M. Adney, Hua Jiang, Kathleen H. Burns, Brian T. Chait, Michael P. Rout, Jef D. Boeke, Lixin Dai. LINE-1s are active human DNA parasites that are agents of genome dynamics in evolution and disease. These streamlined elements require host factors to complete their life cycles, whereas hosts hav....

Ilmin Kwon, Masato Kato, Siheng Xiang, Leeju Wu, Pano Theodoropoulos, Hamid Mirzaei, Tina Han, Shanhai Xie, Jeffry L. Corden, Steven L. McKnight. The low-complexity (LC) domains of the products of the fused in sarcoma (FUS), Ewings sarcoma (EWS), and TAF15 genes are translocated onto a variety of different DNA-binding domains and thereby as....

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RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let-7 miRNA pathways in Caenorhabditis elegans.

Open Biol. 2013;3(11):130151

Authors: Akay A, Craig A, Lehrbach N, Larance M, Pourkarimi E, Wright JE, Lamond A, Miska E, Gartner A

Abstract
Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1, two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways.

PMID: 24258276 [PubMed - as supplied by publisher]

Alu elements: at the crossroads between disease and evolution.

Biochem Soc Trans. 2013 Dec 1;41(6):1532-1535

Authors: Ule J

Abstract
The cost of DNA sequencing is decreasing year by year, and the era of personalized medicine and the $1000 genome seems to be just around the corner. In order to link genetic variation to gene function, however, we need to learn more about the function of the non-coding genomic elements. The advance of high-throughput sequencing enabled rapid progress in mapping the functional elements in our genome. In the present article, I discuss how intronic mutations acting at Alu elements enable formation of new exons. I review the mutations that cause disease when promoting a major increase in the inclusion of Alu exon into mature transcripts. Moreover, I present the mechanism that represses such a major inclusion of Alu exons and instead enables a gradual evolution of Alu elements into new exons.

PMID: 24256249 [PubMed - as supplied by publisher]

Background: In eukaryotic organisms, gene expression is regulated at multiple levels during the processes of transcription and translation. The absence of a tight regulatory network for transcription in the human malaria parasite suggests that gene expression may largely be controlled at post-transcriptional and translational levels. Results: In this study, we compare steady-state mRNA and polysome-associated mRNA levels of Plasmodium falciparum at different time points during its asexual cell cycle. For more than 30% of its genes, we observe a delay in peak transcript abundance in the polysomal fraction as compared to the steady-state mRNA fraction, suggestive of strong translational control. Our data show that key regulatory mechanisms could include inhibitory activity of upstream open reading frames and translational repression of the major virulence gene family by intronic transcripts. In addition, we observe polysomal mRNA-specific alternative splicing events and widespread transcription of non-coding transcripts. Conclusions: These different layers of translational regulation are likely to contribute to a complex network that controls gene expression in this eukaryotic pathogen. Disrupting the mechanisms involved in such translational control could provide novel anti-malarial strategies.

A report on the EMBO/EMBL Symposium on The Non-Coding Genome, held in Heidelberg, Germany, 9-12 October, 2013.

Nature advance online publication 24 November 2013. doi:10.1038/nature12756

Authors: Yiliang Ding, Yin Tang, Chun Kit Kwok, Yu Zhang, Philip C. Bevilacqua & Sarah M. Assmann

RNA structure has critical roles in processes ranging from ligand sensing to the regulation of translation, polyadenylation and splicing. However, a lack of genome-wide in vivo RNA structural data has limited our understanding of how RNA structure regulates gene expression in living cells. Here we present a high-throughput, genome-wide in vivo RNA structure probing method, structure-seq, in which dimethyl sulphate methylation of unprotected adenines and cytosines is identified by next-generation sequencing. Application of this method to Arabidopsis thaliana seedlings yielded the first in vivo genome-wide RNA structure map at nucleotide resolution for any organism, with quantitative structural information across more than 10,000 transcripts. Our analysis reveals a three-nucleotide periodic repeat pattern in the structure of coding regions, as well as a less-structured region immediately upstream of the start codon, and shows that these features are strongly correlated with translation efficiency. We also find patterns of strong and weak secondary structure at sites of alternative polyadenylation, as well as strong secondary structure at 5′ splice sites that correlates with unspliced events. Notably, in vivo structures of messenger RNAs annotated for stress responses are poorly predicted in silico, whereas mRNA structures of genes related to cell function maintenance are well predicted. Global comparison of several structural features between these two categories shows that the mRNAs associated with stress responses tend to have more single-strandedness, longer maximal loop length and higher free energy per nucleotide, features that may allow these RNAs to undergo conformational changes in response to environmental conditions. Structure-seq allows the RNA structurome and its biological roles to be interrogated on a genome-wide scale and should be applicable to any organism.

Long double-stranded RNA may undergo hyper-editing by adenosine deaminases that act on RNA (ADARs), where up to 50% of adenosine residues may be converted to inosine. However, although numerous RNAs may undergo hyper-editing, the role for inosine-containing hyper-edited double-stranded RNA in cells is poorly understood. Nevertheless, editing plays a critical role in mammalian cells, as highlighted by the analysis of ADAR-null mutants. In particular, the long form of ADAR1 (ADAR1^p150) is essential for viability. Moreover, a number of studies have implicated ADAR1^p150 in various stress pathways. We have previously shown that ADAR1^p150 localized to cytoplasmic stress granules in HeLa cells following either oxidative or interferon-induced stress. Here, we show that the Z-DNA-binding domain (Zα^ADAR1) exclusively found in ADAR1^p150 is required for its localization to stress granules. Moreover, we show that fusion of Zα^ADAR1 to either green fluorescent protein (GFP) or polypyrimidine binding protein 4 (PTB4) also results in their localization to stress granules. We additionally show that the Zα domain from other Z-DNA-binding proteins (ZBP1, E3L) is likewise sufficient for localization to stress granules. Finally, we show that Z-RNA or Z-DNA binding is important for stress granule localization. We have thus identified a novel role for Z-DNA-binding domains in mammalian cells.

Navneet Momi, Sukhwinder Kaur, Satyanarayana Rachagani, Apar K. Ganti, Surinder K. Batra.

Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition

Nature 503, 7476 (2013). doi:10.1038/nature12632

Authors: Miler T. Lee, Ashley R. Bonneau, Carter M. Takacs, Ariel A. Bazzini, Kate R. DiVito, Elizabeth S. Fleming & Antonio J. Giraldez

After fertilization, maternal factors direct development and trigger zygotic genome activation (ZGA) at the maternal-to-zygotic transition (MZT). In zebrafish, ZGA is required for gastrulation and clearance of maternal messenger RNAs, which is in part regulated by the conserved microRNA miR-430. However, the factors that activate

Oded Rechavi.

Nature advance online publication 17 November 2013. doi:10.1038/nature12803

Authors: Lijun Zhou, Jing Hang, Yulin Zhou, Ruixue Wan, Guifeng Lu, Ping Yin, Chuangye Yan & Yigong Shi

Splicing of precursor messenger RNA (pre-mRNA) in eukaryotic cells is carried out by the spliceosome, which consists of five small nuclear ribonucleoproteins (snRNPs) and a number of accessory factors and enzymes. Each snRNP contains a ring-shaped subcomplex of seven proteins and a specific RNA molecule. The U6 snRNP contains a unique heptameric Lsm protein complex, which specifically recognizes the U6 small nuclear RNA at its 3′ end. Here we report the crystal structures of the heptameric Lsm complex, both by itself and in complex with a 3′ fragment of U6 snRNA, at 2.8 Å resolution. Each of the seven Lsm proteins interacts with two neighbouring Lsm components to form a doughnut-shaped assembly, with the order Lsm3–2–8–4–7–5–6. The four uridine nucleotides at the 3′ end of U6 snRNA are modularly recognized by Lsm3, Lsm2, Lsm8 and Lsm4, with the uracil base specificity conferred by a highly conserved asparagine residue. The uracil base at the extreme 3′ end is sandwiched by His 36 and Arg 69 from Lsm3, through π–π and cation–π interactions, respectively. The distinctive end-recognition of U6 snRNA by the Lsm complex contrasts with RNA binding by the Sm complex in the other snRNPs. The structural features and associated biochemical analyses deepen mechanistic understanding of the U6 snRNP function in pre-mRNA splicing.

It is well established that protein synthesis occurs with high fidelity. This paper describes a sensitive system to analyze the frequency of all possible missense errors by several tRNAs. The only errors that occur with appreciable frequency are observed when codon–anticodon pairing involves noncanonical base-pairing with geometries similar to canonical Watson–Crick base pairs.

Means to an end: mechanisms of alternative polyadenylation of messenger RNA precursors.

Wiley Interdiscip Rev RNA. 2013 Nov 14;

Authors: Gruber AR, Martin G, Keller W, Zavolan M

Abstract
Expression of mature messenger RNAs (mRNAs) requires appropriate transcription initiation and termination, as well as pre-mRNA processing by capping, splicing, cleavage, and polyadenylation. A core 3'-end processing complex carries out the cleavage and polyadenylation reactions, but many proteins have been implicated in the selection of polyadenylation sites among the multiple alternatives that eukaryotic genes typically have. In recent years, high-throughput approaches to map both the 3'-end processing sites as well as the binding sites of proteins that are involved in the selection of cleavage sites and in the processing reactions have been developed. Here, we review these approaches as well as the insights into the mechanisms of polyadenylation that emerged from genome-wide studies of polyadenylation across a range of cell types and states. Conflict of interest: The authors have declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website.

PMID: 24243805 [PubMed - as supplied by publisher]

Phosphorylation of DGCR8 Increases Its Intracellular Stability and Induces a Progrowth miRNA Profile.

Cell Rep. 2013 Nov 13;

Authors: Herbert KM, Pimienta G, Degregorio SJ, Alexandrov A, Steitz JA

Abstract
During miRNA biogenesis, the microprocessor complex (MC), which is composed minimally of Drosha, an RNase III enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary miRNA (pri-miRNA) in order to release the pre-miRNA stem-loop structure. Using phosphoproteomics, we mapped 23 phosphorylation sites on full-length human DGCR8 expressed in insect or mammalian cells. DGCR8 can be phosphorylated by mitogenic ERK/MAPK, indicating that DGCR8 phosphorylation may respond to and integrate extracellular cues. The expression of phosphomimetic DGCR8 or inhibition of phosphatases increased the cellular levels of DGCR8 and Drosha proteins. Increased levels of phosphomimetic DGCR8 were not due to higher mRNA levels, altered DGCR8 localization, or DGCR8's ability to self-associate, but rather to an increase in protein stability. MCs incorporating phosphomutant or phosphomimetic DGCR8 were not altered in specific processing activity. However, HeLa cells expressing phosphomimetic DGCR8 exhibited a progrowth miRNA expression profile and increased proliferation and scratch closure rates relative to cells expressing phosphomutant DGCR8.

PMID: 24239349 [PubMed - as supplied by publisher]

Glycosyl groups are an essential mediator of molecular interactions in cells and on cellular surfaces. There are very few methods that directly relate sugar-containing molecules to their biosynthetic machineries. Here, we introduce glycogenomics as an experiment-guided genome-mining approach for fast characterization of glycosylated natural products (GNPs) and their biosynthetic pathways...

Daniel Muñoz-Espín, Marta Cañamero, Antonio Maraver, Gonzalo Gómez-López, Julio Contreras, Silvia Murillo-Cuesta, Alfonso Rodríguez-Baeza, Isabel Varela-Nieto, Jesús Ruberte, Manuel Collado, Manuel Serrano. Cellular senescence disables proliferation in damaged cells, and it is relevant for cancer and aging. Here, we show that senescence occurs during mammalian embryonic development at multiple locati....

The EMBO Journal. doi:10.1038/emboj.2013.245

Authors: Julieta Aprea, Silvia Prenninger, Martina Dori, Tanay Ghosh, Laura Sebastian Monasor, Elke Wessendorf, Sara Zocher, Simone Massalini, Dimitra Alexopoulou, Mathias Lesche, Andreas Dahl, Matthias Groszer, Michael Hiller & Federico Calegari

Development of testes in the mammalian embryo requires the formation and assembly of several cell types that allow these organs to achieve their roles in male reproduction and endocrine regulation. Testis development is unusual in that several cell types such as Sertoli, Leydig, and spermatogonial cells arise from bipotential precursors present in the precursor tissue, the genital ridge. These cell types do not differentiate independently but depend on signals from Sertoli cells that differentiate under the influence of transcription factors SRY and SOX9. While these steps are becoming better understood, the origins and roles of many testicular cell types and structures—including peritubular myoid cells, the tunica albuginea, the arterial and venous blood vasculature, lymphatic vessels, macrophages, and nerve cells—have remained unclear. This review synthesizes current knowledge of how the architecture of the testis unfolds and highlights the questions that remain to be explored, thus providing a roadmap for future studies that may help illuminate the causes of XY disorders of sex development, infertility, and testicular cancers.

Nature Reviews Genetics. doi:10.1038/nrg3594

Authors: Vicent Pelechano & Lars M. Steinmetz

Intertwined pathways for Argonaute-mediated microRNA biogenesis in Drosophila.

Nucleic Acids Res. 2013 Nov 12;

Authors: Yang JS, Smibert P, Westholm JO, Jee D, Maurin T, Lai EC

Abstract
Although Dicer is essential for general microRNA (miRNA) biogenesis, vertebrate mir-451 is Dicer independent. Instead, its short pre-miRNA hairpin is 'sliced' by Ago2, then 3'-resected into mature miRNAs. Here, we show that Drosophila cells and animals generate functional small RNAs from mir-451-type precursors. However, their bulk maturation arrests as Ago-cleaved pre-miRNAs, which mostly associate with the RNAi effector AGO2. Routing of pre-mir-451 hairpins to the miRNA effector AGO1 was inhibited by Dicer-1 and its partner Loqs. Loss of these miRNA factors promoted association of pre-mir-451 with AGO1, which sliced them and permitted maturation into ∼23-26 nt products. The difference was due to the 3' modification of single-stranded species in AGO2 by Hen1 methyltransferase, whose depletion permitted 3' trimming of Ago-cleaved pre-miRNAs in AGO2. Surprisingly, Nibbler, a 3'-5' exoribonuclease that trims 'long' mature miRNAs in AGO1, antagonized miR-451 processing. We used an in vitro reconstitution assay to identify a soluble, EDTA-sensitive activity that resects sliced pre-miRNAs in AGO1 complexes. Finally, we use deep sequencing to show that depletion of dicer-1 increases the diversity of small RNAs in AGO1, including some candidate mir-451-like loci. Altogether, we document unexpected aspects of miRNA biogenesis and Ago sorting, and provide insights into maturation of Argonaute-cleaved miRNA substrates.

PMID: 24220090 [PubMed - as supplied by publisher]

Acetylation of p53 stimulates miRNA processing and determines cell survival following genotoxic stress.

EMBO J. 2013 Nov 12;

Authors: Chang J, Davis-Dusenbery BN, Kashima R, Jiang X, Marathe N, Sessa R, Louie J, Gu W, Lagna G, Hata A

Abstract
It is widely accepted that different forms of stress activate a common target, p53, yet different outcomes are triggered in a stress-specific manner. For example, activation of p53 by genotoxic agents, such as camptothecin (CPT), triggers apoptosis, while non-genotoxic activation of p53 by Nutlin-3 (Nut3) leads to cell-cycle arrest without significant apoptosis. Such stimulus-specific responses are attributed to differential transcriptional activation of various promoters by p53. In this study, we demonstrate that CPT, but not Nut3, induces miR-203, which downregulates anti-apoptotic bcl-w and promotes cell death in a p53-dependent manner. We find that acetylation of K120 in the DNA-binding domain of p53 augments its association with the Drosha microprocessor and promotes nuclear primary miRNA processing. Knockdown of human orthologue of Males absent On the First (hMOF), the acetyltransferase that targets K120 in p53, abolishes induction of miR-203 and cell death mediated by CPT. Thus, this study reveals that p53 acetylation at K120 plays a critical role in the regulation of the Drosha microprocessor and that post-transcriptional regulation of gene expression by p53 via miRNAs plays a role in determining stress-specific cellular outcomes.

PMID: 24219989 [PubMed - as supplied by publisher]

by Judith A. Blake

Nature Chemical Biology 9, 754 (2013). doi:10.1038/nchembio.1399

Author: Mirella Bucci

Nature Chemical Biology 9, 754 (2013). doi:10.1038/nchembio.1397

Author: Terry L. Sheppard