Hyeshik Chang

Staufen1 (Stau1) is a ribonucleic acid (RNA)-binding protein involved in the post-transcriptional regulation of gene expression. Recent studies indicate that Stau1-bound messenger RNAs (mRNAs) mainly code for proteins involved in transcription and cell cycle control. Consistently, we report here that Stau1 abundance fluctuates through the cell cycle in HCT116 and U2OS cells: it is high from the S phase to the onset of mitosis and rapidly decreases as cells transit through mitosis. Stau1 down-regulation is mediated by the ubiquitin-proteasome system and the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Stau1 interacts with the APC/C co-activators Cdh1 and Cdc20 via its first 88 N-terminal amino acids. The importance of controlling Stau1⁵⁵ levels is underscored by the observation that its overexpression affects mitosis entry and impairs proliferation of transformed cells. Microarray analyses identified 275 Stau1⁵⁵-bound mRNAs in prometaphase cells, an early mitotic step that just precedes Stau1 degradation. Interestingly, several of these mRNAs are more abundant in Stau1⁵⁵-containing complexes in cells arrested in prometaphase than in asynchronous cells. Our results point out for the first time to the possibility that Stau1 participates in a mechanism of post-transcriptional regulation of gene expression that is linked to cell cycle progression in cancer cells.

LIN28 function is fundamental to the activity and behavior of human embryonic stem cells (hESCs) and induced pluripotent stem cells. Its main roles in these cell types are the regulation of translational efficiency and let-7 miRNA maturation. However, LIN28-associated mRNA cargo shifting and resultant regulation of translational efficiency upon the initiation of differentiation remain unknown. An RNA-immunoprecipitation and microarray analysis protocol, eRIP, that has high specificity and sensitivity was developed to test endogenous LIN28-associated mRNA cargo shifting. A combined eRIP and polysome analysis of early stage differentiation of hESCs with two distinct differentiation cues revealed close similarities between the dynamics of LIN28 association and translational modulation of genes involved in the Wnt signaling, cell cycle, RNA metabolism and proteasomal pathways. Our data demonstrate that change in translational efficiency is a major contributor to early stages of differentiation of hESCs, in which LIN28 plays a central role. This implies that eRIP analysis of LIN28-associated RNA cargoes may be used for rapid functional quality control of pluripotent stem cells under manufacture for therapeutic applications.

Both canonical and alternative splicing of RNAs are governed by intronic sequence elements and produce transient lariat structures fastened by branch points within introns. To map precisely the location of branch points on a genomic scale, we developed LaSSO (Lariat Sequence Site Origin), a data-driven algorithm which utilizes RNA-seq data. Using fission yeast cells lacking the debranching enzyme Dbr1, LaSSO not only accurately identified canonical splicing events, but also pinpointed novel, but rare, exon-skipping events, which may reflect aberrantly spliced transcripts. Compromised intron turnover perturbed gene regulation at multiple levels, including splicing and protein translation. Notably, Dbr1 function was also critical for the expression of mitochondrial genes and for the processing of self-spliced mitochondrial introns. LaSSO showed better sensitivity and accuracy than algorithms used for computational branch-point prediction or for empirical branch-point determination. Even when applied to a human data set acquired in the presence of debranching activity, LaSSO identified both canonical and exon-skipping branch points. LaSSO thus provides an effective approach for defining high-resolution maps of branch-site sequences and intronic elements on a genomic scale. LaSSO should be useful to validate introns and uncover branch-point sequences in any eukaryote, and it could be integrated into RNA-seq pipelines.

We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes. We next characterize their commonalities and differences in endo-siRNA populations. Interestingly, most cell lines exhibit enhanced TE-siRNA production relative to tissues, suggesting this as a common aspect of cell immortalization. We also broadly extend annotations of cis-NAT-siRNA loci, identifying ones with common expression across diverse cells and tissues, as well as cell-restricted loci. Finally, we characterize small RNAs in a set of ovary-derived cell lines, including somatic cells (OSS and OSC) and a mixed germline/somatic cell population (fGS/OSS) that exhibits ping-pong piRNA signatures. Collectively, the ovary data reveal new genic piRNA loci, including unusual configurations of piRNA-generating regions. Together with the companion analysis of mRNAs described in a previous study, these small RNA data provide comprehensive information on the transcriptional landscape of diverse Drosophila cell lines. These data should encourage broader usage of fly cell lines, beyond the few that are presently in common usage.

Publication date: 3 July 2014
Source:Molecular Cell, Volume 55, Issue 1
Author(s): Brian S. Plosky
Very few specific functions have been assigned to ultraconserved regions. In this issue of Molecular Cell, Liz et al. (2014) describe how a lncRNA transcribed from an ultraconserved region can negatively regulate miRNA maturation.

Teaser

Very few specific functions have been assigned to ultraconserved regions. In this issue of Molecular Cell, Liz et al. describe how a lncRNA transcribed from an ultraconserved region can negatively regulate miRNA maturation.

Nature advance online publication 06 July 2014. doi:10.1038/nature13406

Authors: Elizabeth V. Wasmuth, Kurt Januszyk & Christopher D. Lima

Nature Protocols 9, 1825 (2014). doi:10.1038/nprot.2014.103

Authors: Martin Kampmann, Michael C Bassik & Jonathan S Weissman

Systematic genetic interaction maps in microorganisms are powerful tools for identifying functional relationships between genes and for defining the function of uncharacterized genes. We have recently implemented this strategy in mammalian cells as a two-stage approach. First, genes of interest are robustly identified in a

Nature Structural & Molecular Biology 21, 591 (2014). doi:10.1038/nsmb.2834

Authors: Ingmar B Schäfer, Michaela Rode, Fabien Bonneau, Steffen Schüssler & Elena Conti

Nature Structural & Molecular Biology 21, 599 (2014). doi:10.1038/nsmb.2837

Authors: Stefanie Jonas, Mary Christie, Daniel Peter, Dipankar Bhandari, Belinda Loh, Eric Huntzinger, Oliver Weichenrieder & Elisa Izaurralde

Nature Structural & Molecular Biology 21, 641 (2014). doi:10.1038/nsmb.2843

Authors: Caia D S Duncan & Juan Mata

Nature Structural & Molecular Biology 21, 585 (2014). doi:10.1038/nsmb.2842

Authors: Grace X Y Zheng, Brian T Do, Dan E Webster, Paul A Khavari & Howard Y Chang

Nature Structural & Molecular Biology 21, 633 (2014). doi:10.1038/nsmb.2844

Authors: Jessica A Brown, David Bulkley, Jimin Wang, Max L Valenstein, Therese A Yario, Thomas A Steitz & Joan A Steitz

Nature Structural & Molecular Biology 21, 577 (2014). doi:10.1038/nsmb.2850

Authors: Sophie Martin & Jeff Coller

Post-transcriptional mRNA regulation is often attained by lengthening or shortening the 3′ poly(A) tail of a transcript. Eukaryotic mRNAs show a spectrum of deadenylation rates, thus allowing intricate control of gene expression, but the mechanisms that determine such rates are unclear. Three new studies highlight the structural and biochemical features of a key enzyme in removing poly(A) tails, the PAN2–PAN3 complex, providing clues to how different mRNA deadenylation rates can be achieved.

The bone marrow delivers dormancy signals in exosomes to metastatic cancer cells.

This protocol describes the detection of small RNAs (~10–200 nucleotides) by blot hybridization. The RNA samples, denatured in formamide, are separated by denaturing polyacrylamide gel electrophoresis. Because high-percentage polyacrylamide gels are required to separate RNAs in this size range, it is necessary to perform electrophoretic transfer to positively charged nylon membranes. After transfer, the immobilized RNAs are subjected to hybridization with a ³²P-radiolabeled DNA or RNA probe and detected by phosphorimaging or autoradiography. This procedure is commonly used to detect small, U-rich spliceosomal small nuclear RNAs (snRNAs) and miRNAs. It should be possible also to detect most miRNAs using high-percentage (e.g., 15%) urea–polyacrylamide gel electrophoresis.

Cytoplasmic changes in polyA tail length is a key mechanism of translational control and is implicated in germline development, synaptic plasticity, cellular proliferation, senescence, and cancer progression. The presence of a U-rich cytoplasmic polyadenylation element (CPE) in the 3' untranslated regions (UTRs) of the responding mRNAs gives them the selectivity to be regulated by the CPE-binding (CPEB) family of proteins, which recognizes RNA via the tandem RNA recognition motifs (RRMs). Here we report the solution structures of the tandem RRMs of two human paralogs (CPEB1 and CPEB4) in their free and RNA-bound states. The structures reveal an unprecedented arrangement of RRMs in the free state that undergo an original closure motion upon RNA binding that ensures high fidelity. Structural and functional characterization of the ZZ domain (zinc-binding domain) of CPEB1 suggests a role in both protein–protein and protein–RNA interactions. Together with functional studies, the structures reveal how RNA binding by CPEB proteins leads to an optimal positioning of the N-terminal and ZZ domains at the 3' UTR, which favors the nucleation of the functional ribonucleoprotein complexes for translation regulation.

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Influence of trypsinization and alternative procedures for cell preparation before RNA extraction on RNA integrity.

Anal Biochem. 2014 Jun 28;

Authors: Vrtačnik P, Kos S, Bustin SA, Marc J, Ostanek B

Abstract
The accuracy of techniques such as microarrays, reverse transcription polymerase chain reaction and whole transcriptome shotgun sequencing is critically dependent on RNA quality. We have repeatedly observed extensive RNA degradation following trypsinization, a routine procedure used to dissociate adherent tissue culture cells prior to RNA extraction. This study investigates the cause for this degradation and identifies an alternative procedure that enables extraction of intact high quality RNA. Trypsinization as well as several alternative procedures was used to dissociate a range of different cell lines prior to RNA extraction. The contribution of exogenous ribonucleases or induction of endogenous ribonucleases by trypsin reagent proteases on RNA degradation was examined. Trypsinization resulted in a complete degradation of RNA regardless of cell line type, differentiation stage or passage number. This occurred when intact RNA was incubated directly with trypsin and was not suppressed by inhibiting trypsin's protease activity. Prevention of degradation by sodium hypochlorite treatment of trypsin reagent identified the presence of ribonucleases in trypsin derived from animal pancreas. Consistent extraction of high quality RNA requires the use of direct cell lysis with a phenol-guanidine-based reagent or an animal-origin free protease-based dissociation agent if enzymatic detachment prior to RNA extraction cannot be avoided.

PMID: 24983903 [PubMed - as supplied by publisher]

Nature advance online publication 02 July 2014. doi:10.1038/nature13551

Authors: Hong Ma, Robert Morey, Ryan C. O'Neil, Yupeng He, Brittany Daughtry, Matthew D. Schultz, Manoj Hariharan, Joseph R. Nery, Rosa Castanon, Karen Sabatini, Rathi D. Thiagarajan, Masahito Tachibana, Eunju Kang, Rebecca Tippner-Hedges, Riffat Ahmed, Nuria Marti Gutierrez, Crystal Van Dyken, Alim Polat, Atsushi Sugawara, Michelle Sparman, Sumita Gokhale, Paula Amato, Don P.Wolf, Joseph R. Ecker, Louise C. Laurent & Shoukhrat Mitalipov

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Genome activation in bovine embryos: Review of the literature and new insights from RNA sequencing experiments.

Anim Reprod Sci. 2014 Jun 6;

Authors: Graf A, Krebs S, Heininen-Brown M, Zakhartchenko V, Blum H, Wolf E

Abstract
Maternal-to-embryonic transition (MET) is the period in early embryonic development when maternal RNAs and proteins stored in the oocyte are gradually degraded and transcription of the embryonic genome is activated. First insights into the timing of embryonic genome activation (EGA) came from autoradiographic analyses of embryos following incorporation of [(3)H]uridine. These studies identified the eight- to 16-cell stage of bovine embryos as the period of major EGA, but detected first transcriptional activity already in one-cell embryos. Subsequent studies compared the transcriptome profiles of untreated embryos and of embryos incubated with the transcription inhibitor α-amanitin to reveal transcripts of embryonic origin. In addition, candidate gene-based and global gene expression studies over several stages of early development were performed and characteristic profiles were revealed. However, the onset of embryonic transcription was obscured by the presence of maternal transcripts and could only be determined for genes which are not expressed in oocytes. Using RNA sequencing of bovine germinal vesicle and metaphase II oocytes, and of four-cell, eight-cell, 16-cell and blastocyst stage embryos, we established the most comprehensive transcriptome data set of bovine oocyte maturation and early development. EGA was analyzed by (i) detection of embryonic transcripts which are not present in oocytes; (ii) detection of transcripts from the paternal allele; and (iii) detection of primary transcripts with intronic sequences. Using these three approaches we were able to map the onset of embryonic transcription for almost 7400 genes. Genes activated at the four-cell stage or before were functionally related to RNA processing, translation, and transport, preparing the embryo for major EGA at the eight-cell stage, when genes from a broad range of functional categories were found to be activated. These included transcriptional and translational functions as well as protein ubiquitination. The functions of the genes activated at the 16-cell stage were consistent with ongoing transcription and translation, while the genes activated in blastocysts included regulators of early lineage specification. Fine mapping of EGA provides a new layer of information for detecting disturbances of early development due to genetic, epigenetic, and environmental factors.

PMID: 24975847 [PubMed - as supplied by publisher]

Background: RNA-seq is a powerful technique for identifying and quantifying transcription and splicing events, both known and novel. However, given its recent development and the proliferation of library construction methods, understanding the bias it introduces is incomplete but critical to realizing its value. Results: We present a method, in vitro transcription sequencing (IVT-seq), for identifying and assessing the technical biases in RNA-seq library generation and sequencing at scale. We created a pool of over 1,000 in vitro transcribed RNAs from a full-length human cDNA library and sequenced them with polyA and total RNA-seq, the most common protocols. Because each cDNA is full length, and we show in vitro transcription is incredibly processive, each base in each transcript should be equivalently represented. However, with common RNA-seq applications and platforms, we find 50% of transcripts have more than two-fold and 10% have more than 10-fold differences in within-transcript sequence coverage. We also find greater than 6% of transcripts have regions of dramatically unpredictable sequencing coverage between samples, confounding accurate determination of their expression. We use a combination of experimental and computational approaches to show rRNA depletion is responsible for the most significant variability in coverage, and several sequence determinants also strongly influence representation. Conclusions: These results show the utility of IVT-seq for promoting better understanding of bias introduced by RNA-seq. We find rRNA depletion is responsible for substantial, unappreciated biases in coverage introduced during library preparation. These biases suggest exon-level expression analysis may be inadvisable, and we recommend caution when interpreting RNA-seq results.

All mammals above 3 kilograms take the same amount of time to urinate

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Characterization and analysis of the composition and dynamics of the mammalian riboproteome.

Cell Rep. 2013 Sep 26;4(6):1276-87

Authors: Reschke M, Clohessy JG, Seitzer N, Goldstein DP, Breitkopf SB, Schmolze DB, Ala U, Asara JM, Beck AH, Pandolfi PP

Abstract
Increasing evidence points to an important role for the ribosome in the regulation of biological processes and as a target for deregulation in disease. Here, we describe a SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry approach to probing mammalian riboproteomes. Using a panel of cell lines, as well as genetic and pharmacological perturbations, we obtained a comparative characterization of the cellular riboproteome. This analysis identified a set of riboproteome components, consisting of a diverse array of proteins with a strong enrichment for RNA-binding proteins. Importantly, this global analysis uncovers a high incidence of genetic alterations to riboproteome components in cancer, with a distinct bias toward genetic amplification. We further validated association with polyribosomes for several riboproteome components and demonstrate that enrichment at the riboproteome can depend on cell type, genetics, or cellular stimulus. Our results have important implications for the understanding of how ribosomes function and provide a platform for uncovering regulators of translation.

PMID: 24055062 [PubMed - indexed for MEDLINE]

Nature Methods 11, 699 (2014). doi:10.1038/nmeth.3005

Authors: Martin Krzywinski & Naomi Altman

Good experimental designs mitigate experimental error and the impact of factors not under study.

Publication date: 10 July 2014
Source:Cell Reports, Volume 8, Issue 1
Author(s): Schraga Schwartz , Maxwell R. Mumbach , Marko Jovanovic , Tim Wang , Karolina Maciag , G. Guy Bushkin , Philipp Mertins , Dmitry Ter-Ovanesyan , Naomi Habib , Davide Cacchiarelli , Neville E. Sanjana , Elizaveta Freinkman , Michael E. Pacold , Rahul Satija , Tarjei S. Mikkelsen , Nir Hacohen , Feng Zhang , Steven A. Carr , Eric S. Lander , Aviv Regev
N6-methyladenosine (m6A) is a common modification of mRNA with potential roles in fine-tuning the RNA life cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of the methyltransferase complex, and show that three of them (WTAP, METTL14, and KIAA1429) are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps and their classification into WTAP-dependent and -independent sites. WTAP-dependent sites are located at internal positions in transcripts, topologically static across a variety of systems we surveyed, and inversely correlated with mRNA stability, consistent with a role in establishing “basal” degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data shed light on the proteomic and transcriptional underpinnings of this RNA modification.

Graphical abstract

Teaser

N6-methyladenosine (m6A) is a highly abundant modification of mRNA. Schwartz et al. identify and validate a network of proteins required for mRNA methylation in mammalian cells. They define two distinct classes of methylation sites. The majority of sites depend on the identified proteins, are located at internal positions in transcripts, and inversely correlate with mRNA stability. Sites independent of these proteins form at the first transcribed base as part of the cap structure, forming a previously unappreciated layer of transcriptome complexity.

Long Noncoding RNAs in Imprinting and X Chromosome Inactivation.

Biomolecules. 2014;4(1):76-100

Authors: Autuoro JM, Pirnie SP, Carmichael GG

Abstract
The field of long noncoding RNA (lncRNA) research has been rapidly advancing in recent years. Technological advancements and deep-sequencing of the transcriptome have facilitated the identification of numerous new lncRNAs, many with unusual properties, however, the function of most of these molecules is still largely unknown. Some evidence suggests that several of these lncRNAs may regulate their own transcription in cis, and that of nearby genes, by recruiting remodeling factors to local chromatin. Notably, lncRNAs are known to exist at many imprinted gene clusters. Genomic imprinting is a complex and highly regulated process resulting in the monoallelic silencing of certain genes, based on the parent-of-origin of the allele. It is thought that lncRNAs may regulate many imprinted loci, however, the mechanism by which they exert such influence is poorly understood. This review will discuss what is known about the lncRNAs of major imprinted loci, and the roles they play in the regulation of imprinting.

PMID: 24970206 [PubMed]

Nature advance online publication 29 June 2014. doi:10.1038/nature13468

Authors: Qingfeng Zhang, T. Nicolai Siegel, Rafael M. Martins, Fei Wang, Jun Cao, Qi Gao, Xiu Cheng, Lubin Jiang, Chung-Chau Hon, Christine Scheidig-Benatar, Hiroshi Sakamoto, Louise Turner, Anja T. R. Jensen, Aurelie Claes, Julien Guizetti, Nicholas A. Malmquist & Artur Scherf

Antigenic variation of the Plasmodium falciparum multicopy var gene family enables parasite evasion of immune destruction by host antibodies. Expression of a particular var subgroup, termed upsA, is linked to the obstruction of blood vessels in the brain and to the pathogenesis of human cerebral malaria. The mechanism determining upsA activation remains unknown. Here we show that an entirely new type of gene silencing mechanism involving an exonuclease-mediated degradation of nascent RNA controls the silencing of genes linked to severe malaria. We identify a novel chromatin-associated exoribonuclease, termed PfRNase II, that controls the silencing of upsAvar genes by marking their transcription start site and intron-promoter regions leading to short-lived cryptic RNA. Parasites carrying a deficient PfRNase II gene produce full-length upsAvar transcripts and intron-derived antisense long non-coding RNA. The presence of stable upsAvar transcripts overcomes monoallelic expression, resulting in the simultaneous expression of both upsA and upsC type PfEMP1 proteins on the surface of individual infected red blood cells. In addition, we observe an inverse relationship between transcript levels of PfRNase II and upsA-type var genes in parasites from severe malaria patients, implying a crucial role of PfRNase II in severe malaria. Our results uncover a previously unknown type of post-transcriptional gene silencing mechanism in malaria parasites with repercussions for other organisms. Additionally, the identification of RNase II as a parasite protein controlling the expression of virulence genes involved in pathogenesis in patients with severe malaria may provide new strategies for reducing malaria mortality.

Nature advance online publication 29 June 2014. doi:10.1038/nature13448

Authors: Sam Behjati, Meritxell Huch, Ruben van Boxtel, Wouter Karthaus, David C. Wedge, Asif U. Tamuri, Iñigo Martincorena, Mia Petljak, Ludmil B. Alexandrov, Gunes Gundem, Patrick S. Tarpey, Sophie Roerink, Joyce Blokker, Mark Maddison, Laura Mudie, Ben Robinson, Serena Nik-Zainal, Peter Campbell, Nick Goldman, Marc van de Wetering, Edwin Cuppen, Hans Clevers & Michael R. Stratton

The somatic mutations present in the genome of a cell accumulate over the lifetime of a multicellular organism. These mutations can provide insights into the developmental lineage tree, the number of divisions that each cell has undergone and the mutational processes that have been operative. Here we describe whole genomes of clonal lines derived from multiple tissues of healthy mice. Using somatic base substitutions, we reconstructed the early cell divisions of each animal, demonstrating the contributions of embryonic cells to adult tissues. Differences were observed between tissues in the numbers and types of mutations accumulated by each cell, which likely reflect differences in the number of cell divisions they have undergone and varying contributions of different mutational processes. If somatic mutation rates are similar to those in mice, the results indicate that precise insights into development and mutagenesis of normal human cells will be possible.

Publication date: August 2014
Source:Trends in Genetics, Volume 30, Issue 8
Author(s): Keith W. Vance , Chris P. Ponting
Several nuclear localised intergenic long noncoding RNAs (lncRNAs) have been ascribed regulatory roles in transcriptional control and their number is growing rapidly. Initially, these transcripts were shown to function locally, near their sites of synthesis, by regulating the expression of neighbouring genes. More recently, lncRNAs have been demonstrated to interact with chromatin at several thousand different locations across multiple chromosomes and to modulate large-scale gene expression programs. Although the molecular mechanisms involved in targeting lncRNAs to distal binding sites remain poorly understood, the spatial organisation of the genome may have a role in specifying lncRNA function. Recent advances indicate that intergenic lncRNAs may exert more widespread effects on gene regulation than previously anticipated.

by Philip E. Bourne

Motivation: Computer-assisted studies of structure, function and evolution of viruses remains a neglected area of research. The attention of bioinformaticians to this interesting and challenging field is far from commensurate with its medical and biotechnological importance. It is telling that out of >200 talks held at ISMB 2013, the largest international bioinformatics conference, only one presentation explicitly dealt with viruses. In contrast to many broad, established and well-organized bioinformatics communities (e.g. structural genomics, ontologies, next-generation sequencing, expression analysis), research groups focusing on viruses can probably be counted on the fingers of two hands.

Results: The purpose of this review is to increase awareness among bioinformatics researchers about the pressing needs and unsolved problems of computational virology. We focus primarily on RNA viruses that pose problems to many standard bioinformatics analyses owing to their compact genome organization, fast mutation rate and low evolutionary conservation. We provide an overview of tools and algorithms for handling viral sequencing data, detecting functionally important RNA structures, classifying viral proteins into families and investigating the origin and evolution of viruses.

Contact: manja@uni-jena.de

Supplementary information: Supplementary data are available at Bioinformatics online. The references for this article can be found in the Supplementary Material.