Hyeshik Chang

High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy

Nature 504, 7479 (2013). doi:10.1038/nature12748

Authors: Samuel A. Hasson, Lesley A. Kane, Koji Yamano, Chiu-Hui Huang, Danielle A. Sliter, Eugen Buehler, Chunxin Wang, Sabrina M. Heman-Ackah, Tara Hessa, Rajarshi Guha, Scott E. Martin & Richard J. Youle

An increasing body of evidence points to mitochondrial dysfunction as a contributor to the molecular pathogenesis of neurodegenerative diseases such as Parkinson’s disease. Recent studies of the Parkinson’s disease associated genes PINK1 (ref. 2) and parkin (PARK2, ref. 3) indicate that they may act in a quality control pathway preventing the accumulation of dysfunctional mitochondria. Here we elucidate regulators that have an impact on parkin translocation to damaged mitochondria with genome-wide small interfering RNA (siRNA) screens coupled to high-content microscopy. Screening yielded gene candidates involved in diverse cellular processes that were subsequently validated in low-throughput assays. This led to characterization of TOMM7 as essential for stabilizing PINK1 on the outer mitochondrial membrane following mitochondrial damage. We also discovered that HSPA1L (HSP70 family member) and BAG4 have mutually opposing roles in the regulation of parkin translocation. The screens revealed that SIAH3, found to localize to mitochondria, inhibits PINK1 accumulation after mitochondrial insult, reducing parkin translocation. Overall, our screens provide a rich resource to understand mitochondrial quality control.

Computing: Out of the hood

Nature 504, 7479 (2013). doi:10.1038/nj7479-319a

Author: Roberta Kwok

Biologists frustrated with wet-lab work can find rewards in a move to computational research.

Background: Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, this assay has not been adequately validated in primates. Thus, the objectives of the current study were to assess the suitability of lactating rhesus macaques as a model for lactating humans and to determine whether RNA extracted from milk fractions is representative of RNA extracted from mammary tissue for the purpose of studying the transcriptome of milk-producing cells. Results: We confirmed that macaque milk contains cytoplasmic crescents and that ample high-quality RNA can be obtained for sequencing. Using RNA sequencing, RNA extracted from macaque milk fat and milk cell fractions more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from whole mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat, whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from milk-producing mammary epithelial cells. Conclusions: RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells in a non-human primate. However, this sample type clearly requires protocols that minimize RNA degradation. Overall, we validated the use of RNA extracted from human and macaque milk and provided evidence to support the use of lactating macaques as a model for human lactation.

Nature Protocols 9, 90 (2014). doi:10.1038/nprot.2013.167

Authors: H Benjamin Larman, Anthony C Liang, Stephen J Elledge & Jian Zhu

Parallel analysis of translated open reading frames (ORFs) (PLATO) can be used for the unbiased discovery of interactions between full-length proteins encoded by a library of 'prey' ORFs and surface-immobilized 'bait' antibodies, polypeptides or small-molecular-weight compounds. PLATO uses ribosome display (RD) to link ORF-derived mRNA

Article

The long non-coding RNA HOTAIR acts as a scaffold to bring together chromatin silencing complexes in the nucleus. Here, the authors show that HOTAIR also serves as a scaffold between E3 ubiquitin ligases and their substrates and thereby enhances their ubiquitination.

Nature Communications doi: 10.1038/ncomms3939

Authors: Je-Hyun Yoon, Kotb Abdelmohsen, Jiyoung Kim, Xiaoling Yang, Jennifer L. Martindale, Kumiko Tominaga-Yamanaka, Elizabeth J. White, Arturo V. Orjalo, John L. Rinn, Stefan G. Kreft, Gerald M. Wilson, Myriam Gorospe

Article

RNA is central to many cellular functions, but in vivo structures of most RNAs are unknown. Here Kwok et al . present a universally applicable method, DMS/SHAPE-LMPCR, to identify structures of low-abundance transcripts in living cells, which reveals important features that are uniquely present in vivo .

Nature Communications doi: 10.1038/ncomms3971

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

Nature Biotechnology 31, 1133 (2013). doi:10.1038/nbt.2701

Authors: Eric M Mendenhall, Kaylyn E Williamson, Deepak Reyon, James Y Zou, Oren Ram, J Keith Joung & Bradley E Bernstein

Mammalian gene regulation is dependent on tissue-specific enhancers that can act across large distances to influence transcriptional activity. Mapping experiments have identified hundreds of thousands of putative enhancers whose functionality is supported by cell type–specific chromatin signatures and striking enrichments for disease-associated sequence variants. However, these studies did not address the in vivo functions of the putative elements or their chromatin states and did not determine which genes, if any, a given enhancer regulates. Here we present a strategy to investigate endogenous regulatory elements by selectively altering their chromatin state using programmable reagents. Transcription activator–like (TAL) effector repeat domains fused to the LSD1 histone demethylase efficiently remove enhancer-associated chromatin modifications from target loci, without affecting control regions. We find that inactivation of enhancer chromatin by these fusion proteins frequently causes downregulation of proximal genes, revealing enhancer target genes. Our study demonstrates the potential of epigenome editing tools to characterize an important class of functional genomic elements.

Nature Biotechnology 31, 1126 (2013). doi:10.1038/nbt.2720

Authors: Jeff Gole, Athurva Gore, Andrew Richards, Yu-Jui Chiu, Ho-Lim Fung, Diane Bushman, Hsin-I Chiang, Jerold Chun, Yu-Hwa Lo & Kun Zhang

Nature advance online publication 11 December 2013. doi:10.1038/nature12820

Authors: Lawrence A. David, Corinne F. Maurice, Rachel N. Carmody, David B. Gootenberg, Julie E. Button, Benjamin E. Wolfe, Alisha V. Ling, A. Sloan Devlin, Yug Varma, Michael A. Fischbach, Sudha B. Biddinger, Rachel J. Dutton & Peter J. Turnbaugh

Long-term dietary intake influences the structure and activity of the trillions of microorganisms residing in the human gut, but it remains unclear how rapidly and reproducibly the human gut microbiome responds to short-term macronutrient change. Here we show that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression. The animal-based diet increased the abundance of bile-tolerant microorganisms (Alistipes, Bilophila and Bacteroides) and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia, Eubacterium rectale and Ruminococcus bromii). Microbial activity mirrored differences between herbivorous and carnivorous mammals, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi and even viruses. Finally, increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids and the outgrowth of microorganisms capable of triggering inflammatory bowel disease. In concert, these results demonstrate that the gut microbiome can rapidly respond to altered diet, potentially facilitating the diversity of human dietary lifestyles.

In silico prediction of genomic long non-coding RNAs (lncRNAs) is prerequisite to the construction and elucidation of non-coding regulatory network. Chromatin modifications marked by chromatin regulators are important epigenetic features, which can be captured by prevailing high-throughput approaches such as ChIP sequencing. We demonstrate that the accuracy of lncRNA predictions can be greatly improved when incorporating high-throughput chromatin modifications over mouse embryonic stem differentiation toward adult Cerebellum by logistic regression with LASSO regularization. The discriminating features include H3K9me3, H3K27ac, H3K4me1, open reading frames and several repeat elements. Importantly, chromatin information is suggested to be complementary to genomic sequence information, highlighting the importance of an integrated model. Applying integrated model, we obtain a list of putative lncRNAs based on uncharacterized fragments from transcriptome assembly. We demonstrate that the putative lncRNAs have regulatory roles in vicinity of known gene loci by expression and Gene Ontology enrichment analysis. We also show that the lncRNA expression specificity can be efficiently modeled by the chromatin data with same developmental stage. The study not only supports the biological hypothesis that chromatin can regulate expression of tissue-specific or developmental stage-specific lncRNAs but also reveals the discriminating features between lncRNA and coding genes, which would guide further lncRNA identifications and characterizations.

Short interfering RNAs (siRNAs) and microRNAs (miRNAs) have been widely used in mammalian tissue culture and model organisms to selectively silence genes of interest. One limitation of this technology is the lack of precise external control over the gene-silencing event. The use of photocleavable protecting groups installed on nucleobases is a promising strategy to circumvent this limitation, providing high spatial and temporal control over siRNA or miRNA activation. Here, we have designed, synthesized and site-specifically incorporated new photocaged guanosine and uridine RNA phosphoramidites into short RNA duplexes. We demonstrated the applicability of these photocaged siRNAs in the light-regulation of the expression of an exogenous green fluorescent protein reporter gene and an endogenous target gene, the mitosis motor protein, Eg5. Two different approaches were investigated with the caged RNA molecules: the light-regulation of catalytic RNA cleavage by RISC and the light-regulation of seed region recognition. The ability to regulate both functions with light enables the application of this optochemical methodology to a wide range of small regulatory RNA molecules.

Related Articles

Poly-combing the genome for RNA.

Nat Struct Mol Biol. 2013 Dec;20(12):1344-6

Authors: Goff LA, Rinn JL

PMID: 24304912 [PubMed - in process]

Related Articles

Scaffold function of long non-coding RNA HOTAIR in protein ubiquitination.

Nat Commun. 2013 Dec 11;4:2939

Authors: Yoon JH, Abdelmohsen K, Kim J, Yang X, Martindale JL, Tominaga-Yamanaka K, White EJ, Orjalo AV, Rinn JL, Kreft SG, Wilson GM, Gorospe M

Abstract
Although mammalian long non-coding (lnc)RNAs are best known for modulating transcription, their post-transcriptional influence on mRNA splicing, stability and translation is emerging. Here we report a post-translational function for the lncRNA HOTAIR as an inducer of ubiquitin-mediated proteolysis. HOTAIR associates with E3 ubiquitin ligases bearing RNA-binding domains, Dzip3 and Mex3b, as well as with their respective ubiquitination substrates, Ataxin-1 and Snurportin-1. In this manner, HOTAIR facilitates the ubiquitination of Ataxin-1 by Dzip3 and Snurportin-1 by Mex3b in cells and in vitro, and accelerates their degradation. HOTAIR levels are highly upregulated in senescent cells, causing rapid decay of targets Ataxin-1 and Snurportin-1, and preventing premature senescence. These results uncover a role for a lncRNA, HOTAIR, as a platform for protein ubiquitination.

PMID: 24326307 [PubMed - in process]

Genetic Variation in DROSHA 3'UTR Regulated by hsa-miR-27b Is Associated with Bladder Cancer Risk.

PLoS One. 2013;8(11):e81524

Authors: Yuan L, Chu H, Wang M, Gu X, Shi D, Ma L, Zhong D, Du M, Li P, Tong N, Fu G, Qin C, Yin C, Zhang Z

Abstract
PURPOSE: miRNAs can regulate the biological processes, including differentiation, proliferation and apoptosis. DICER and DROSHA are two members of RNase III family, playing pivotal roles in the pathway of miRNAs biogenesis. In this study, we hypothesized that genetic variations of the DICER and DROSHA genes were associated with the bladder cancer risk.
EXPERIMENTAL DESIGN: We performed a case-control study of 685 bladder cancer cases and 730 controls to investigate the association between the seven functional SNPs of DICER and DROSHA genes and bladder cancer risk. We then evaluated the functionality of the important SNPs.
RESULTS: We found that rs10719T>C polymorphism located in 3' untranslated region (UTR) of DROSHA gene was associated with the increased risk of bladder cancer. Stratified analysis suggested that rs10719TC/CC genotype can increase risk of bladder cancer among male patients (Adjusted OR = 1.34, 95% CI = 1.05-1.70, P = 0.018), and ever smokers (1.56, 1.14-2.14, 0.006), compared with TT genotype. Furthermore, DROSHA rs10719T>C polymorphism was predicted to regulate the binding activity of hsa-miR-27a/b. Luciferase reported gene assay confirmed that rs10719 T to G substitution disrupted the binding site for hsa-miR-27b, resulting the increased levels of DROSHA protein.
CONCLUSIONS: Taken together, these findings suggested that DROSHA rs10719T>C polymorphism may be associated with bladder cancer risk in a Chinese population, and hsa-miR-27b can influence the expression of DROSHA protein by binding with 3'UTR.

PMID: 24312312 [PubMed - in process]

Related Articles

MicroRNA: key gene expression regulators.

Fertil Steril. 2013 Dec 4;

Authors: Moreno-Moya JM, Vilella F, Simón C

Abstract
MicroRNAs, also called miRNAs, are small 19-22 nucleotide (nt) sequences of noncoding RNA that work as endogenous epigenetic gene expression regulators. They are transcribed as large primary miRNAs or pre-miRNAs by RNA polymerase II and III, and are subsequently processed by the ribonucleases Drosha and Dicer to give rise to their mature forms. These mature miRNAs are then incorporated into the RISC complex (RNA-induced silencing complex) where they bind to the 3'-UTR mRNA complementary region, which induces their degradation or inhibits their translation, resulting in gene silencing. MicroRNAs are essential for embryo, cell, and tissue development, regulating cell differentiation, proliferation, and apoptosis, hence their importance in human reproduction. Currently, methods of detecting these molecules include real-time polymerase chain reaction, microarrays, in situ hybridization, and deep sequencing as well as novel approaches such as Nanostring nCounter. However, functional characterization is still required to confirm their biologic roles. Furthermore, miRNAs are not only found in cells but also have been identified in most biologic fluids, including serum, plasma, and saliva. Once miRNAs are secreted by cells, they are either incorporated into microvesicles or become associated with proteins, which protect them from RNase degradation so that they may remain intact for long periods of time. This suggests that they might also mediate paracrine signaling via different pathways and could therefore represent potential new biomarkers. Indeed, many pharmaceutic companies have recently started to investigate these molecules as possible routes to develop new human disease treatments.

PMID: 24314918 [PubMed - as supplied by publisher]

Related Articles

The role of replicates for error mitigation in next-generation sequencing.

Nat Rev Genet. 2013 Dec 10;

Authors: Robasky K, Lewis NE, Church GM

Abstract
Advances in next-generation sequencing (NGS) technologies have rapidly improved sequencing fidelity and substantially decreased sequencing error rates. However, given that there are billions of nucleotides in a human genome, even low experimental error rates yield many errors in variant calls. Erroneous variants can mimic true somatic and rare variants, thus requiring costly confirmatory experiments to minimize the number of false positives. Here, we discuss sources of experimental errors in NGS and how replicates can be used to abate such errors.

PMID: 24322726 [PubMed - as supplied by publisher]

by Thomas D. Pollard

For a wide breadth of human noncoding RNAs, including intergenic, intronic, and antisense RNAs, the authors investigate connections between tissue specificity and level of expression, evolutionary conservation, and locus chromatin signature. Uncharacterized ncRNAs are benchmarked against ncRNAs with known cellular roles. It will be of interest to test the authors’ prediction that as yet poorly characterized ncRNAs with profiles of expression, conservation, and locus chromatin similar to known functional ncRNAs are a set enriched for ncRNAs with biological function.

Fan Yang, Huafeng Zhang, Yide Mei, Mian Wu. Hypoxia has long been linked to the Warburg effect, yet the underlying mechanism remains largely unclear. It is also not known if lncRNAs are involved in the contribution of hypoxia to the Warburg....

Microprocessor [Drosha–DGCR8 (DiGeorge syndrome critical region gene 8) complex] processing of primary microRNA (pri-miRNA) is the critical first step in miRNA biogenesis, but how the Drosha cleavage site is determined has been unclear. Previous models proposed that the Drosha–DGCR8 complex measures either ∼22 nt from the upper stem–single-stranded RNA (ssRNA,...

This paper describes a convenient system for highly specific and highly sensitive detection of miRNA expression. The authors show that their method can discriminate mature miRNAs from the precursors using synthetic miRNAs and in vitro transcribed precursors as well as in tissue samples. It also allows for the discrimination of closely related miRNAs of the same family, with individual members differing in sequence by a single nucleotide.

Related Articles

Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo.

Nature. 2013 Dec 15;

Authors: Rouskin S, Zubradt M, Washietl S, Kellis M, Weissman JS

Abstract
RNA has a dual role as an informational molecule and a direct effector of biological tasks. The latter function is enabled by RNA's ability to adopt complex secondary and tertiary folds and thus has motivated extensive computational and experimental efforts for determining RNA structures. Existing approaches for evaluating RNA structure have been largely limited to in vitro systems, yet the thermodynamic forces which drive RNA folding in vitro may not be sufficient to predict stable RNA structures in vivo. Indeed, the presence of RNA-binding proteins and ATP-dependent helicases can influence which structures are present inside cells. Here we present an approach for globally monitoring RNA structure in native conditions in vivo with single-nucleotide precision. This method is based on in vivo modification with dimethyl sulphate (DMS), which reacts with unpaired adenine and cytosine residues, followed by deep sequencing to monitor modifications. Our data from yeast and mammalian cells are in excellent agreement with known messenger RNA structures and with the high-resolution crystal structure of the Saccharomyces cerevisiae ribosome. Comparison between in vivo and in vitro data reveals that in rapidly dividing cells there are vastly fewer structured mRNA regions in vivo than in vitro. Even thermostable RNA structures are often denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Indeed, analysis of mRNA structure under ATP-depleted conditions in yeast shows that energy-dependent processes strongly contribute to the predominantly unfolded state of mRNAs inside cells. Our studies broadly enable the functional analysis of physiological RNA structures and reveal that, in contrast to the Anfinsen view of protein folding whereby the structure formed is the most thermodynamically favourable, thermodynamics have an incomplete role in determining mRNA structure in vivo.

PMID: 24336214 [PubMed - as supplied by publisher]

Related Articles

Dodging two bullets with one dsRNA-binding protein.

Cell Cycle. 2014 Feb 1;13(3):345-6

Authors: Elbarbary RA, Maquat LE

PMID: 24335473 [PubMed - indexed for MEDLINE]

Nature advance online publication 18 December 2013. doi:10.1038/nature12869

Authors: Derek W. Trobaugh, Christina L. Gardner, Chengqun Sun, Andrew D. Haddow, Eryu Wang, Elik Chapnik, Alexander Mildner, Scott C. Weaver, Kate D. Ryman & William B. Klimstra

Currently, there is little evidence for a notable role of the vertebrate microRNA (miRNA) system in the pathogenesis of RNA viruses. This is primarily attributed to the ease with which these viruses mutate to disrupt recognition and growth suppression by host miRNAs. Here we report that the haematopoietic-cell-specific miRNA miR-142-3p potently restricts the replication of the mosquito-borne North American eastern equine encephalitis virus in myeloid-lineage cells by binding to sites in the 3′ non-translated region of its RNA genome. However, by limiting myeloid cell tropism and consequent innate immunity induction, this restriction directly promotes neurologic disease manifestations characteristic of eastern equine encephalitis virus infection in humans. Furthermore, the region containing the miR-142-3p binding sites is essential for efficient virus infection of mosquito vectors. We propose that RNA viruses can adapt to use antiviral properties of vertebrate miRNAs to limit replication in particular cell types and that this restriction can lead to exacerbation of disease severity.

Molecular biology: RNAs leave yeast poised for action

Nature 504, 7478 (2013). doi:10.1038/504011c

One way in which long, non-coding RNAs control gene expression in yeast is to accelerate the activation of protein-coding genes.Elizabeth Tran and her colleagues at Purdue University in West Lafayette, Indiana, studied various strains of Saccharomyces cerevisiae, in which the GAL genes

Research: All journals need to correct errors

Nature 504, 7478 (2013). doi:10.1038/504033d

Author: Jonathan D. Linton

Innocent errors are more commonplace than fraud in research papers, so they need to be identified and corrected promptly. We call for more journals to accept their responsibility to ensure that this happens.We searched for errors in 107 papers in the fields of engineering,

Related Articles

Timescales and bottlenecks in miRNA-dependent gene regulation.

Mol Syst Biol. 2013;9:711

Authors: Hausser J, Syed AP, Selevsek N, van Nimwegen E, Jaskiewicz L, Aebersold R, Zavolan M

Abstract
MiRNAs are post-transcriptional regulators that contribute to the establishment and maintenance of gene expression patterns. Although their biogenesis and decay appear to be under complex control, the implications of miRNA expression dynamics for the processes that they regulate are not well understood. We derived a mathematical model of miRNA-mediated gene regulation, inferred its parameters from experimental data sets, and found that the model describes well time-dependent changes in mRNA, protein and ribosome density levels measured upon miRNA transfection and induction. The inferred parameters indicate that the timescale of miRNA-dependent regulation is slower than initially thought. Delays in miRNA loading into Argonaute proteins and the slow decay of proteins relative to mRNAs can explain the typically small changes in protein levels observed upon miRNA transfection. For miRNAs to regulate protein expression on the timescale of a day, as miRNAs involved in cell-cycle regulation do, accelerated miRNA turnover is necessary.

PMID: 24301800 [PubMed - in process]

Nature Protocols 9, 11 (2014). doi:10.1038/nprot.2013.165

Authors: Ashish Kala, Piyush K Jain, Dipu Karunakaran, Samit Shah & Simon H Friedman

Light-activated RNA interference (LARI) is an effective way to control gene expression with light. This, in turn, allows for the spacing, timing and degree of gene expression to be controlled by the spacing, timing and amount of light irradiation. The key mediators of this process

A microRNA expressed in adult neurons affects movement by modulating neuronal signaling networks and excitability. Authors: Chan Lek Tan, Joshua L. Plotkin, Morten T. Venø, Melanie von Schimmelmann, Philip Feinberg, Silas Mann, Annie Handler, Jørgen Kjems, D. James Surmeier, Dónal O’Carroll, Paul Greengard, Anne Schaefer