Hyeshik Chang

Publication date: 10 November 2014
Source:Cancer Cell, Volume 26, Issue 5
Author(s): Sonia A. Melo , Hikaru Sugimoto , Joyce T. O’Connell , Noritoshi Kato , Alberto Villanueva , August Vidal , Le Qiu , Edward Vitkin , Lev T. Perelman , Carlos A. Melo , Anthony Lucci , Cristina Ivan , George A. Calin , Raghu Kalluri
Exosomes are secreted by all cell types and contain proteins and nucleic acids. Here, we report that breast cancer associated exosomes contain microRNAs (miRNAs) associated with the RISC-Loading Complex (RLC) and display cell-independent capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes derived from cells and sera of patients with breast cancer instigate nontumorigenic epithelial cells to form tumors in a Dicer-dependent manner. These findings offer opportunities for the development of exosomes based biomarkers and therapies.

Teaser

Melo et al. report that breast cancer cells secrete exosomes with CD43-mediated accumulation of Dicer and capacity for cell-independent microRNA biogenesis. Cancer exosomes promote tumor formation of nontumorigenic epithelial cells by altering the transcriptome of target cells.

Identification of 4438 novel lincRNAs involved in mouse pre-implantation embryonic development.

Mol Genet Genomics. 2014 Nov 27;

Authors: Lv J, Liu H, Yu S, Liu H, Cui W, Gao Y, Zheng T, Qin G, Guo J, Zeng T, Han Z, Zhang Y, Wu Q

Abstract
Long intergenic non-coding RNAs (lincRNAs) as a key group of non-coding RNAs have gained substantial attention. Though lincRNAs have been systematically explored in various mouse tissues and cell lines, large-scale identification of lincRNAs in mouse pre-implantation embryonic development (PED) process has not be documented previously. Therefore, it is important to identify and characterize novel lincRNAs that may be involved in PED. In this paper, we performed transcriptome assembly based on published single-cell RNA-seq data during mouse PED and identified 4,438 putative lincRNAs. Combining these with Ensembl lincRNAs, we established a reference catalog of 5,808 transcribed lincRNAs in PED. We then systematically analyzed the lincRNAs in this reference catalog and revealed that the identified novel PED lincRNAs are generally comparable with known Ensembl lincRNAs in genomic aspects. In addition, the global expression patterns can be separated by zygote first cleavage division in clustering analysis and we further identified and analyzed differentially expressed lincRNAs involved in this process. The expression of lincRNAs involved in the process is negatively correlated with promoter methylation in trend. The identified lincRNAs involved in zygote first cleavage division could have important roles in mouse early embryonic development and need further functional studies. Altogether, a novel reference catalog of mouse PED lincRNAs is provided and characterized, which would be a valuable resource for further functional analyses and may help elucidate the pre-implantation regulatory mechanism.

PMID: 25428585 [PubMed - as supplied by publisher]

Mining diverse small RNA species in the deep transcriptome.

Trends Biochem Sci. 2014 Nov 27;

Authors: Vickers KC, Roteta LA, Hucheson-Dilks H, Han L, Guo Y

Abstract
Transcriptomes of many species are proving to be exquisitely diverse, and many investigators are now using high-throughput sequencing to quantify non-protein-coding RNAs, namely small RNAs (sRNA). Unfortunately, most studies are focused solely on microRNA changes, and many investigators are not analyzing the full compendium of sRNA species present in their large datasets. We provide here a rationale to include all types of sRNAs in sRNA sequencing analyses, which will aid in the discovery of their biological functions and physiological relevance.

PMID: 25435401 [PubMed - as supplied by publisher]

m(6)A RNA Modification Controls Cell Fate Transition in Mammalian Embryonic Stem Cells.

Cell Stem Cell. 2014 Oct 16;15(6):707-719

Authors: Batista PJ, Molinie B, Wang J, Qu K, Zhang J, Li L, Bouley DM, Lujan E, Haddad B, Daneshvar K, Carter AC, Flynn RA, Zhou C, Lim KS, Dedon P, Wernig M, Mullen AC, Xing Y, Giallourakis CC, Chang HY

Abstract
N6-methyl-adenosine (m(6)A) is the most abundant modification on messenger RNAs and is linked to human diseases, but its functions in mammalian development are poorly understood. Here we reveal the evolutionary conservation and function of m(6)A by mapping the m(6)A methylome in mouse and human embryonic stem cells. Thousands of messenger and long noncoding RNAs show conserved m(6)A modification, including transcripts encoding core pluripotency transcription factors. m(6)A is enriched over 3' untranslated regions at defined sequence motifs and marks unstable transcripts, including transcripts turned over upon differentiation. Genetic inactivation or depletion of mouse and human Mettl3, one of the m(6)A methylases, led to m(6)A erasure on select target genes, prolonged Nanog expression upon differentiation, and impaired ESC exit from self-renewal toward differentiation into several lineages in vitro and in vivo. Thus, m(6)A is a mark of transcriptome flexibility required for stem cells to differentiate to specific lineages.

PMID: 25456834 [PubMed - as supplied by publisher]

by Jacopo Marino, Melanie I. Stefan, Sarah Blackford

Publication date: March 2015
Source:Trends in Cell Biology, Volume 25, Issue 3
Author(s): Joana A. Vidigal, Andrea Ventura
Despite their clear importance as a class of regulatory molecules, pinpointing the relevance of individual miRNAs has been challenging. Studies querying miRNA functions by overexpressing or silencing specific miRNAs have yielded data that are often at odds with those collected from loss-of-functions models. In addition, knockout studies suggest that many conserved miRNAs are dispensable for animal development or viability. In this review, we discuss these observations in the context of our current knowledge of miRNA biology and review the evidence implicating miRNA-mediated gene regulation in the mechanisms that ensure biological robustness.

Gene expression levels are determined by the balance between rates of mRNA transcription and decay, and genetic variation in either of these processes can result in heritable differences in transcript abundance. Although the genetics of gene expression has been a subject of intense interest, the contribution of heritable variation in mRNA decay rates to gene expression variation has received far less attention. To this end, we developed a novel statistical framework and measured allele-specific differences in mRNA decay rates in a diploid yeast hybrid created by mating two genetically diverse parental strains. We estimate that 31% of genes exhibit allelic differences in mRNA decay rates, of which 350 can be identified at a false discovery rate of 10%. Genes with significant allele-specific differences in mRNA decay rates have higher levels of polymorphism compared to other genes, with all gene regions contributing to allelic differences in mRNA decay rates. Strikingly, we find widespread evidence for compensatory evolution, such that variants influencing transcriptional initiation and decay have opposite effects, suggesting that steady-state gene expression levels are subject to pervasive stabilizing selection. Our results demonstrate that heritable differences in mRNA decay rates are widespread and are an important target for natural selection to maintain or fine-tune steady-state gene expression levels.

The binding of a protein to an RNA sequence protects the region of the RNA from cleavage by chemicals or RNases; this protected region is known as the protein's "footprint." In the footprinting protocol presented here, end-labeled RNAs with and without bound protein are cleaved using chemical methods. Fe(II)–EDTA is used to generate hydroxyl radicals in the presence of a reducing agent. These hydroxyl radicals indiscriminately cleave ribose groups in regions of the ribose–phosphate backbone that are exposed to solvent. After termination of cleavage, the resulting RNA fragments are analyzed by gel electrophoresis on denaturing polyacrylamide gels. Because hydroxyl radicals are smaller and cleave less specifically than RNases, this approach, if feasible, is often the method of choice for monitoring sites of RNA–protein interactions.

Footprinting methods are used to determine the binding site of a protein on an RNA. They are based on the fact that a protein bound to an RNA protects the RNA from cleavage by chemicals or nucleases. The footprinting method described here relies on the ability of iodoethanol to cleave the backbone of RNA when a phosphodiester bond contains sulfur in place of a nonbridging oxygen. A potential advantage of using iodoethanol for cleavage is that one can prepare RNAs that contain selective thiol substitutions such that the resulting cleavage patterns contain fewer bands, making quantification "easier" and the results cleaner. For example, a population of RNAs that only contains nonbridging thiol substitutions 5' to each adenine can be prepared by including αS ATP in the transcription reaction. In this protocol, all positions on an RNA are surveyed. First, a series of RNAs is synthesized by transcription in the presence of αS ATP, αS CTP, αS UTP, or αS GTP. Each of the selectively substituted RNAs is probed with the binding protein of interest. The portion of the RNA that is not bound by protein is accessible and vulnerable to cleavage by iodoethanol. Finally, the cleavage products are analyzed by gel electrophoresis on denaturing polyacrylamide gels.

While the human transcriptome contains a large number of circular RNAs (circRNAs), the functions of most circRNAs remain unclear. Sequence annotation suggests that most circRNAs are generated from splicing in reversed orders across exons. However, the mechanisms of this backsplicing are largely unknown. Here we constructed a single exon minigene containing split GFP, and found that the pre-mRNA indeed produces circRNA through efficient backsplicing in human and Drosophila cells. The backsplicing is enhanced by complementary introns that form double-stranded RNA structure to bring splice sites in proximity, but such structure is not required. Moreover, backsplicing is regulated by general splicing factors and cis-elements, but with regulatory rules distinct from canonical splicing. The resulting circRNA can be translated to generate functional proteins. Unlike linear mRNA, poly-adenosine or poly-thymidine in 3' UTR can inhibit circular mRNA translation. This study revealed that backsplicing can occur efficiently in diverse eukaryotes to generate circular mRNAs.

Publication date: 4 December 2014
Source:Cell, Volume 159, Issue 6
Author(s): Jaechul Lim , Minju Ha , Hyeshik Chang , S. Chul Kwon , Dhirendra K. Simanshu , Dinshaw J. Patel , V. Narry Kim
Uridylation occurs pervasively on mRNAs, yet its mechanism and significance remain unknown. By applying TAIL-seq, we identify TUT4 and TUT7 (TUT4/7), also known as ZCCHC11 and ZCCHC6, respectively, as mRNA uridylation enzymes. Uridylation readily occurs on deadenylated mRNAs in cells. Consistently, purified TUT4/7 selectively recognize and uridylate RNAs with short A-tails (less than ∼25 nt) in vitro. PABPC1 antagonizes uridylation of polyadenylated mRNAs, contributing to the specificity for short A-tails. In cells depleted of TUT4/7, the vast majority of mRNAs lose the oligo-U-tails, and their half-lives are extended. Suppression of mRNA decay factors leads to the accumulation of oligo-uridylated mRNAs. In line with this, microRNA induces uridylation of its targets, and TUT4/7 are required for enhanced decay of microRNA targets. Our study explains the mechanism underlying selective uridylation of deadenylated mRNAs and demonstrates a fundamental role of oligo-U-tail as a molecular mark for global mRNA decay.

Graphical abstract

Teaser

Two enzymes, TUT4 and TUT7, selectively recognize and uridylate cellular mRNAs with shortened poly(A) tails to facilitate decay, thus establishing the oligo-U-tail as a general molecular mark for mRNA decay.

High-throughput assays, such as RNA-seq, to detect differential abundance are widely used. Variable performance across statistical tests, normalizations, and conditions leads to resource wastage and reduced sensitivity. EDDA represents a first, general design tool for RNA-seq, Nanostring, and metagenomic analysis, that rationally selects tests, predicts performance, and plans experiments to minimize resource wastage. Case studies highlight EDDA’s ability to model single-cell RNA-seq, suggesting ways to reduce sequencing costs up to five-fold and improving metagenomic biomarker detection through improved test selection. EDDA’s novel mode-based normalization for detecting differential abundance improves robustness by 10% to 20% and precision by up to 140%.

Article

Flow cytometry allows high-throughput analysis of multiple proteins in individual cells, but relies on availability of antibodies. Here Porichis et al. report a sensitive method for multi-parameter flow cytometric and imaging detection of proteins together with mRNA or miRNA at the single-cell level.

Nature Communications doi: 10.1038/ncomms6641

Authors: Filippos Porichis, Meghan G. Hart, Morgane Griesbeck, Holly L. Everett, Muska Hassan, Amy E. Baxter, Madelene Lindqvist, Sara M. Miller, Damien Z. Soghoian, Daniel G. Kavanagh, Susan Reynolds, Brett Norris, Scott K. Mordecai, Quan Nguyen, Chunfai Lai, Daniel E. Kaufmann

Nature Methods 11, 1187 (2014). doi:10.1038/nmeth.3180

Authors: Martin Krzywinski & Naomi Altman

When multiple factors can affect a system, allowing for interaction can increase sensitivity.

Hierarchical interactions among alleles in a mustard plant explain how self-incompatibility evolved and is maintained. Authors: Eléonore Durand, Raphaël Méheust, Marion Soucaze, Pauline M. Goubet, Sophie Gallina, Céline Poux, Isabelle Fobis-Loisy, Eline Guillon, Thierry Gaude, Alexis Sarazin, Martin Figeac, Elisa Prat, William Marande, Hélène Bergès, Xavier Vekemans, Sylvain Billiard, Vincent Castric

Publication date: 4 December 2014
Source:Molecular Cell, Volume 56, Issue 5
Author(s): Yuka W. Iwasaki , Haruhiko Siomi
MicroRNAs (miRNAs) reshape spatiotemporal gene expression by both modulating the levels of actively transcribed genes and accelerating the clearance of previously transcribed messages, thereby promoting the transition from a preceding stage to subsequent processes during development. Lee et al. (2014) now demonstrate that maternal miRNAs are adenylated by Wispy, which leads to clearing of maternal miRNAs during early embryogenesis.

Teaser

MicroRNAs (miRNAs) reshape spatiotemporal gene expression by both modulating the levels of actively transcribed genes and accelerating the clearance of previously transcribed messages, thereby promoting the transition from a preceding stage to subsequent processes during development. Lee et al. now demonstrate that maternal miRNAs are adenylated by Wispy, which leads to clearing of maternal miRNAs during early embryogenesis.

A molecular explanation is given for how sweet domestic cucumbers were derived from bitter wild cucumbers. Authors: Yi Shang, Yongshuo Ma, Yuan Zhou, Huimin Zhang, Lixin Duan, Huiming Chen, Jianguo Zeng, Qian Zhou, Shenhao Wang, Wenjia Gu, Min Liu, Jinwei Ren, Xingfang Gu, Shengping Zhang, Ye Wang, Ken Yasukawa, Harro J. Bouwmeester, Xiaoquan Qi, Zhonghua Zhang, William J. Lucas, Sanwen Huang

We present a discriminative learning method for pattern discovery of binding sites in nucleic acid sequences based on hidden Markov models. Sets of positive and negative example sequences are mined for sequence motifs whose occurrence frequency varies between the sets. The method offers several objective functions, but we concentrate on mutual information of condition and motif occurrence. We perform a systematic comparison of our method and numerous published motif-finding tools. Our method achieves the highest motif discovery performance, while being faster than most published methods. We present case studies of data from various technologies, including ChIP-Seq, RIP-Chip and PAR-CLIP, of embryonic stem cell transcription factors and of RNA-binding proteins, demonstrating practicality and utility of the method. For the alternative splicing factor RBM10, our analysis finds motifs known to be splicing-relevant.

The motif discovery method is implemented in the free software package Discrover. It is applicable to genome- and transcriptome-scale data, makes use of available repeat experiments and aside from binary contrasts also more complex data configurations can be utilized.

Many critical processes in the cell involve direct binding between RNAs and proteins, making it imperative to fully understand the physicochemical principles behind such interactions at the atomistic level. Here, we use molecular dynamics simulations and 15 μs of sampling to study the behavior of amino acids and amino acid sidechain analogs in high-concentration aqueous solutions of standard RNA nucleobases. Structural and energetic analysis of simulated systems allows us to derive interaction propensity scales for different amino acid/nucleobase combinations. The derived scales closely match and greatly extend the available experimental data, providing a comprehensive foundation for studying RNA–protein interactions in different contexts. By using these scales, we demonstrate a statistically significant connection between nucleobase composition of human mRNA coding sequences and nucleobase interaction propensities of their cognate protein sequences. For example, pyrimidine density profiles of mRNAs match uracil-propensity profiles of their cognate proteins with a median Pearson correlation coefficient of R = –0.70. Our results provide support for the recently proposed hypotheses that mRNAs and their cognate proteins may be physicochemically complementary to each other and bind, especially if unstructured, with the complementarity level being negatively influenced by mRNA adenine content. Finally, we utilize the derived scales to refine the complementarity hypothesis and closely examine its physicochemical underpinnings.

Trim25 Is an RNA-Specific Activator of Lin28a/TuT4-Mediated Uridylation.

Cell Rep. 2014 Nov 6;9(4):1265-1272

Authors: Choudhury NR, Nowak JS, Zuo J, Rappsilber J, Spoel SH, Michlewski G

Abstract
RNA binding proteins have thousands of cellular RNA targets and often exhibit opposite or passive molecular functions. Lin28a is a conserved RNA binding protein involved in pluripotency and tumorigenesis that was previously shown to trigger TuT4-mediated pre-let-7 uridylation, inhibiting its processing and targeting it for degradation. Surprisingly, despite binding to other pre-microRNAs (pre-miRNAs), only pre-let-7 is efficiently uridylated by TuT4. Thus, we hypothesized the existence of substrate-specific cofactors that stimulate Lin28a-mediated pre-let-7 uridylation or restrict its functionality on non-let-7 pre-miRNAs. Through RNA pull-downs coupled with quantitative mass spectrometry, we identified the E3 ligase Trim25 as an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation. We show that Trim25 binds to the conserved terminal loop (CTL) of pre-let-7 and activates TuT4, allowing for more efficient Lin28a-mediated uridylation. These findings reveal that protein-modifying enzymes, only recently shown to bind RNA, can guide the function of canonical ribonucleoprotein (RNP) complexes in cis, thereby providing an additional level of specificity.

PMID: 25457611 [PubMed - as supplied by publisher]

Nature Protocols 9, 2732 (2014). doi:10.1038/nprot.2014.186

Authors: Ivan Bedzhov, Chuen Yan Leung, Monika Bialecka & Magdalena Zernicka-Goetz

The implanting mouse blastocyst invades the uterine stroma and undergoes a dramatic transformation into an egg cylinder. The morphogenetic and signaling events during this transition are largely unexplored, as the uterine tissues engulf the embryo. Here we describe a protocol supporting the development of the

Neurobiology: A molecular knife to dice depression

Nature 516, 7529 (2014). doi:10.1038/nature13942

Authors: Gerhard Schratt

Chronic stress can cause depression in some individuals, but leaves others untouched. Engagement of a molecular pathway controlling the production of tiny RNA snippets might help to explain the difference. See Article p.51

Nature advance online publication 12 November 2014. doi:10.1038/nature13976

Authors: Caroline Dias, Jian Feng, Haosheng Sun, Ning yi Shao, Michelle S. Mazei-Robison, Diane Damez-Werno, Kimberly Scobie, Rosemary Bagot, Benoit LaBonté, Efrain Ribeiro, XiaoChuan Liu, Pamela Kennedy, Vincent Vialou, Deveroux Ferguson, Catherine Peña, Erin S. Calipari, Ja Wook Koo, Ezekiell Mouzon, Subroto Ghose, Carol Tamminga, Rachael Neve, Li Shen & Eric J. Nestler

Nature advance online publication 19 November 2014. doi:10.1038/nature14010

Authors: Shifeng Xue, Siqi Tian, Kotaro Fujii, Wipapat Kladwang, Rhiju Das & Maria Barna

Nature advance online publication 24 November 2014. doi:10.1038/nature13923

Authors: Eliezer Calo, Ryan A. Flynn, Lance Martin, Robert C. Spitale, Howard Y. Chang & Joanna Wysocka

DEAD-box RNA helicases are vital for the regulation of various aspects of the RNA life cycle, but the molecular underpinnings of their involvement, particularly in mammalian cells, remain poorly understood. Here we show that the DEAD-box RNA helicase DDX21 can sense the transcriptional status of both RNA polymerase (Pol) I and II to control multiple steps of ribosome biogenesis in human cells. We demonstrate that DDX21 widely associates with Pol I- and Pol II-transcribed genes and with diverse species of RNA, most prominently with non-coding RNAs involved in the formation of ribonucleoprotein complexes, including ribosomal RNA, small nucleolar RNAs (snoRNAs) and 7SK RNA. Although broad, these molecular interactions, both at the chromatin and RNA level, exhibit remarkable specificity for the regulation of ribosomal genes. In the nucleolus, DDX21 occupies the transcribed rDNA locus, directly contacts both rRNA and snoRNAs, and promotes rRNA transcription, processing and modification. In the nucleoplasm, DDX21 binds 7SK RNA and, as a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, is recruited to the promoters of Pol II-transcribed genes encoding ribosomal proteins and snoRNAs. Promoter-bound DDX21 facilitates the release of the positive transcription elongation factor b (P-TEFb) from the 7SK snRNP in a manner that is dependent on its helicase activity, thereby promoting transcription of its target genes. Our results uncover the multifaceted role of DDX21 in multiple steps of ribosome biogenesis, and provide evidence implicating a mammalian RNA helicase in RNA modification and Pol II elongation control.

Nature advance online publication 01 December 2014. doi:10.1038/nature13975

Authors: Linyan Meng, Amanda J. Ward, Seung Chun, C. Frank Bennett, Arthur L. Beaudet & Frank Rigo

Angelman syndrome is a single-gene disorder characterized by intellectual disability, developmental delay, behavioural uniqueness, speech impairment, seizures and ataxia. It is caused by maternal deficiency of the imprinted gene UBE3A, encoding an E3 ubiquitin ligase. All patients carry at least one copy of paternal UBE3A, which is intact but silenced by a nuclear-localized long non-coding RNA, UBE3A antisense transcript (UBE3A-ATS). Murine Ube3a-ATS reduction by either transcription termination or topoisomerase I inhibition has been shown to increase paternal Ube3a expression. Despite a clear understanding of the disease-causing event in Angelman syndrome and the potential to harness the intact paternal allele to correct the disease, no gene-specific treatment exists for patients. Here we developed a potential therapeutic intervention for Angelman syndrome by reducing Ube3a-ATS with antisense oligonucleotides (ASOs). ASO treatment achieved specific reduction of Ube3a-ATS and sustained unsilencing of paternal Ube3a in neurons in vitro and in vivo. Partial restoration of UBE3A protein in an Angelman syndrome mouse model ameliorated some cognitive deficits associated with the disease. Although additional studies of phenotypic correction are needed, we have developed a sequence-specific and clinically feasible method to activate expression of the paternal Ube3a allele.

Nature Structural & Molecular Biology 21, 945 (2014). doi:10.1038/nsmb.2911

Author: Samie R Jaffrey

The fate of an mRNA is regulated by internal base modifications that generate the modified nucleotides N6-methyladenosine, 5-methylcytosine and inosine. Three new studies show that yeast and human mRNAs also contain pseudouridine residues and that pseudouridylation is induced in various stress states, hinting at a new pathway for post-transcriptional control of mRNA.

Nature Reviews Genetics. doi:10.1038/nrg3832

Authors: Nicola Crosetto, Magda Bienko & Alexander van Oudenaarden

Nature Genetics 46, 1311 (2014). doi:10.1038/ng.3142

Authors: Leighton J Core, André L Martins, Charles G Danko, Colin T Waters, Adam Siepel & John T Lis

Gene translation modeling and prediction is a fundamental problem that has numerous biomedical implementations. In this work we present a novel user friendly tool/index for calculating the Mean of the Typical Decoding Rates (MTDR), which enables predicting translation elongation efficiency of protein coding genes for different tissue types, developmental stages, and experimental conditions. The suggested translation efficiency index is based on the analysis of the organism's ribosome profiling data. This index could be used for example to predict changes in translation elongation efficiency of lowly expressed genes that usually have relatively low and/or biased ribosomal densities and protein levels measurements, or can be used for example for predicting translation efficiency of new genetically engineered genes. We demonstrate the usability of this index via the analysis of six organisms in different tissues and developmental stages. Distributable cross platform application and guideline are available for download at: http://www.cs.tau.ac.il/~tamirtul/MTDR/MTDR_Install.html