Hyeshik Chang

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Fragmentation of whole-transcriptome RNA using E. coli RNase III.

Cold Spring Harb Protoc. 2013 May;2013(5):479-81

Authors: Ares M

Abstract
High-throughput sequencing (HTS) methods can provide short sequence reads for many millions of individual molecules in a sample, allowing the use of sequencing to measure the abundance of RNA molecules. To quantify the amount of a particular sequence in a sample of large RNAs (e.g., mRNAs), it is important to fragment the RNA into short pieces that can be ligated to oligonucleotides that allow polymerase chain reaction (PCR) amplification and sequencing. The most desired end structure of RNA for such ligation steps is a 5' phosphate and a 3' OH. Thus, enzymes that leave these groups after cleavage are of particular utility, avoiding the need to dephosphorylate the 3' end with phosphatases or phosphorylate the 5' end with kinase before proceeding. One such enzyme, RNase III, is widely available. Although it primarily cuts duplex RNA, this specificity is salt- and concentration-dependent, and many RNAs that lack strong extended duplexes are nonetheless susceptible to cleavage at many spots. RNA fragmentation by RNase III does not seem to grossly affect the distribution of RNA sequencing reads. Thus, it has become a standard method for creating nominally representative pools of transcriptome sequences with 5' phosphates and 3' OH for library construction. Three steps in preparing fragmented transcriptome RNA for sequencing library construction are described here: (1) fragmenting the RNA with RNase III to the extent that ~60-100-nucleotide fragments are created, (2) purifying the RNA from the RNase III reaction, and (3) analyzing the digestion products for their suitability in library production.

PMID: 23637372 [PubMed - indexed for MEDLINE]

Finding the target sites of RNA-binding proteins.

Wiley Interdiscip Rev RNA. 2013 Nov 11;

Authors: Li X, Kazan H, Lipshitz HD, Morris QD

Abstract
RNA-protein interactions differ from DNA-protein interactions because of the central role of RNA secondary structure. Some RNA-binding domains (RBDs) recognize their target sites mainly by their shape and geometry and others are sequence-specific but are sensitive to secondary structure context. A number of small- and large-scale experimental approaches have been developed to measure RNAs associated in vitro and in vivo with RNA-binding proteins (RBPs). Generalizing outside of the experimental conditions tested by these assays requires computational motif finding. Often RBP motif finding is done by adapting DNA motif finding methods; but modeling secondary structure context leads to better recovery of RBP-binding preferences. Genome-wide assessment of mRNA secondary structure has recently become possible, but these data must be combined with computational predictions of secondary structure before they add value in predicting in vivo binding. There are two main approaches to incorporating structural information into motif models: supplementing primary sequence motif models with preferred secondary structure contexts (e.g., MEMERIS and RNAcontext) and directly modeling secondary structure recognized by the RBP using stochastic context-free grammars (e.g., CMfinder and RNApromo). The former better reconstruct known binding preferences for sequence-specific RBPs but are not suitable for modeling RBPs that recognize shape and geometry of RNAs. Future work in RBP motif finding should incorporate interactions between multiple RBDs and multiple RBPs in binding to RNA. 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: 24217996 [PubMed - as supplied by publisher]

RNA catalyses nuclear pre-mRNA splicing

Nature 503, 7475 (2013). doi:10.1038/nature12734

Authors: Sebastian M. Fica, Nicole Tuttle, Thaddeus Novak, Nan-Sheng Li, Jun Lu, Prakash Koodathingal, Qing Dai, Jonathan P. Staley & Joseph A. Piccirilli

In nuclear pre-messenger RNA splicing, introns are excised by the spliceosome, a dynamic machine composed of both proteins and small nuclear RNAs (snRNAs). Over thirty years ago, after the discovery of self-splicing group II intron RNAs, the snRNAs were proposed to catalyse splicing. However, no

In this study, the authors focus on the biogenesis of intronic miRNA clusters. They find the primary transcripts of miR 106/93/25 and miR 23b/27b/24-1 are transcribed independently from the host genes. Moreover, alternative splicing can give rise to transcripts that contain individual miRNAs from each cluster. This is important since it demonstrates the existence of yet another mechanism to regulate individual expression of miRNAs within clusters.

Background: Deep RNA sequencing (RNAseq) has opened a new horizon for understanding global gene expression. The functional annotation of non-model mammalian genomes including bovines is still poor compared to that of human and mouse. This particularly applies to tissues without direct significance for milk and meat production, like skin, in spite of its multifunctional relevance for the individual. Thus, applying an RNAseq approach, we performed a whole transcriptome analysis of pigmented and nonpigmented bovine skin to describe the comprehensive transcript catalogue of this tissue. Results: A total of 39,577 unique primary skin transcripts were mapped to the bovine reference genome assembly. The majority of the transcripts were mapped to known transcriptional units (65%). In addition to the reannotation of known genes, a substantial number (10,884) of unknown transcripts (UTs) were discovered, which had not previously been annotated. The classification of UTs was based on the prediction of their coding potential and comparative sequence analysis, subsequently followed by meticulous manual curation. The classification analysis and experimental validation of selected UTs confirmed that RNAseq data can be used to amend the annotation of known genes by providing evidence for additional exons, untranslated regions or splice variants, by approving genes predicted in silico and by identifying novel bovine loci. A large group of UTs (4,848) was predicted to potentially represent long noncoding RNA (lncRNA). Predominantly, potential lncRNAs mapped in intergenic chromosome regions (4,365) and therefore, were classified as potential intergenic lncRNA. Our analysis revealed that only about 6% of all UTs displayed interspecies conservation and discovered a variety of unknown transcripts without interspecies homology but specific expression in bovine skin. Conclusions: The results of our study demonstrate a complex transcript pattern for bovine skin and suggest a possible functional relevance of novel transcripts, including lncRNA, in the modulation of pigmentation processes. The results also indicate that the comprehensive identification and annotation of unknown transcripts from whole transcriptome analysis using RNAseq data remains a tremendous future challenge.

Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges.

Nat Struct Mol Biol. 2013 Nov 10;

Authors: Lovci MT, Ghanem D, Marr H, Arnold J, Gee S, Parra M, Liang TY, Stark TJ, Gehman LT, Hoon S, Massirer KB, Pratt GA, Black DL, Gray JW, Conboy JG, Yeo GW

Abstract
Alternative splicing (AS) enables programmed diversity of gene expression across tissues and development. We show here that binding in distal intronic regions (>500 nucleotides (nt) from any exon) by Rbfox splicing factors important in development is extensive and is an active mode of splicing regulation. Similarly to exon-proximal sites, distal sites contain evolutionarily conserved GCATG sequences and are associated with AS activation and repression upon modulation of Rbfox abundance in human and mouse experimental systems. As a proof of principle, we validated the activity of two specific Rbfox enhancers in KIF21A and ENAH distal introns and showed that a conserved long-range RNA-RNA base-pairing interaction (an RNA bridge) is necessary for Rbfox-mediated exon inclusion in the ENAH gene. Thus we demonstrate a previously unknown RNA-mediated mechanism for AS control by distally bound RNA-binding proteins.

PMID: 24213538 [PubMed - as supplied by publisher]

Poly(A)-Specific Ribonuclease Mediates 3'-End Trimming of Argonaute2-Cleaved Precursor MicroRNAs.

Cell Rep. 2013 Oct 23;

Authors: Yoda M, Cifuentes D, Izumi N, Sakaguchi Y, Suzuki T, Giraldez AJ, Tomari Y

Abstract
MicroRNAs (miRNAs) are typically generated as ∼22-nucleotide double-stranded RNAs via the processing of precursor hairpins by the ribonuclease III enzyme Dicer, after which they are loaded into Argonaute (Ago) proteins to form an RNA-induced silencing complex (RISC). However, the biogenesis of miR-451, an erythropoietic miRNA conserved in vertebrates, occurs independently of Dicer and instead requires cleavage of the 3' arm of the pre-miR-451 precursor hairpin by Ago2. The 3' end of the Ago2-cleaved pre-miR-451 intermediate is then trimmed to the mature length by an unknown nuclease. Here, using a classical chromatographic approach, we identified poly(A)-specific ribonuclease (PARN) as the enzyme responsible for the 3'-5' exonucleolytic trimming of Ago2-cleaved pre-miR-451. Surprisingly, our data show that trimming of Ago2-cleaved precursor miRNAs is not essential for target silencing, indicating that RISC is functional with miRNAs longer than the mature length. Our findings define the maturation step in the miRNA biogenesis pathway that depends on Ago2-mediated cleavage.

PMID: 24209750 [PubMed - as supplied by publisher]

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TALEN-based knockout library for human microRNAs.

Nat Struct Mol Biol. 2013 Nov 10;

Authors: Kim YK, Wee G, Park J, Kim J, Baek D, Kim JS, Kim VN

Abstract
Various technical tools have been developed to probe the functions of microRNAs (miRNAs), yet their application has been limited by low efficacy and specificity. To overcome the limitations, we used transcription activator-like effector nucleases (TALENs) to knock out human miRNA genes. We designed and produced a library of 540 pairs of TALENs for 274 miRNA loci, focusing on potentially important miRNAs. The knockout procedure takes only 2-4 weeks and can be applied to any cell type. As a case study, we generated knockout cells for two related miRNAs, miR-141 and miR-200c, which belong to the highly conserved miR-200 family. Interestingly, miR-141 and miR-200c, despite their overall similarity, suppress largely nonoverlapping groups of targets, thus suggesting that functional miRNA-target interaction requires strict seed-pairing. Our study illustrates the potency of TALEN technology and provides useful resources for miRNA research.

PMID: 24213537 [PubMed - as supplied by publisher]

The EMBO Journal. doi:10.1038/emboj.2013.242

Authors: Jonathan Chang, Brandi N Davis-Dusenbery, Risa Kashima, Xuan Jiang, Nisha Marathe, Roberto Sessa, Justin Louie, Wei Gu, Giorgio Lagna & Akiko Hata

The enzyme ribonucleotide reductase (RNR) plays a critical role in the production of deoxynucleoside-5′-triphosphates (dNTPs), the building blocks for DNA synthesis and replication. The levels of the cellular dNTPs are tightly controlled, in large part through allosteric control of RNR. One important reason for controlling the dNTPs relates to their...

Nature Reviews Genetics. doi:10.1038/nrg3628

Author: Isabel Lokody

During the last 3 years, a number of approaches for the normalization of RNA sequencing data have emerged in the literature, differing both in the type of bias adjustment and in the statistical strategy adopted. However, as data continue to accumulate, there has been no clear consensus on the appropriate normalization method to be used or the impact of a chosen method on the downstream analysis. In this work, we focus on a comprehensive comparison of seven recently proposed normalization methods for the differential analysis of RNA-seq data, with an emphasis on the use of varied real and simulated datasets involving different species and experimental designs to represent data characteristics commonly observed in practice. Based on this comparison study, we propose practical recommendations on the appropriate normalization method to be used and its impact on the differential analysis of RNA-seq data.

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Measurements of the impact of 3' end sequences on gene expression reveal wide range and sequence dependent effects.

PLoS Comput Biol. 2013;9(3):e1002934

Authors: Shalem O, Carey L, Zeevi D, Sharon E, Keren L, Weinberger A, Dahan O, Pilpel Y, Segal E

Abstract
A full understanding of gene regulation requires an understanding of the contributions that the various regulatory regions have on gene expression. Although it is well established that sequences downstream of the main promoter can affect expression, our understanding of the scale of this effect and how it is encoded in the DNA is limited. Here, to measure the effect of native S. cerevisiae 3' end sequences on expression, we constructed a library of 85 fluorescent reporter strains that differ only in their 3' end region. Notably, despite being driven by the same strong promoter, our library spans a continuous twelve-fold range of expression values. These measurements correlate with endogenous mRNA levels, suggesting that the 3' end contributes to constitutive differences in mRNA levels. We used deep sequencing to map the 3'UTR ends of our strains and show that determination of polyadenylation sites is intrinsic to the local 3' end sequence. Polyadenylation mapping was followed by sequence analysis, we found that increased A/T content upstream of the main polyadenylation site correlates with higher expression, both in the library and genome-wide, suggesting that native genes differ by the encoded efficiency of 3' end processing. Finally, we use single cells fluorescence measurements, in different promoter activation levels, to show that 3' end sequences modulate protein expression dynamics differently than promoters, by predominantly affecting the size of protein production bursts as opposed to the frequency at which these bursts occur. Altogether, our results lead to a more complete understanding of gene regulation by demonstrating that 3' end regions have a unique and sequence dependent effect on gene expression.

PMID: 23505350 [PubMed - indexed for MEDLINE]

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RNaseIII and T4 polynucleotide Kinase sequence biases and solutions during RNA-seq library construction.

Biol Direct. 2013;8(1):16

Authors: Lee C, Harris RA, Wall JK, Mayfield RD, Wilke CO

Abstract
BACKGROUND: RNA-seq is a next generation sequencing method with a wide range of applications including single nucleotide polymorphism (SNP) detection, splice junction identification, and gene expression level measurement. However, the RNA-seq sequence data can be biased during library constructions resulting in incorrect data for SNP, splice junction, and gene expression studies. Here, we developed new library preparation methods to limit such biases.
RESULTS: A whole transcriptome library prepared for the SOLiD system displayed numerous read duplications (pile-ups) and gaps in known exons. The pile-ups and gaps of the whole transcriptome library caused a loss of SNP and splice junction information and reduced the quality of gene expression results. Further, we found clear sequence biases for both 5' and 3' end reads in the whole transcriptome library. To remove this bias, RNaseIII fragmentation was replaced with heat fragmentation. For adaptor ligation, T4 Polynucleotide Kinase (T4PNK) was used following heat fragmentation. However, its kinase and phosphatase activities introduced additional sequence biases. To minimize them, we used OptiKinase before T4PNK. Our study further revealed the specific target sequences of RNaseIII and T4PNK.
CONCLUSIONS: Our results suggest that the heat fragmentation removed the RNaseIII sequence bias and significantly reduced the pile-ups and gaps. OptiKinase minimized the T4PNK sequence biases and removed most of the remaining pile-ups and gaps, thus maximizing the quality of RNA-seq data.

PMID: 23826734 [PubMed - indexed for MEDLINE]

Nature Reviews Genetics. doi:10.1038/nrg3574

Authors: Colm J. Ryan, Peter Cimermančič, Zachary A. Szpiech, Andrej Sali, Ryan D. Hernandez & Nevan J. Krogan

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Nature Biotechnology 31, 1015 (2013). doi:10.1038/nbt.2702

Authors: Peter A C 't Hoen, Marc R Friedländer, Jonas Almlöf, Michael Sammeth, Irina Pulyakhina, Seyed Yahya Anvar, Jeroen F J Laros, Henk P J Buermans, Olof Karlberg, Mathias Brännvall, Gert-Jan B van Ommen, Xavier Estivill, Roderic Guigó, Ann-Christine Syvänen, Ivo G Gut, Emmanouil T Dermitzakis, Stylianos E Antonarakis, Alvis Brazma, Paul Flicek, Stefan Schreiber, Philip Rosenstiel, Thomas Meitinger, Tim M Strom, Hans Lehrach, Ralf Sudbrak, Angel Carracedo, Peter A C 't Hoen, Irina Pulyakhina, Seyed Yahya Anvar, Jeroen F J Laros, Henk P J Buermans, Maarten van Iterson, Marc R Friedländer, Jean Monlong, Esther Lizano, Gabrielle Bertier, Pedro G Ferreira, Michael Sammeth, Jonas Almlöf, Olof Karlberg, Mathias Brännvall, Paolo Ribeca, Thasso Griebel, Sergi Beltran, Marta Gut, Katja Kahlem, Tuuli Lappalainen, Thomas Giger, Halit Ongen, Ismael Padioleau, Helena Kilpinen, Mar Gonzàlez-Porta, Natalja Kurbatova, Andrew Tikhonov, Liliana Greger, Matthias Barann, Daniela Esser, Robert Häsler, Thomas Wieland, Thomas Schwarzmayr, Marc Sultan, Vyacheslav Amstislavskiy, Johan T den Dunnen, Gert-Jan B van Ommen, Ivo G Gut, Roderic Guigó, Xavier Estivill, Ann-Christine Syvänen, Emmanouil T Dermitzakis & Tuuli Lappalainen

Nature Biotechnology 31, 1009 (2013). doi:10.1038/nbt.2705

Authors: Donald Sharon, Hagen Tilgner, Fabian Grubert & Michael Snyder

Nature Biotechnology 31, 965 (2013). doi:10.1038/nbt.2737

Authors: Brent Dickinson, Yuanji Zhang, Jay S Petrick, Gregory Heck, Sergey Ivashuta & William S Marshall

ChIP-seq is an established manually-performed method for identifying DNA-protein interactions genome-wide. Here, we describe a protocol for automated high-throughput (AHT) ChIP-seq. To demonstrate the quality of data obtained using AHT-ChIP-seq, we applied it to five proteins in mouse livers using a single 96-well plate, demonstrating an extremely high degree of qualitative and quantitative reproducibility among biological and technical replicates. We estimated the optimum and minimum recommended cell numbers required to perform AHT-ChIP-seq by running an additional plate using HepG2 and MCF7 cells. With this protocol, commercially available robotics can perform four hundred experiments in five days.

Peter W. Reddien. Embryos and juveniles in many organisms repair tissue injuries better than adults. In this issue, Shyh-Chang et al. find that postnatal activation of Lin28a, a gene typically active in embr....

Daniel Schulz, Bjoern Schwalb, Anja Kiesel, Carlo Baejen, Phillipp Torkler, Julien Gagneur, Johannes Soeding, Patrick Cramer. Pervasive transcription of eukaryotic genomes stems to a large extent from bidirectional promoters that synthesize mRNA and divergent noncoding RNA (ncRNA). Here, we show that ncRNA transcription ....

Michael H. Hastings. The purpose of m⁶A methylation of RNA, first observed in the 1970s, has been a longstanding mystery. Fustin et al. now show that it regulates RNA processing and determines the period an....

Tae-Min Kim, Peter W. Laird, Peter J. Park. Microsatellites—simple tandem repeats present at millions of sites in the human genome—can shorten or lengthen due to a defect in DNA mismatch repair. We present here a comprehensive genome-wide a....

Jean-Michel Fustin, Masao Doi, Yoshiaki Yamaguchi, Hayashi Hida, Shinichi Nishimura, Minoru Yoshida, Takayuki Isagawa, Masaki Suimye Morioka, Hideaki Kakeya, Ichiro Manabe, Hitoshi Okamura. The eukaryotic biological clock involves a negative transcription-translation feedback loop in which clock genes regulate their own transcription and that of output genes of metabolic significance....

Xinchen Teng, Margaret Dayhoff-Brannigan, Wen-Chih Cheng, Catherine E. Gilbert, Cierra N. Sing, Nicola L. Diny, Sarah J. Wheelan, Maitreya J. Dunham, Jef D. Boeke, Fernando J. Pineda, J. Marie Hardwick. Loss or duplication of chromosome segments can lead to further genomic changes associated with cancer. However, it is not known whether only a select subset of genes is responsible for driving fur....

Nature Structural & Molecular Biology 20, 1250 (2013). doi:10.1038/nsmb.2679

Authors: Chen Davidovich, Leon Zheng, Karen J Goodrich & Thomas R Cech

Nature Structural & Molecular Biology 20, 1325 (2013). doi:10.1038/nsmb.2678

Authors: Anders Jacobsen, Joachim Silber, Girish Harinath, Jason T Huse, Nikolaus Schultz & Chris Sander

Nature Structural & Molecular Biology 20, 1289 (2013). doi:10.1038/nsmb.2681

Authors: Andreas Boland, Ying Chen, Tobias Raisch, Stefanie Jonas, Duygu Kuzuoğlu-Öztürk, Lara Wohlbold, Oliver Weichenrieder & Elisa Izaurralde

Nature Structural & Molecular Biology 20, 1258 (2013). doi:10.1038/nsmb.2700

Authors: Syuzo Kaneko, Jinsook Son, Steven S Shen, Danny Reinberg & Roberto Bonasio