Hyeshik Chang

Nature Reviews Genetics. doi:10.1038/nrg3724

Authors: Ye Fu, Dan Dominissini, Gideon Rechavi & Chuan He

Systematic genetic perturbation screening in human cells remains technically challenging. Typically, large libraries of chemically synthesized siRNA oligonucleotides are used, each designed to degrade a specific cellular mRNA via the RNA interference (RNAi) mechanism. Here, we report on data from three genome-wide siRNA screens, conducted to uncover host factors required...

Contact: poirazi@imbb.forth.gr

Supplementary information: Supplementary data are available at Bioinformatics online.

Related Articles

Widespread context-dependency of microRNA-mediated regulation.

Genome Res. 2014 Mar 25;

Authors: Erhard F, Haas J, Lieber D, Malterer G, Jaskiewicz L, Zavolan M, Dölken L, Zimmer R

Abstract
Gene expression is regulated in a context-dependent, cell-type specific manner. Condition-specific transcription is dependent on the presence of transcription factors (TFs) that can activate or inhibit its target genes (global context). Additional factors such as chromatin structure, histone or DNA modifications also influence the activity of individual target genes (individual context). The role of the global and individual context for post-transcriptional regulation has not systematically been investigated on a large-scale and is poorly understood. Here we show that global and individual context-dependency is a pervasive feature of microRNA-mediated regulation. Our comprehensive and highly consistent dataset from several high-throughput technologies (PAR-CLIP, RIP-Chip, 4sU-tagging and SILAC) provides strong evidence that context-dependent microRNA target sites (CDTS) are as frequent and functionally relevant as constitutive target sites (CTS). Furthermore, we found the global context to be insufficient to explain the CDTS and that flanking sequence motifs provide individual context that is an equally important factor. Our results demonstrate that, similar to TF-mediated regulation, global and individual context-dependency are prevalent in microRNA-mediated gene regulation implying a much more complex post-transcriptional regulatory network than currently known. The necessary tools to unravel post-transcriptional regulations and mechanisms need to be much more involved and much more data will be needed for particular cell types and cellular conditions to understand microRNA-mediated regulation and the context-dependent post-transcriptional regulatory network.

PMID: 24668909 [PubMed - as supplied by publisher]

While gene expression is a fundamental and tightly controlled cellular process that is regulated at multiple steps, the exact contribution of each step remains unknown in any organism. The absence of transcription initiation regulation for RNA polymerase II in the protozoan parasite Trypanosoma brucei greatly simplifies the task of elucidating the contribution of translation to global gene expression. Therefore, we have sequenced ribosome-protected mRNA fragments in T. brucei, permitting the genome-wide analysis of RNA translation and translational efficiency. We find that the latter varies greatly between life cycle stages of the parasite and ~100-fold between genes, thus contributing to gene expression to a similar extent as RNA stability. The ability to map ribosome positions at sub-codon resolution revealed extensive translation from upstream open reading frames located within 5' UTRs and enabled the identification of hundreds of previously un-annotated putative coding sequences (CDSs). Evaluation of existing proteomics and genome-wide RNAi data confirmed the translation of previously un-annotated CDSs and suggested an important role for >200 of those CDSs in parasite survival, especially in the form that is infective to mammals. Overall our data show that translational control plays a prevalent and important role in different parasite life cycle stages of T. brucei.

Related Articles

mirTools 2.0 for non-coding RNA discovery, profiling, and functional annotation based on high-throughput sequencing.

RNA Biol. 2013 Jul;10(7):1087-92

Authors: Wu J, Liu Q, Wang X, Zheng J, Wang T, You M, Sheng Sun Z, Shi Q

Abstract
Next-generation sequencing has been widely applied to understand the complexity of non-coding RNAs (ncRNAs) in a cost-effective way. In this study, we developed mirTools 2.0, an updated version of mirTools 1.0, which includes the following new features. (1) From miRNA discovery in mirTools 1.0, mirTools 2.0 allows users to detect and profile various types of ncRNAs, such as miRNA, tRNA, snRNA, snoRNA, rRNA, and piRNA. (2) From miRNA profiling in mirTools 1.0, mirTools 2.0 allows users to identify miRNA-targeted genes and performs detailed functional annotation of miRNA targets, including Gene Ontology, KEGG pathway and protein-protein interaction. (3) From comparison of two samples for differentially expressed miRNAs in mirTools 1.0, mirTools 2.0 allows users to detect differentially expressed ncRNAs between two experimental groups or among multiple samples. (4) Other significant improvements include strategies used to detect novel miRNAs and piRNAs, more taxonomy categories to discover more known miRNAs and a stand-alone version of mirTools 2.0. In conclusion, we believe that mirTools 2.0 (122.228.158.106/mr2_dev and centre.bioinformatics.zj.cn/mr2_dev) will provide researchers with more detailed insight into small RNA transcriptomes.

PMID: 23778453 [PubMed - indexed for MEDLINE]

Related Articles

Genome-wide analysis of c-MYC-regulated mRNAs and miRNAs, and c-MYC DNA binding by next-generation sequencing.

Methods Mol Biol. 2013;1012:145-85

Authors: Jackstadt R, Menssen A, Hermeking H

Abstract
The c-MYC oncogene is activated in ~50 % of all tumors, and its product, the c-MYC transcription factor, regulates numerous processes, which contribute to tumor initiation and progression. Therefore, the genome-wide characterization of c-MYC targets and their role in different tumor entities is a recurrent theme in cancer research. Recently, next-generation sequencing (NGS) has become a powerful tool to analyze mRNA and miRNA expression, as well as DNA binding of proteins in a genome-wide manner with an extremely high resolution and coverage. Since the c-MYC transcription factor regulates mRNA and miRNA expression by binding to specific DNA elements in the vicinity of promoters, NGS can be used to generate integrated representations of c-MYC-mediated regulations of gene transcription and chromatin modifications. Here, we provide protocols and examples of NGS-based analyses of c-MYC-regulated mRNA and miRNA expression, as well as of DNA binding by c-MYC. Furthermore, the validation of single c-MYC targets identified by NGS is described. Taken together, these approaches allow an accelerated and comprehensive analysis of c-MYC function in numerous cellular contexts which will further illuminate the role of this important oncogene.

PMID: 24006064 [PubMed - indexed for MEDLINE]

Related Articles

Polysome analysis of mammalian cells.

Methods Enzymol. 2013;530:183-92

Authors: He SL, Green R

Abstract
To assess the global translational level of mammalian cells (see similar protocols for bacteria and yeast on Analysis of polysomes from bacteria, Polysome Profile Analysis - Yeast and Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae).

PMID: 24034322 [PubMed - indexed for MEDLINE]

Computational chemistry: Targeting microRNA with small molecules.

Nat Rev Drug Discov. 2014 Apr 1;13(4):258

Authors: Harrison C

PMID: 24687063 [PubMed - in process]

Related Articles

An expanding world of small RNAs.

Dev Cell. 2014 Jan 27;28(2):111-2

Authors: Neeb ZT, Zahler AM

Abstract
In ciliated protozoans, small RNAs (sRNAs) are integral to guiding large-scale genomic rearrangements after mating. Sandoval et al. (2014) report in this issue of Developmental Cell the discovery of a class of Paramecium sRNAs, produced by a unique Dicer-like enzyme, that likely provides late stage quality control in this process.

PMID: 24480639 [PubMed - indexed for MEDLINE]

This method paper describes a quantitative dual fluorescence approach to analyze protein–mRNA interactions in vivo.

This paper provides an analysis of the origin of multiple RNA recognition motifs (RRMs) within single RNA-binding proteins. One main finding is that such RRMs (sibling RRMs) have significantly higher similarity than expected by chance. These results suggest that sibling RRMs arose through domain duplication.

This paper examines the function of the mammalian protein ZC3H14, which is a zinc finger polyadenosine binding protein that may be orthologous to the yeast protein Nab2. Studies in a neuronal cell line demonstrate that the protein controls poly(A) tail length. Furthermore, complementation analyses in Drosophila indicate that ZC3H14 plays an evolutionarily conserved role in controlling poly(A) tail length in neurons.

This manuscript describes the design of a versatile reporter that exhibits, via multicolor flow cytometry, distinctive signatures for defects in a wide variety of processes involved in gene expression. The reporter is capable of performing high-throughput, rapid, and large-scale screens in budding yeast.

In this paper, the authors analyze how the secondary structure of primary microRNAs (pri-miRNAs) affects their processing by the Microprocessor. They show that pri-miRNAs with central mismatches in the upper stem are more sensitive to changes in Drosha level. They conclude that the secondary structure of pri-miRNA affects Drosha processing efficiency when Drosha is limiting. This raises the possibility that differentiating cells may use this as a means of miRNA regulation.

This protocol uses T4 polynucleotide kinase to catalyze the transfer of a radiolabeled, terminal () phosphate of ATP to the 5'-hydroxyl terminus of a DNA or RNA molecule. The reaction is very efficient and hence is used as a general method for phosphorylating polynucleotides or oligonucleotides.

This protocol is used to radiolabel the 3' ends of RNAs, either synthesized by in vitro transcription or purified from cells or tissues, by ligation of [5'-³²P]cytidine 3',5'-bis(phosphate) (pCp). [5'-³²P]pCp can be obtained commercially or prepared in the laboratory using polynucleotide kinase to phosphorylate cytidine-3'-monophosphate (Cp) with [-³²P]ATP. "Homemade" [5'-³²P]pCp is considerably cheaper and has a higher final concentration than that obtained from commercial sources. The labeling protocol uses T4 RNA ligase 1, which covalently joins [5'-³²P]pCp to the free 3' hydroxyl of RNA. For best labeling, [5'-³²P]pCp should be at least equimolar or higher to available 3'-hydroxyl ends. The reaction requires overnight incubation at low temperature. At the end of the procedure, the reaction is desalted by gel filtration to remove any unincorporated [5'-³²P]pCp.

The electrophoretic mobility shift assay (EMSA), or gel mobility shift assay, is a popular and powerful technique for the detection of RNA–protein interactions. It relies on the fact that naked RNA has certain mobility on nondenaturing gels, but if the RNA is bound by protein, the mobility of the RNA is reduced. Therefore, the binding of protein results in a characteristic upward shift of the RNA on a gel, as monitored using radiolabeled RNA. For reasons that are not completely understood, most RNA–protein complexes—particularly those that result from high-affinity interactions—do not dissociate during the prolonged times required for electrophoretic separation. Because high-affinity interactions are more stable, it is often possible to identify specific interactions over a "background" of weak interactions. Accordingly, EMSAs can be performed using complex mixtures of proteins such as cell extracts. They can be used to investigate a wide range of RNA–protein interactions—from single protein-binding events to assembly of large complexes such as the spliceosome. EMSAs can also be useful for determining kinetic parameters (such as affinity constants) for RNA–protein interactions.

Long intergenic noncoding RNAs (lincRNAs) play diverse regulatory roles in human development and disease, but little is known about their evolutionary history and constraint. Here, we characterize human lincRNA expression patterns in nine tissues across six mammalian species and multiple individuals. Of the 1898 human lincRNAs expressed in these tissues, we find orthologous transcripts for 80% in chimpanzee, 63% in rhesus, 39% in cow, 38% in mouse, and 35% in rat. Mammalian-expressed lincRNAs show remarkably strong conservation of tissue specificity, suggesting that it is selectively maintained. In contrast, abundant splice-site turnover suggests that exact splice sites are not critical. Relative to evolutionarily young lincRNAs, mammalian-expressed lincRNAs show higher primary sequence conservation in their promoters and exons, increased proximity to protein-coding genes enriched for tissue-specific functions, fewer repeat elements, and more frequent single-exon transcripts. Remarkably, we find that ~20% of human lincRNAs are not expressed beyond chimpanzee and are undetectable even in rhesus. These hominid-specific lincRNAs are more tissue specific, enriched for testis, and faster evolving within the human lineage.

In bilaterians, which comprise most of extant animals, microRNAs (miRNAs) regulate the majority of messenger RNAs (mRNAs) via base-pairing of a short sequence (the miRNA "seed") to the target, subsequently promoting translational inhibition and transcript instability. In plants, many miRNAs guide endonucleolytic cleavage of highly complementary targets. Because little is known about miRNA function in nonbilaterian animals, we investigated the repertoire and biological activity of miRNAs in the sea anemone Nematostella vectensis, a representative of Cnidaria, the sister phylum of Bilateria. Our work uncovers scores of novel miRNAs in Nematostella, increasing the total miRNA gene count to 87. Yet only a handful are conserved in corals and hydras, suggesting that microRNA gene turnover in Cnidaria greatly exceeds that of other metazoan groups. We further show that Nematostella miRNAs frequently direct the cleavage of their mRNA targets via nearly perfect complementarity. This mode of action resembles that of small interfering RNAs (siRNAs) and plant miRNAs. It appears to be common in Cnidaria, as several of the miRNA target sites are conserved among distantly related anemone species, and we also detected miRNA-directed cleavage in Hydra. Unlike in bilaterians, Nematostella miRNAs are commonly coexpressed with their target transcripts. In light of these findings, we propose that post-transcriptional regulation by miRNAs functions differently in Cnidaria and Bilateria. The similar, siRNA-like mode of action of miRNAs in Cnidaria and plants suggests that this may be an ancestral state.

Andreas Pircher, Kamilla Bakowska-Zywicka, Lukas Schneider, Marek Zywicki, Norbert Polacek. The structural and functional repertoire of small non-protein-coding RNAs (ncRNAs) is central for establishing gene regulation networks in cells and organisms. Here, we show that an mRNA-derived 1....

Eukaryotic gene regulation involves a balance between packaging of the genome into nucleosomes and enabling access to regulatory proteins and RNA polymerase. Nucleosomes are integral components of gene regulation that restrict access to both regulatory sequences and the underlying template. Whereas canonical histones package the newly replicated genome, they can be replaced with histone variants that alter nucleosome structure, stability, dynamics, and, ultimately, DNA accessibility. Here we consider how histone variants and their interacting partners are involved in transcriptional regulation through the creation of unique chromatin states.

The multifunctional CCCTC-binding factor (CTCF) protein exhibits a broad range of functions, including that of insulator and higher-order chromatin organizer. We found that CTCF comprises a previously unrecognized region that is necessary and sufficient to bind RNA (RNA-binding region [RBR]) and is distinct from its DNA-binding domain. Depletion of cellular CTCF led to a decrease in not only levels of p53 mRNA, as expected, but also those of Wrap53 RNA, an antisense transcript originated from the p53 locus. PAR-CLIP-seq (photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation [PAR-CLIP] combined with deep sequencing) analyses indicate that CTCF binds a multitude of transcripts genome-wide as well as to Wrap53 RNA. Apart from its established role at the p53 promoter, CTCF regulates p53 expression through its physical interaction with Wrap53 RNA. Cells harboring a CTCF mutant in its RBR exhibit a defective p53 response to DNA damage. Moreover, the RBR facilitates CTCF multimerization in an RNA-dependent manner, which may bear directly on its role in establishing higher-order chromatin structures in vivo.

Nature advance online publication 02 April 2014. doi:10.1038/nature13186

Authors: Seung Wook Oh, Julie A. Harris, Lydia Ng, Brent Winslow, Nicholas Cain, Stefan Mihalas, Quanxin Wang, Chris Lau, Leonard Kuan, Alex M. Henry, Marty T. Mortrud, Benjamin Ouellette, Thuc Nghi Nguyen, Staci A. Sorensen, Clifford R. Slaughterbeck, Wayne Wakeman, Yang Li, David Feng, Anh Ho, Eric Nicholas, Karla E. Hirokawa, Phillip Bohn, Kevin M. Joines, Hanchuan Peng, Michael J. Hawrylycz, John W. Phillips, John G. Hohmann, Paul Wohnoutka, Charles R. Gerfen, Christof Koch, Amy Bernard, Chinh Dang, Allan R. Jones & Hongkui Zeng

Nature Methods 11, 369 (2014). doi:10.1038/nmeth.2905

Nature Methods 11, 370 (2014). doi:10.1038/nmeth.2906

Nature Methods 11, 355 (2014). doi:10.1038/nmeth.2900

Authors: Martin Krzywinski & Naomi Altman

When a large number of tests are performed, P values must be interpreted differently.

Nature Methods 11, 360 (2014). doi:10.1038/nmeth.2892

Authors: Eric Lubeck, Ahmet F Coskun, Timur Zhiyentayev, Mubhij Ahmad & Long Cai

Nature advance online publication 26 March 2014. doi:10.1038/nature13134

Authors: Eunju Kang, Guangming Wu, Hong Ma, Ying Li, Rebecca Tippner-Hedges, Masahito Tachibana, Michelle Sparman, Don P. Wolf, Hans R. Schöler & Shoukhrat Mitalipov

Successful mammalian cloning using somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II (MII)-arrested oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing totipotency in somatic cell nuclei. However, these poorly defined maternal factors presumably decline sharply after fertilization, as the cytoplasm of pronuclear-stage zygotes is reportedly inactive. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase, can also support derivation of embryonic stem (ES) cells after SCNT, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in the metaphase but not in the interphase cytoplasm are ‘trapped’ inside the nucleus during interphase and effectively removed during enucleation. Here we investigated the presence of reprogramming activity in the cytoplasm of interphase two-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated metaphase and interphase zygotes and two-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Second, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ES cell, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ES cells capable of contributing to traditional germline and tetraploid chimaeras. Last, direct transfer of cloned embryos, reconstructed with ES cell nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming, with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to use interphase cytoplasm in SCNT could aid efforts to generate autologous human ES cells for regenerative applications, as donated or discarded embryos are more accessible than unfertilized MII oocytes.