Hyeshik Chang

Our knowledge of the role of higher-order chromatin structures in transcription of microRNA genes (MIRs) is evolving rapidly. Here we investigate the effect of 3D architecture of chromatin on the transcriptional regulation of MIRs. We demonstrate that MIRs have transcriptional features that are similar to protein-coding genes. RNA polymerase II–associated ChIA-PET data reveal that many groups of MIRs and protein-coding genes are organized into functionally compartmentalized chromatin communities and undergo coordinated expression when their genomic loci are spatially colocated. We observe that MIRs display widespread communication in those transcriptionally active communities. Moreover, miRNA–target interactions are significantly enriched among communities with functional homogeneity while depleted from the same community from which they originated, suggesting MIRs coordinating function-related pathways at posttranscriptional level. Further investigation demonstrates the existence of spatial MIR–MIR chromatin interacting networks. We show that groups of spatially coordinated MIRs are frequently from the same family and involved in the same disease category. The spatial interaction network possesses both common and cell-specific subnetwork modules that result from the spatial organization of chromatin within different cell types. Together, our study unveils an entirely unexplored layer of MIR regulation throughout the human genome that links the spatial coordination of MIRs to their co-expression and function.

The addition of uridine nucleotide by the poly(U) polymerase (PUP) enzymes has a demonstrated impact on various classes of RNAs such as microRNAs (miRNAs), histone-encoding RNAs and messenger RNAs. Cid1 protein is a member of the PUP family. We solved the crystal structure of Cid1 in complex with non-hydrolyzable UMPNPP and a short dinucleotide compound ApU. These structures revealed new residues involved in substrate/product stabilization. In particular, one of the three catalytic aspartate residues explains the RNA dependence of its PUP activity. Moreover, other residues such as residue N165 or the β-trapdoor are shown to be critical for Cid1 activity. We finally suggest that the length and sequence of Cid1 substrate RNA influence the balance between Cid1's processive and distributive activities. We propose that particular processes regulated by PUPs require the enzymes to switch between the two types of activity as shown for the miRNA biogenesis where PUPs can either promote DICER cleavage via short U-tail or trigger miRNA degradation by adding longer poly(U) tail. The enzymatic properties of these enzymes may be critical for determining their particular function in vivo.

A key player in translation initiation is eIF4E, the mRNA 5' cap-binding protein. 4E-Transporter (4E-T) is a recently characterized eIF4E-binding protein, which regulates specific mRNAs in several developmental model systems. Here, we first investigated the role of its enrichment in P-bodies and eIF4E-binding in translational regulation in mammalian cells. Identification of the conserved C-terminal sequences that target 4E-T to P-bodies was enabled by comparison of vertebrate proteins with homologues in Drosophila (Cup and CG32016) and Caenorhabditis elegans by sequence and cellular distribution. In tether function assays, 4E-T represses bound mRNA translation, in a manner independent of these localization sequences, or of endogenous P-bodies. Quantitative polymerase chain reaction and northern blot analysis verified that bound mRNA remained intact and polyadenylated. Ectopic 4E-T reduces translation globally in a manner dependent on eIF4E binding its consensus Y³⁰X₄L site. In contrast, tethered 4E-T continued to repress translation when eIF4E-binding was prevented by mutagenesis of YX₄L, and modestly enhanced the decay of bound mRNA, compared with wild-type 4E-T, mediated by increased binding of CNOT1/7 deadenylase subunits. As depleting 4E-T from HeLa cells increased steady-state translation, in part due to relief of microRNA-mediated silencing, this work demonstrates the conserved yet unconventional mechanism of 4E-T silencing of particular subsets of mRNAs.

In eukaryotic cells, the shortening and removal of the poly(A) tail of cytoplasmic mRNA by deadenylase enzymes is a critical step in post-transcriptional gene regulation. The ribonuclease activity of deadenylase enzymes is attributed to either a DEDD (Asp-Glu-Asp-Asp) or an endonuclease–exonuclease–phosphatase domain. Both domains require the presence of two Mg²⁺ ions in the active site. To facilitate the biochemical analysis of deadenylase enzymes, we have developed a fluorescence-based deadenylase assay. The assay is based on end-point measurement, suitable for quantitative analysis and can be adapted for 96- and 384-well microplate formats. We demonstrate the utility of the assay by screening a chemical compound library, resulting in the identification of non-nucleoside inhibitors of the Caf1/CNOT7 enzyme, a catalytic subunit of the Ccr4–Not deadenylase complex. These compounds may be useful tools for the biochemical analysis of the Caf1/CNOT7 deadenylase subunit of the Ccr4–Not complex and indicate the feasibility of developing selective inhibitors of deadenylase enzymes using the fluorescence-based assay.

Thermodynamic data are reported revealing that pseudouridine () can stabilize RNA duplexes when replacing U and forming -A, -G, -U and -C pairs. Stabilization is dependent on type of base pair, position of within the RNA duplex, and type and orientation of adjacent Watson–Crick pairs. NMR spectra demonstrate that for internal -A, -G and -U pairs, the N3 imino proton is hydrogen bonded to the opposite strand nucleotide and the N1 imino proton may also be hydrogen bonded. CD spectra show that general A-helix structure is preserved, but there is some shifting of peaks and changing of intensities. has two hydrogen donors (N1 and N3 imino protons) and two hydrogen bond acceptors because the glycosidic bond is C-C rather than C-N as in uridine. This greater structural potential may allow to behave as a kind of structurally driven universal base because it can enhance stability relative to U when paired with A, G, U or C inside a double helix. These structural and thermodynamic properties may contribute to the biological functions of .

C9orf72 nucleotide repeat structures initiate molecular cascades of disease

Nature 507, 7491 (2014). doi:10.1038/nature13124

Authors: Aaron R. Haeusler, Christopher J. Donnelly, Goran Periz, Eric A. J. Simko, Patrick G. Shaw, Min-Sik Kim, Nicholas J. Maragakis, Juan C. Troncoso, Akhilesh Pandey, Rita Sattler, Jeffrey D. Rothstein & Jiou Wang

A hexanucleotide repeat expansion (HRE), (GGGGCC)n, in C9orf72 is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we identify a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD

Statistics: Biomedicine must look beyond P values

Nature 507, 7491 (2014). doi:10.1038/507169b

Authors: Anne-Louise Ponsonby & Terence Dwyer

Establishing statistical validity for study findings goes beyond a consideration of P values alone (R.NuzzoNature506, 150–152; 10.1038/506150a2014). In the era of big data, we now have many biological measures available for assessing how likely

Impairment of RNA editing at a handful of coding sites causes severe disorders, prompting the view that coding RNA editing is highly advantageous. Recent genomic studies have expanded the list of human coding RNA editing sites by more than 100 times, raising the question of how common advantageous RNA editing...

See related comment on picking a graduate studentship, http://genomebiology.com/2013/14/4/114

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Detection of Uridylated mRNAs.

Methods Mol Biol. 2014;1125:43-51

Authors: Sement FM, Gagliardi D

Abstract
Uridine addition at the 3' end of RNAs (i.e., uridylation) emerges as a critical posttranscriptional modification promoting RNA degradation. Uridylation has been notably linked to the degradation of small RNAs, correlated with the 5' shortening of RISC-cleaved transcripts and the degradation of mRNAs. We describe here a method based on 3' RACE (3' Rapid Amplification of cDNA End) PCR that has been successfully used to investigate nucleotide addition at the 3' end of RISC-cleaved transcripts and full-length mRNAs in plants.

PMID: 24590778 [PubMed - in process]

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Global and Quantitative Profiling of Polyadenylated RNAs Using PAS-seq.

Methods Mol Biol. 2014;1125:179-85

Authors: Yao C, Shi Y

Abstract
mRNA alternative polyadenylation (APA) has been increasingly recognized as a widespread and evolutionarily conserved mechanism for eukaryotic gene regulation. Here we describe a method called poly(A) site sequencing that can not only map RNA polyadenylation sites on a transcriptome level but also provide quantitative information on the relative abundance of polyadenylated RNAs. This method has been successfully used for both global APA analysis and digital gene expression profiling.

PMID: 24590790 [PubMed - in process]

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Using Klenow-mediated extension to measure poly(a)-tail length and position in the transcriptome.

Methods Mol Biol. 2014;1125:25-42

Authors: Lee MC, Jänicke A, Beilharz TH

Abstract
The poly(A)-tail that terminates most mRNA and many noncoding RNA is a convenient "hook" to isolate mRNA. However the length of this tail and its position within the primary RNA transcript can also hold diagnostic value for RNA metabolism. In general, mRNA with a long poly(A)-tail is well translated, whereas a short poly(A)-tail can indicate translational silencing. A short poly(A)-tail is also appended to RNA-decay intermediates via the TRAMP complex. A number of approaches have been developed to measure the length and position of the poly(A)-tail. Here, we describe a simple method to "tag" adenylated RNA using the native function of DNA polymerase I to extend an RNA primer on a DNA template in second-strand DNA synthesis. This function can be harnessed as a means to purify, visualize, and quantitate poly(A)-dynamics of individual RNA and the transcriptome en masse.

PMID: 24590777 [PubMed - in process]

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Determining the RNA Specificity and Targets of RNA-Binding Proteins using a Three-Hybrid System.

Methods Enzymol. 2014;539:163-81

Authors: Koh YY, Wickens M

Abstract
The three-hybrid system can be used to identify RNA sequences that bind a specific protein by screening a hybrid RNA library with a protein-activation domain fusion as 'bait.' These screens complement biochemical techniques, for example, SELEX, co-immunoprecipitation, and cross-linking experiments (see UV crosslinking of interacting RNA and protein in cultured cells and PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation): a step-by-step protocol to the transcriptome-wide identification of binding sites of RNA-binding proteins).

PMID: 24581443 [PubMed - in process]

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Dissecting a Known RNA-Protein Interaction using a Yeast Three-Hybrid System.

Methods Enzymol. 2014;539:183-93

Authors: Koh YY, Wickens M

Abstract
The yeast three-hybrid system has been applied to known protein-RNA interactions for a variety of purposes. For instance, protein and RNA mutants with altered or relaxed binding specificities can be identified. Mutant RNAs can also be analyzed to better understand RNA-binding specificity of a specific protein. Furthermore, this system complements other biochemical techniques, for example, SELEX, co-immunoprecipitation and cross-linking experiments (see UV crosslinking of interacting RNA and protein in cultured cells and PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation): a step-by-step protocol to the transcriptome-wide identification of binding sites of RNA-binding proteins).

PMID: 24581444 [PubMed - in process]

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MARIS: Method for Analyzing RNA following Intracellular Sorting.

PLoS One. 2014;9(3):e89459

Authors: Hrvatin S, Deng F, O'Donnell CW, Gifford DK, Melton DA

Abstract
Transcriptional profiling is a key technique in the study of cell biology that is limited by the availability of reagents to uniquely identify specific cell types and isolate high quality RNA from them. We report a Method for Analyzing RNA following Intracellular Sorting (MARIS) that generates high quality RNA for transcriptome profiling following cellular fixation, intracellular immunofluorescent staining and FACS. MARIS can therefore be used to isolate high quality RNA from many otherwise inaccessible cell types simply based on immunofluorescent tagging of unique intracellular proteins. As proof of principle, we isolate RNA from sorted human embryonic stem cell-derived insulin-expressing cells as well as adult human β cells. MARIS is a basic molecular biology technique that could be used across several biological disciplines.

PMID: 24594682 [PubMed - in process]

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RNA polymerase II pausing during development.

Development. 2014 Mar;141(6):1179-83

Authors: Gaertner B, Zeitlinger J

Abstract
The rapid expansion of genomics methods has enabled developmental biologists to address fundamental questions of developmental gene regulation on a genome-wide scale. These efforts have demonstrated that transcription of developmental control genes by RNA polymerase II (Pol II) is commonly regulated at the transition to productive elongation, resulting in the promoter-proximal accumulation of transcriptionally engaged but paused Pol II prior to gene induction. Here we review the mechanisms and possible functions of Pol II pausing and their implications for development.

PMID: 24595285 [PubMed - in process]

Sebastien M. Weyn-Vanhentenryck, Aldo Mele, Qinghong Yan, Shuying Sun, Natalie Farny, Zuo Zhang, Chenghai Xue, Margaret Herre, Pamela A. Silver, Michael Q. Zhang, Adrian R. Krainer, Robert B. Darnell, Chaolin Zhang. The RNA binding proteins Rbfox1/2/3 regulate alternative splicing in the nervous system, and disruption of Rbfox1 has been implicated in autism. However, comprehensive identification of functional....

Nature Structural & Molecular Biology 21, 228 (2014). doi:10.1038/nsmb.2779

Authors: Brian J Conti, Johannes Elferich, Zhongying Yang, Ujwal Shinde & William R Skach

Nature Structural & Molecular Biology 21, 207 (2014). doi:10.1038/nsmb.2782

Authors: Shinichi Nakagawa & Tatsuya Hirano

Long noncoding RNAs (lncRNAs) have divergent roles in the nuclei of higher eukaryotes, including chromatin modification and regulation of nuclear bodies. A new study adds a new lncRNA function to the current list: serving as a platform for trans-chromosomal associations. At least three gene loci located on different chromosomes are brought together around the transcription site of a lncRNA termed functional intergenic repeating RNA element (Firre).

Nature Structural & Molecular Biology 21, 209 (2014). doi:10.1038/nsmb.2791

Author: Arianne Heinrichs

Contraction of biomedical research labs may have begun due to 5% sequester cut to NIH budget

Sven Diederichs.

Yan Zhang, Rachel A. Mooney, Jeffrey A. Grass, Priya Sivaramakrishnan, Christophe Herman, Robert Landick, Jue D. Wang. In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inh....

Teemu P. Miettinen, Heli K.J. Pessa, Matias J. Caldez, Tobias Fuhrer, M. Kasim Diril, Uwe Sauer, Philipp Kaldis, Mikael Björklund. BackgroundRegulation of cell size requires coordination of growth and proliferation. Conditional loss of cyclin-dependent kinase 1 in mice permits hepatocyte growth without cell division, allowing....

Nature advance online publication 09 March 2014. doi:10.1038/nature13079

Authors: Pengda Liu, Michael Begley, Wojciech Michowski, Hiroyuki Inuzuka, Miriam Ginzberg, Daming Gao, Peiling Tsou, Wenjian Gan, Antonella Papa, Byeong Mo Kim, Lixin Wan, Amrik Singh, Bo Zhai, Min Yuan, Zhiwei Wang, Steven P. Gygi, Tae Ho Lee, Kun-Ping Lu, Alex Toker, Pier Paolo Pandolfi, John M. Asara, Marc W. Kirschner, Piotr Sicinski, Lewis Cantley & Wenyi Wei

Akt, also known as protein kinase B, plays key roles in cell proliferation, survival and metabolism. Akt hyperactivation contributes to many pathophysiological conditions, including human cancers, and is closely associated with poor prognosis and chemo- or radiotherapeutic resistance. Phosphorylation of Akt at S473 (ref. 5) and T308 (ref. 6) activates Akt. However, it remains unclear whether further mechanisms account for full Akt activation, and whether Akt hyperactivation is linked to misregulated cell cycle progression, another cancer hallmark. Here we report that Akt activity fluctuates across the cell cycle, mirroring cyclin A expression. Mechanistically, phosphorylation of S477 and T479 at the Akt extreme carboxy terminus by cyclin-dependent kinase 2 (Cdk2)/cyclin A or mTORC2, under distinct physiological conditions, promotes Akt activation through facilitating, or functionally compensating for, S473 phosphorylation. Furthermore, deletion of the cyclin A2 allele in the mouse olfactory bulb leads to reduced S477/T479 phosphorylation and elevated cellular apoptosis. Notably, cyclin A2-deletion-induced cellular apoptosis in mouse embryonic stem cells is partly rescued by S477D/T479E-Akt1, supporting a physiological role for cyclin A2 in governing Akt activation. Together, the results of our study show Akt S477/T479 phosphorylation to be an essential layer of the Akt activation mechanism to regulate its physiological functions, thereby providing a new mechanistic link between aberrant cell cycle progression and Akt hyperactivation in cancer.

Nature Protocols 9, 751 (2014). doi:10.1038/nprot.2014.051

Authors: Ranen Aviner, Tamar Geiger & Orna Elroy-Stein

Regulation of mRNA translation has a pivotal role in modulating protein levels, and the genome-wide identification of proteins synthesized at a given time is indispensable to our understanding of gene expression. This protocol describes the mass-spectrometric analysis of newly synthesized proteins from cultured cells or

The three-dimensional structures of large biomolecules important in the function and mechanistic pathways of all living systems and viruses can be determined by x-ray diffraction from crystals of these molecules and their complexes. This area of crystallography is continually expanding and evolving, and the introduction of new methods that use the latest technology is allowing the elucidation of ever larger and more complex biological systems, which are now becoming tractable to structure solution. This review looks back at what has been achieved and forward at how current and future developments may allow technical challenges to be overcome. Author: Elspeth F. Garman

by Lit-Hsin Loo, Danai Laksameethanasan, Yi-Ling Tung

Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein functions and how these functions were acquired in cells from different organisms or species. A public web interface of PLAST is available at http://plast.bii.a-star.edu.sg.

by Tatsunori B. Hashimoto, Matthew D. Edwards, David K. Gifford

We show that existing RNA-seq, DNase-seq, and ChIP-seq data exhibit overdispersed per-base read count distributions that are not matched to existing computational method assumptions. To compensate for this overdispersion we introduce a nonparametric and universal method for processing per-base sequencing read count data called Fixseq. We demonstrate that Fixseq substantially improves the performance of existing RNA-seq, DNase-seq, and ChIP-seq analysis tools when compared with existing alternatives.

This year, to introduce the 2014 update to his Next Generation Sequencing Field Guide—perennially our most-accessed community resource—Travis Glenn has a bit more to say than just what goes in the tables. So here it is as a guest post!

Welcome to the 2014 update of the NGS Field Guide. Previously, I have presented the data & left the interpretation of those data to the readers, other than my “grades” about the utility of specific platforms for specific applications (i.e., Tables 1a, 1b, and 1c). However, many of the people that I have corresponded with have asked me to cut to the chase and just make a recommendation. So, by popular demand, in this year’s edition I am being much more direct about my recommendations.

Overall, if you are in the market for a next generation DNA sequencer in early 2014, the data indicate one clear inexorable trend – think Illumina. For fans of the Brady Bunch – Illumina, Illumina, Illumina! For fans of Star Trek – Prepare to be assimilated by one of Illumina’s Borg-like cubes. For fans of Henry Ford – You can have any NSG instrument you want, so long as it’s an Illumina.

OK, it’s not quite that clear-cut, but it isn’t far from it. For the average scientists who just want to get stuff done, then there are few compelling reasons to do anything other than use Illumina sequencers, especially with the introduction of the anti-proton NextSeq 500. The biggest problem with Illumina instruments is that they take a good while to collect all that data and that frequently creates long queues to get samples run (up to months at the lowest-cost high-quality sequencing facilities). The relatively low cost and low maintenance of the Illumina MiSeq and NextSeq 500 are likely to be especially attractive purchases for molecular ecologists who can afford them to avoid the queues.

To be fair to the other companies, there are some situations where it can make sense to purchase and use an Ion Torrent instrument or to use a PacBio. The Ion Torrent PGM and Proton can deliver data significantly faster than any Illumina and the PacBio delivers reads even faster and that are much longer than any other current instrument. Additionally, there is great anticipation for sequencers in development, such as the offerings from Oxford Nanopore, which may have run times and read lengths that could outperform PacBio by quantum leaps. Oxford Nanopore has released sufficient information that some forecasts can be made, but other companies (such as Genia and Genapsys) are developing perhaps even better technologies, but have kept silent about details other than stating their goal of delivering a human genome sequence for $100.

As much as I or anyone else wants to see a good diversity of platforms to help keep the pressure on Illumina so that it doesn’t do an Orwellian Animal Farmesque transformation into ABI or Microsoft, it’s clear that other platforms are currently side shows compared to the Illumina juggernaut. Helicose is gone. Roche has announced an end to support for the 454 platform and it’s only a matter of time until Life Technologies does the same for SOLiD. Illumina’s NextSeq 500 puts a serious dent in the arguments to switch to Ion Torrent.

Until the nanopore or electronic sequencers arrive en mass, Illumina appears set to be the dominant NGS platform. Even after they arrive, Illumina will likely continue to be dominant, then competitive and then co-exist for quite some time. Illumina is currently the big dawg, and if you peruse these tables, you will understand why.