Hyeshik Chang

In nonsense-mediated mRNA decay (NMD), an exon-junction complex (EJC) located downstream from a termination codon acts as an enhancing signal, but is generally not required. We compared "EJC-enhanced" and "EJC-independent" modes of NMD with regard to their requirement for seven known NMD factors in human cells. This study suggests the existence of several mechanistically distinct branches of NMD and reveals a redundancy of the endo- and exonucleolytic mRNA degradation pathways in both modes of NMD.

Monitoring protein synthesis is essential to our understanding of gene expression regulation, as protein abundance is thought to be predominantly controlled at the level of translation. Mass-spectrometric and RNA sequencing methods have been recently developed for investigating mRNA translation at a global level, but these still involve technical limitations and are not widely applicable. In this study, we describe a novel system-wide proteomic approach for direct monitoring of translation, termed puromycin-associated nascent chain proteomics (PUNCH-P), which is based on incorporation of biotinylated puromycin into newly synthesized proteins under cell-free conditions followed by streptavidin affinity purification and liquid chromatography-tandem mass spectrometry analysis. Using PUNCH-P, we measured cell cycle-specific fluctuations in synthesis for >5000 proteins in mammalian cells, identified proteins not previously implicated in cell cycle processes, and generated the first translational profile of a whole mouse brain. This simple and economical technique is broadly applicable to any cell type and tissue, enabling the identification and quantification of rapid proteome responses under various biological conditions.

Polyadenylation of pre-mRNAs is critical for efficient nuclear export, stability, and translation of the mature mRNAs, and thus for gene expression. The bulk of pre-mRNAs are processed by canonical nuclear poly(A) polymerase (PAPS). Both vertebrate and higher-plant genomes encode more than one isoform of this enzyme, and these are coexpressed...

More investigation into the role of lncRNAs in blood cell development and blood cell cancers is warranted.

Nature advance online publication 14 August 2013. doi:10.1038/nature12477

Authors: Ludmil B. Alexandrov, Serena Nik-Zainal, David C. Wedge, Samuel A. J. R. Aparicio, Sam Behjati, Andrew V. Biankin, Graham R. Bignell, Niccolò Bolli, Ake Borg, Anne-Lise Børresen-Dale, Sandrine Boyault, Birgit Burkhardt, Adam P. Butler, Carlos Caldas, Helen R. Davies, Christine Desmedt, Roland Eils, Jórunn Erla Eyfjörd, John A. Foekens, Mel Greaves, Fumie Hosoda, Barbara Hutter, Tomislav Ilicic, Sandrine Imbeaud, Marcin Imielinsk, Natalie Jäger, David T. W. Jones, David Jones, Stian Knappskog, Marcel Kool, Sunil R. Lakhani, Carlos López-Otín, Sancha Martin, Nikhil C. Munshi, Hiromi Nakamura, Paul A. Northcott, Marina Pajic, Elli Papaemmanuil, Angelo Paradiso, John V. Pearson, Xose S. Puente, Keiran Raine, Manasa Ramakrishna, Andrea L. Richardson, Julia Richter, Philip Rosenstiel, Matthias Schlesner, Ton N. Schumacher, Paul N. Span, Jon W. Teague, Yasushi Totoki, Andrew N. J. Tutt, Rafael Valdés-Mas, Marit M. van Buuren, Laura van ’t Veer, Anne Vincent-Salomon, Nicola Waddell, Lucy R. Yates, Jessica Zucman-Rossi, P. Andrew Futreal, Ultan McDermott, Peter Lichter, Matthew Meyerson, Sean M. Grimmond, Reiner Siebert, Elías Campo, Tatsuhiro Shibata, Stefan M. Pfister, Peter J. Campbell & Michael R. Stratton

Nature advance online publication 14 August 2013. doi:10.1038/nature12548

Authors: Adam M. Schmitt & Howard Y. Chang

The discovery of long non-coding RNAs that control the liaisons between a transcription factor with a key role in prostate cancer and its target genes sheds light on how RNAs dictate information flow in the cell nucleus.

The regulation of intragenic miRNAs by their own intronic promoters is one of the open problems of miRNA biogenesis. Here, we describe PROmiRNA, a new approach for miRNA promoter annotation based on a semi-supervised statistical model trained on deepCAGE data and sequence features. We validate our results with existing annotation, PolII occupancy data and read coverage from RNA-seq data. Compared to previous methods PROmiRNA increases the detection rate of intronic promoters by 30%, allowing us to perform a large-scale analysis of their genomic features, as well as elucidate their contribution to tissue-specific regulation. PROmiRNA can be downloaded from http://promirna.molgen.mpg.de.

Fluorescence-Based High-Throughput Screening of Dicer Cleavage Activity.

J Biomol Screen. 2013 Aug 14;

Authors: Podolska K, Sedlak D, Bartunek P, Svoboda P

Abstract
Production of small RNAs by ribonuclease III Dicer is a key step in microRNA and RNA interference pathways, which employ Dicer-produced small RNAs as sequence-specific silencing guides. Further studies and manipulations of microRNA and RNA interference pathways would benefit from identification of small-molecule modulators. Here, we report a study of a fluorescence-based in vitro Dicer cleavage assay, which was adapted for high-throughput screening. The kinetic assay can be performed under single-turnover conditions (35 nM substrate and 70 nM Dicer) in a small volume (5 µL), which makes it suitable for high-throughput screening in a 1536-well format. As a proof of principle, a small library of bioactive compounds was analyzed, demonstrating potential of the assay.

PMID: 23945873 [PubMed - as supplied by publisher]

Genome-wide analysis of Staufen-associated mRNAs identifies secondary structures that confer target specificity.

Nucleic Acids Res. 2013 Aug 13;

Authors: Laver JD, Li X, Ancevicius K, Westwood JT, Smibert CA, Morris QD, Lipshitz HD

Abstract
Despite studies that have investigated the interactions of double-stranded RNA-binding proteins like Staufen with RNA in vitro, how they achieve target specificity in vivo remains uncertain. We performed RNA co-immunoprecipitations followed by microarray analysis to identify Staufen-associated mRNAs in early Drosophila embryos. Analysis of the localization and functions of these transcripts revealed a number of potentially novel roles for Staufen. Using computational methods, we identified two sequence features that distinguish Staufen's target transcripts from non-targets. First, these Drosophila transcripts, as well as those human transcripts bound by human Staufen1 and 2, have 3' untranslated regions (UTRs) that are 3-4-fold longer than unbound transcripts. Second, the 3'UTRs of Staufen-bound transcripts are highly enriched for three types of secondary structures. These structures map with high precision to previously identified Staufen-binding regions in Drosophila bicoid and human ARF1 3'UTRs. Our results provide the first systematic genome-wide analysis showing how a double-stranded RNA-binding protein achieves target specificity.

PMID: 23945942 [PubMed - as supplied by publisher]

Mani Ramaswami, J. Paul Taylor, Roy Parker. The molecular processes that contribute to degenerative diseases are not well understood. Recent observations suggest that some degenerative diseases are promoted by the accumulation of nuclear or....

Taekjip Ha. Enormous mechanistic insight has been gained by studying the behavior of single molecules. The same approaches used to study proteins in isolation are now being leveraged to examine the changes in....

The EMBO Journal. doi:10.1038/emboj.2013.185

Author: Ramin Shiekhattar

EMBO J advance online publication, 13 August 2013; doi: 10.1038/emboj.2013.182Long noncoding RNAs are emerging as a critical component of transcriptional activation. Over the past few years, there has been a number of examples where long noncoding RNAs expressed from distal sites exert their activating functions on neighbouring protein-coding genes. A new study in The EMBO Journal (Lu et al, 2013) now extends this paradigm by revealing a role for the transcription factor Yin Yang 1 (YY1) in the regulation of the expression of long noncoding RNAs (termed Yams) during myogenesis. Importantly, the authors demonstrate a functional role for Yam-1 in muscle differentiation through activation of a neighbouring micoRNA-715 leading to downregulation of Wnt7b.

The Long Noncoding RNA RMST Interacts with SOX2 to Regulate Neurogenesis.

Mol Cell. 2013 Aug 8;51(3):349-59

Authors: Ng SY, Bogu GK, Soh BS, Stanton LW

Abstract
Long noncoding RNAs (lncRNAs) are abundant in the mammalian transcriptome, and many are specifically expressed in the brain. We have identified a group of lncRNAs, including rhabdomyosarcoma 2-associated transcript (RMST), which are indispensable for neurogenesis. Here, we provide mechanistic insight into the role of human RMST in modulating neurogenesis. RMST expression is specific to the brain, regulated by the transcriptional repressor REST, and increases during neuronal differentiation, indicating a role in neurogenesis. RMST physically interacts with SOX2, a transcription factor known to regulate neural fate. RMST and SOX2 coregulate a large pool of downstream genes implicated in neurogenesis. Through RNA interference and genome-wide SOX2 binding studies, we found that RMST is required for the binding of SOX2 to promoter regions of neurogenic transcription factors. These results establish the role of RMST as a transcriptional coregulator of SOX2 and a key player in the regulation of neural stem cell fate.

PMID: 23932716 [PubMed - in process]

High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.

Nat Biotechnol. 2013 Aug 11;

Authors: Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR

Abstract
The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.

PMID: 23934178 [PubMed - as supplied by publisher]

8,5′-cyclo-2'-deoxyadenosine (cdA) and 8,5′-cyclo-2'-deoxyguanosine generated in DNA by both endogenous oxidative stress and ionizing radiation are helix-distorting lesions and strong blocks for DNA replication and transcription. In duplex DNA, these lesions are repaired in the nucleotide excision repair (NER) pathway. However, lesions at DNA strand breaks are most likely poor...

Genetic studies and pharmacological studies produce conflicting results regarding a role for retinoic acid in meiosis of the female germ cells.

let-7 and miR-140 microRNAs coordinately regulate skeletal development.

Proc Natl Acad Sci U S A. 2013 Aug 12;

Authors: Papaioannou G, Inloes JB, Nakamura Y, Paltrinieri E, Kobayashi T

Abstract
MicroRNAs (miRNAs) play critical roles in multiple processes of skeletal development. A global reduction of miRNAs in growth plate chondrocytes results in defects in both proliferation and differentiation; however, specific microRNAs responsible for these defects have not been identified. In this study, we provide evidence that let-7 miRNAs and microRNA-140 (miR-140), among other miRNAs expressed in chondrocytes, play major roles in endochondral bone development. We overexpressed lin-28 homolog A (Lin28a) to inhibit let-7 miRNA biogenesis in growth plate chondrocytes. Lin28a overexpression efficiently and specifically reduced let-7 miRNAs and up-regulated let-7 target genes. However, unlike the previous notion that let-7 miRNAs inhibit proliferation and growth, suppression of let-7 miRNAs via Lin28a overexpression decreased proliferation in growth plate chondrocytes, likely through up-regulation of the let-7 target cell cycle regulators cell division cycle 34 (Cdc34) and E2F transcription factor 5 (E2F5). Deficiency of the chondrocyte-specific miRNA, miR-140, causes a differentiation defect in growth plate chondrocytes. Although either Lin28a overexpression or miR-140 deficiency alone caused only mild growth impairment, mice with both miR-140 deficiency and Lin28a overexpression in chondrocytes showed a dramatic growth defect. Deregulation of distinct processes in the absence of these miRNAs synergistically decreased the proliferating chondrocyte mass; miR-140 deficiency reduced differentiation into proliferating chondrocytes, whereas Lin28a overexpression decreased proliferation per se.

PMID: 23940373 [PubMed - as supplied by publisher]

Related Articles

Formaldehyde and epigenetic alterations: microRNA changes in the nasal epithelium of nonhuman primates.

Environ Health Perspect. 2013 Mar;121(3):339-44

Authors: Rager JE, Moeller BC, Doyle-Eisele M, Kracko D, Swenberg JA, Fry RC

Abstract
BACKGROUND: Formaldehyde is an air pollutant present in both indoor and outdoor atmospheres. Because of its ubiquitous nature, it is imperative to understand the mechanisms underlying formaldehyde-induced toxicity and carcinogenicity. MicroRNAs (miRNAs) can influence disease caused by environmental exposures, yet miRNAs are understudied in relation to formaldehyde. Our previous investigation demonstrated that formaldehyde exposure in human lung cells caused disruptions in miRNA expression profiles in vitro.
OBJECTIVES: Using an in vivo model, we set out to test the hypothesis that formaldehyde inhalation exposure significantly alters miRNA expression profiles within the nasal epithelium of nonhuman primates.
METHODS: Cynomolgus macaques were exposed by inhalation to approximately 0, 2, or 6 ppm formaldehyde for 6 hr/day for 2 consecutive days. Small RNAs were extracted from nasal samples and assessed for genome-wide miRNA expression levels. Transcriptional targets of formaldehyde-altered miRNAs were computationally predicted, analyzed at the systems level, and assessed using real-time reverse transcriptase polymerase chain reaction (RT-PCR).
RESULTS: Expression analysis revealed that 3 and 13 miRNAs were dysregulated in response to 2 and 6 ppm formaldehyde, respectively. Transcriptional targets of the miRNA with the greatest increase (miR-125b) and decrease (miR-142-3p) in expression were predicted and analyzed at the systems level. Enrichment was identified for miR-125b targeting genes involved in apoptosis signaling. The apoptosis-related targets were functionally tested using RT-PCR, where all targets showed decreased expression in formaldehyde-exposed samples.
CONCLUSIONS: Formaldehyde exposure significantly disrupts miRNA expression profiles within the nasal epithelium, and these alterations likely influence apoptosis signaling.

PMID: 23322811 [PubMed - indexed for MEDLINE]

Nature Protocols 8, 1743 (2013). doi:10.1038/nprot.2013.109

Authors: Anna Lyubimova, Shalev Itzkovitz, Jan Philipp Junker, Zi Peng Fan, Xuebing Wu & Alexander van Oudenaarden

We present a protocol for visualizing and quantifying single mRNA molecules in mammalian (mouse and human) tissues. In the approach described here, sets of about 50 short oligonucleotides, each labeled with a single fluorophore, are hybridized to target mRNAs in tissue sections. Each set binds

At first glance, Botryllus schlosseri has very little in common with humans. The small sea creature fuses together with others to form colonies that look like psychedelic blobs, encrusting rocks and seaweeds. It can reproduce asexually, and an entire individual can be regenerated from its blood vessels alone.

And yet, Botryllus is humans’ closest living invertebrate relative. (Invertebrates lack a spinal column.) Now, a group led by Stanford scientists has sequenced its genome, making it possible to find the genetic basis for some of the animal’s amazing regenerative abilities and immunity features, which potentially could be applied to human medicine.

Read more

In mice, a broadly acting RNA, lincRNA-Cox2, regulates the circuit that controls the inflammatory response. Authors: Susan Carpenter, Daniel Aiello, Maninjay K. Atianand, Emiliano P. Ricci, Pallavi Gandhi, Lisa L. Hall, Meg Byron, Brian Monks, Meabh Henry-Bezy, Jeanne B. Lawrence, Luke A. J. O’Neill, Melissa J. Moore, Daniel R. Caffrey, Katherine A. Fitzgerald

A large noncoding RNA uses folds within the chromosome to drive the spread of a chromatin repressive complex. [Also see Perspective by Dimond and Fraser] Authors: Jesse M. Engreitz, Amy Pandya-Jones, Patrick McDonel, Alexander Shishkin, Klara Sirokman, Christine Surka, Sabah Kadri, Jeffrey Xing, Alon Goren, Eric S. Lander, Kathrin Plath, Mitchell Guttman

A new generation of genetically modified crops will kill insects by silencing their genes. Author: Kai Kupferschmidt

A universal method for classifying neuronal subtypes will increase our understanding of the human brain. Authors: Hynek Wichterle, David Gifford, Esteba Mazzoni

The three-dimensional organization of the mammalian genome spatially guides the binding of an RNA to loci for silencing gene expression. [Also see Research Article by Engreitz et al.] Authors: Andrew Dimond, Peter Fraser

Shi-Yan Ng, Gireesh K. Bogu, Boon Seng Soh, Lawrence W. Stanton. Long noncoding RNAs (lncRNAs) are abundant in the mammalian transcriptome, and many are specifically expressed in the brain. We have identified a group of lncRNAs, including rhabdomyosarcoma 2-ass....

Mary Christie, Andreas Boland, Eric Huntzinger, Oliver Weichenrieder, Elisa Izaurralde. The PAN2-PAN3 deadenylase complex functions in general and miRNA-mediated mRNA degradation and is specifically recruited to miRNA targets by GW182/TNRC6 proteins. We describe the PAN3 adaptor prot....

Nature Biotechnology 31, 684 (2013). doi:10.1038/nbt.2652

Authors: Wei Li, Fei Teng, Tianda Li & Qi Zhou