Hyeshik Chang

Related Articles

Dissecting noncoding and pathogen RNA-protein interactomes.

RNA. 2015 Jan;21(1):135-43

Authors: Flynn RA, Martin L, Spitale RC, Do BT, Sagan SM, Zarnegar B, Qu K, Khavari PA, Quake SR, Sarnow P, Chang HY

Abstract
RNA-protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA-protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA-protein recognition across the diversity of human and pathogen RNAs.

PMID: 25411354 [PubMed - indexed for MEDLINE]

Background: MicroRNAs (miRNAs) represent new and potentially informative diagnostic targets for disease detection and prognosis. However, little work exists documenting the effect of TRIzol, a common viral inactivation and nucleic acid extraction reagent, on miRNA purification. Here, we developed an optimized protocol for miRNA extraction from plasma samples by evaluating five different RNA extraction kits, TRIzol phase separation, purification additives, and initial plasma sample volume. This method was then used for downstream profiling of plasma miRNAs found in archived samples from one nonhuman primate (NHPs) experimentally challenged with Ebola virus by the aerosol route. Results: Comparison of real-time RT-PCR results for spiked-in and endogenous miRNA sequences determined extraction efficiency from five different RNA purification kits. These experiments showed that 50 μL plasma processed using the QIAGEN miRNeasy Mini Kit with 5 μg of glycogen as a co-precipitant yielded the highest recovery of endogenous miRNAs. Using this optimized protocol, miRNAs from archived plasma samples of one rhesus macaque challenged with aerosolized Ebola virus was profiled using a targeted real-time PCR array. A total of 519 of the 752 unique miRNAs assayed were present in the plasma samples at day 0 and day 7 (time of death) post-exposure. Statistical analyses revealed 25 sequences significantly up- or down-regulated between day 0 and day 7 post infection, validating the utility of the extraction method for plasma miRNA profiling. Conclusions: This study contributes to the knowledgebase of circulating miRNA extraction methods and expands on the potential applications of cell-free miRNA profiling for diagnostics and pathogenesis studies. Specifically, we optimized an extraction protocol for miRNAs from TRIzol-inactivated plasma samples that can be used for highly pathogenic viruses.

Background: RNA-Seq has become increasingly popular in transcriptome profiling. One aspect of transcriptome research is to quantify the expression levels of genomic elements, such as genes, their transcripts and exons. Acquiring a transcriptome expression profile requires genomic elements to be defined in the context of the genome. Multiple human genome annotation databases exist, including RefGene (RefSeq Gene), Ensembl, and the UCSC annotation database. The impact of the choice of an annotation on estimating gene expression remains insufficiently investigated. Results: In this paper, we systematically characterized the impact of genome annotation choice on read mapping and transcriptome quantification by analyzing a RNA-Seq dataset generated by the Human Body Map 2.0 Project. The impact of a gene model on mapping of non-junction reads is different from junction reads. For the RNA-Seq dataset with a read length of 75 bp, on average, 95% of non-junction reads were mapped to exactly the same genomic location regardless of which gene models was used. By contrast, this percentage dropped to 53% for junction reads. In addition, about 30% of junction reads failed to align without the assistance of a gene model, while 10–15% mapped alternatively. There are 21,958 common genes among RefGene, Ensembl, and UCSC annotations. When we compared the gene quantification results in RefGene and Ensembl annotations, 20% of genes are not expressed, and thus have a zero count in both annotations. Surprisingly, identical gene quantification results were obtained for only 16.3% (about one sixth) of genes. Approximately 28.1% of genes’ expression levels differed by 5% or higher, and of those, the relative expression levels for 9.3% of genes (equivalent to 2038) differed by 50% or greater. The case studies revealed that the gene definition differences in gene models frequently result in inconsistency in gene quantification. Conclusions: We demonstrated that the choice of a gene model has a dramatic effect on both gene quantification and differential analysis. Our research will help RNA-Seq data analysts to make an informed choice of gene model in practical RNA-Seq data analysis.

Background: In studies of development and aging, the expression of many genes has been shown to undergo drastic changes at mRNA and protein levels. The connection between mRNA and protein expression level changes, as well as the role of posttranscriptional regulation in controlling expression level changes in postnatal development and aging, remains largely unexplored. Results: Here, we survey mRNA and protein expression changes in the prefrontal cortex of humans and rhesus macaques over developmental and aging intervals of both species’ lifespans. We find substantial decoupling of mRNA and protein expression levels in aging, but not in development. Genes showing increased mRNA/protein disparity in primate brain aging form expression patterns conserved between humans and macaques are enriched in specific functions involving mammalian target of rapamycin (mTOR) signaling, mitochondrial function and neurodegeneration. Mechanistically, aging-dependent mRNA/protein expression decoupling could be linked to a specific set of RNA binding proteins and, to a lesser extent, to specific microRNAs. Conclusions: Increased decoupling of mRNA and protein expression profiles observed in human and macaque brain aging results in specific co-expression profiles composed of genes with shared functions and shared regulatory signals linked to specific posttranscriptional regulators. Genes targeted and predicted to be targeted by the aging-dependent posttranscriptional regulation are associated with biological processes known to play important roles in aging and lifespan extension. These results indicate the potential importance of posttranscriptional regulation in modulating aging-dependent changes in humans and other species.

An animal that can transfer gene-regulatory information from somatic cells to germ cells may be able to communicate changes in the soma from one generation to the next. In the worm Caenorhabditis elegans, expression of double-stranded RNA (dsRNA) in neurons can result in the export of dsRNA-derived mobile RNAs to...

In a functional genomics screen of mouse embryonic stem cells (ESCs) with nested hemizygous chromosomal deletions, we reveal that ribosomal protein (RP) genes are the most significant haploinsufficient determinants for embryoid body (EB) formation. Hemizygocity for three RP genes (Rps5, Rps14, or Rps28), distinguished by the proximity of their corresponding...

Nature journals offer double-blind review

Nature 518, 7539 (2015). doi:10.1038/518274b

Starting in March, Nature and the monthly Nature research journals will offer an alternative to conventional peer review. Authors will be able to request that their names and affiliations are withheld from reviewers of their papers — a form of peer review known as

Article

DNA methylation undergoes dynamic changes during development and cell differentiation. Here, by comparing DNA methylomes from different stages of embryonic development of the zebrafish, the authors suggest that developmental enhancers are a major target of DNA methylation changes during embryogenesis.

Nature Communications doi: 10.1038/ncomms7315

Authors: Hyung Joo Lee, Rebecca F. Lowdon, Brett Maricque, Bo Zhang, Michael Stevens, Daofeng Li, Stephen L. Johnson, Ting Wang

Article

Roquin is an RNA-binding protein that promotes the degradation of specific mRNAs and is crucial for the maintenance of peripheral immune tolerance. Here the authors show that, in addition to its target mRNAs, Roquin can bind miR-146a and the RISC component Ago2 to control homeostasis of both RNA species.

Nature Communications doi: 10.1038/ncomms7253

Authors: Monika Srivastava, Guowen Duan, Nadia J. Kershaw, Vicki Athanasopoulos, Janet H. C. Yeo, Toyoyuki Ose, Desheng Hu, Simon H. J. Brown, Slobodan Jergic, Hardip R. Patel, Alvin Pratama, Sashika Richards, Anil Verma, E. Yvonne Jones, Vigo Heissmeyer, Thomas Preiss, Nicholas E. Dixon, Mark M. W. Chong, Jeffrey J. Babon, Carola G. Vinuesa

Publication date: March 2015
Source:Trends in Genetics, Volume 31, Issue 3
Author(s): J.R. Mellin, Pascale Cossart
Bacterial riboswitches are elements present in the 5′-untranslated regions (UTRs) of mRNA molecules that bind to ligands and regulate the expression of downstream genes. Riboswitches typically regulate the expression of protein-coding genes. However, mechanisms of riboswitch-mediated regulation have recently been shown to be more diverse than originally thought, with reports showing that riboswitches can regulate the expression of noncoding RNAs and control the access of proteins, such as transcription termination factor Rho and RNase E, to a nascent RNA. Riboswitches are also increasingly used in biotechnology, with advances in the engineering of synthetic riboswitches and the development of riboswitch-based sensors. In this review we address the emerging roles and mechanisms of riboswitch-mediated regulation in natura and recent progress in the development of riboswitch-based technology.

Publication date: 19 February 2015
Source:Molecular Cell, Volume 57, Issue 4
Author(s): Jenna L. Jewell , Fabian Flores , Kun-Liang Guan
In this issue of Molecular Cell, Ye et al. (2015) demonstrate that mTORC1 globally regulates miRNA biogenesis under nutrient-rich conditions via the E3 ubiquitin ligase Mdm2, which promotes Drosha degradation.

Publication date: 19 March 2015
Source:Molecular Cell, Volume 57, Issue 6
Author(s): Daniel Peter , Cátia Igreja , Ramona Weber , Lara Wohlbold , Catrin Weiler , Linda Ebertsch , Oliver Weichenrieder , Elisa Izaurralde
The eIF4E-binding proteins (4E-BPs) represent a diverse class of translation inhibitors that are often deregulated in cancer cells. 4E-BPs inhibit translation by competing with eIF4G for binding to eIF4E through an interface that consists of canonical and non-canonical eIF4E-binding motifs connected by a linker. The lack of high-resolution structures including the linkers, which contain phosphorylation sites, limits our understanding of how phosphorylation inhibits complex formation. Furthermore, the binding mechanism of the non-canonical motifs is poorly understood. Here, we present structures of human eIF4E bound to 4E-BP1 and fly eIF4E bound to Thor, 4E-T, and eIF4G. These structures reveal architectural elements that are unique to 4E-BPs and provide insight into the consequences of phosphorylation. Guided by these structures, we designed and crystallized a 4E-BP mimic that shows increased repressive activity. Our studies pave the way for the rational design of 4E-BP mimics as therapeutic tools to decrease translation during oncogenic transformation.

Graphical abstract

Teaser

The eIF4E-binding proteins (4E-BPs) repress translation by competing with eIF4G for binding to eIF4E. Peter et al. crystallized complexes of several 4E-BPs bound to eIF4E, revealing common binding principles and providing a molecular explanation for competition efficiency and for the negative effect of phosphorylation on complex formation.

Publication date: 26 February 2015
Source:Cell, Volume 160, Issue 5
Author(s): Shannon Yan , Jin-Der Wen , Carlos Bustamante , Ignacio Tinoco Jr.
Programmed ribosomal frameshifting produces alternative proteins from a single transcript. −1 frameshifting occurs on Escherichia coli’s dnaX mRNA containing a slippery sequence AAAAAAG and peripheral mRNA structural barriers. Here, we reveal hidden aspects of the frameshifting process, including its exact location on the mRNA and its timing within the translation cycle. Mass spectrometry of translated products shows that ribosomes enter the −1 frame from not one specific codon but various codons along the slippery sequence and slip by not just −1 but also −4 or +2 nucleotides. Single-ribosome translation trajectories detect distinctive codon-scale fluctuations in ribosome-mRNA displacement across the slippery sequence, representing multiple ribosomal translocation attempts during frameshifting. Flanking mRNA structural barriers mechanically stimulate the ribosome to undergo back-and-forth translocation excursions, broadly exploring reading frames. Both experiments reveal aborted translation around mutant slippery sequences, indicating that subsequent fidelity checks on newly adopted codon position base pairings lead to either resumed translation or early termination.

Graphical abstract

Teaser

Programmed frameshifting allows the translation of alternative protein products from a single transcript. To achieve this, E. coli ribosomes undergo several translocation excursions to shift reading frames and access a range of codon positions.

Publication date: 17 February 2015
Source:Cell Reports, Volume 10, Issue 6
Author(s): Hana Antonicka , Eric A. Shoubridge
Cytoplasmic RNA granules play a central role in mRNA metabolism, but the importance of mitochondrial RNA granules remains relatively unexplored. We characterized their proteome and found that they contain a large toolbox of proteins dedicated to RNA metabolism. Investigation of four uncharacterized putative RNA-binding proteins—two RNA helicases, DHX30 and DDX28, and two proteins of the Fas-activated serine-threonine kinase (FASTKD) family, FASTKD2 and FASTKD5—demonstrated that both helicases and FASTKD2 are required for mitochondrial ribosome biogenesis. RNA-sequencing (RNA-seq) analysis showed that DDX28 and FASTKD2 bound the 16S rRNA. FASTKD5 is required for maturing precursor mRNAs that are not flanked by tRNAs and that therefore cannot be processed by the canonical mRNA maturation pathway. Silencing FASTKD5 rendered mature COX I mRNA almost undetectable, which severely reduced the synthesis of COX I, resulting in a complex IV assembly defect. These data demonstrate that mitochondrial RNA granules are centers for posttranscriptional RNA processing and the biogenesis of mitochondrial ribosomes.

Graphical abstract

Teaser

Antonicka and Shoubridge show that mitochondrial RNA granules contain a collection of proteins involved in RNA metabolism. Characterization of four RNA-binding proteins demonstrates that DDX28, DHX30, and FASTKD2 are necessary for ribosome biogenesis and that FASTKD5 is required for maturation of mRNAs that cannot be processed by the canonical pathway.

Related Articles

Regulatory small RNAs from the 3' regions of bacterial mRNAs.

Curr Opin Microbiol. 2015 Feb 9;24C:132-139

Authors: Miyakoshi M, Chao Y, Vogel J

Abstract
Most studies of small regulatory RNAs in bacteria have focussed on conserved transcripts in intergenic regions. However, several recent developments including single-nucleotide resolution transcriptome profiling by RNA-seq and increased knowledge of the cellular targets of the RNA chaperone Hfq suggest that the bacterial world of functional small RNAs is more diverse. One emerging class are small RNAs that are identical to the 3' regions of known mRNAs, but are produced either by transcription from internal promoters or by mRNA processing. Using several recently discovered examples of such sRNAs, we discuss their biogenesis and modes of action, and illustrate how they can facilitate mRNA crosstalk in various physiological processes.

PMID: 25677420 [PubMed - as supplied by publisher]

Related Articles

mTOR Signaling Regulates DROSHA Stability and miRNA Biogenesis.

Cancer Discov. 2015 Feb 12;

Authors:

Abstract
mTORC1 inhibits miRNA biogenesis in response to nutrient deprivation by decreasing DROSHA stability.

PMID: 25678580 [PubMed - as supplied by publisher]

Related Articles

Transcriptome Wide Annotation of Eukaryotic RNase III Reactivity and Degradation Signals.

PLoS Genet. 2015 Feb;11(2):e1005000

Authors: Gagnon J, Lavoie M, Catala M, Malenfant F, Elela SA

Abstract
Detection and validation of the RNA degradation signals controlling transcriptome stability are essential steps for understanding how cells regulate gene expression. Here we present complete genomic and biochemical annotations of the signals required for RNA degradation by the dsRNA specific ribonuclease III (Rnt1p) and examine its impact on transcriptome expression. Rnt1p cleavage signals are randomly distributed in the yeast genome, and encompass a wide variety of sequences, indicating that transcriptome stability is not determined by the recurrence of a fixed cleavage motif. Instead, RNA reactivity is defined by the sequence and structural context in which the cleavage sites are located. Reactive signals are often associated with transiently expressed genes, and their impact on RNA expression is linked to growth conditions. Together, the data suggest that Rnt1p reactivity is triggered by malleable RNA degradation signals that permit dynamic response to changes in growth conditions.

PMID: 25680180 [PubMed - as supplied by publisher]

Publication date: 26 February 2015
Source:Cell, Volume 160, Issue 5
Author(s): Itamar Harel , Bérénice A. Benayoun , Ben Machado , Param Priya Singh , Chi-Kuo Hu , Matthew F. Pech , Dario Riccardo Valenzano , Elisa Zhang , Sabrina C. Sharp , Steven E. Artandi , Anne Brunet
Aging is a complex process that affects multiple organs. Modeling aging and age-related diseases in the lab is challenging because classical vertebrate models have relatively long lifespans. Here, we develop the first platform for rapid exploration of age-dependent traits and diseases in vertebrates, using the naturally short-lived African turquoise killifish. We provide an integrative genomic and genome-editing toolkit in this organism using our de-novo-assembled genome and the CRISPR/Cas9 technology. We mutate many genes encompassing the hallmarks of aging, and for a subset, we produce stable lines within 2–3 months. As a proof of principle, we show that fish deficient for the protein subunit of telomerase exhibit the fastest onset of telomere-related pathologies among vertebrates. We further demonstrate the feasibility of creating specific genetic variants. This genome-to-phenotype platform represents a unique resource for studying vertebrate aging and disease in a high-throughput manner and for investigating candidates arising from human genome-wide studies. Video Abstract

Graphical abstract

Teaser

Aging is the number one risk factor for many human pathologies, yet it is challenging to study as existing vertebrate models are relatively long lived. The development of an integrative genome-to-phenotype platform in a naturally short-lived vertebrate, the African turquoise killifish, opens the door to high-throughput in vivo modeling of vertebrate aging and complex human diseases.

Publication date: 12 February 2015
Source:Cell, Volume 160, Issue 4
Author(s): Julien Pompon , Mariano A. Garcia-Blanco
Noncoding RNAs have regulatory capabilities that evolution harnesses to fulfill diverse functions. Lee et al. show that a noncoding RNA from Epstein-Barr virus recruits a host transcription factor to silence virus gene expression and propose that it does this through base-pairing with nascent viral transcripts.

Teaser

Noncoding RNAs have regulatory capabilities that evolution harnesses to fulfill diverse functions. Lee et al. show that a noncoding RNA from Epstein-Barr virus recruits a host transcription factor to silence virus gene expression and propose that it does this through base-pairing with nascent viral transcripts.

Related Articles

Signaling-Mediated Regulation of MicroRNA Processing.

Cancer Res. 2015 Feb 6;

Authors: Shen J, Hung MC

Abstract
miRNAs are important regulatory elements for gene expression that are involved in diverse physiologic and pathologic processes. Canonical miRNA biogenesis consists of a two-step processing, from primary transcripts (pri-miRNA) to precursor miRNAs (pre-miRNA) mediated by Drosha in the nucleus and from pre-miRNAs to mature miRNAs mediated by Dicer in the cytoplasm. Various routes of miRNA maturation that are tightly regulated by signaling cascades and specific to an individual or a subclass of miRNAs have been recently identified. Here, we review the current findings in signaling-mediated miRNA processing as well as their potential clinical relevance in cancer. Cancer Res; 75(5); 1-9. ©2014 AACR.

PMID: 25660948 [PubMed - as supplied by publisher]

RNA helicase DDX21 coordinates transcription and ribosomal RNA processing

Nature 518, 7538 (2015). 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.

Bigger is not better when it comes to lab size

Nature 518, 7538 (2015). doi:10.1038/518141f

Author: Chris Woolston

An analysis linking the number of researchers in a lab to productivity spurs online debate.

Publication date: 9 February 2015
Source:Cancer Cell, Volume 27, Issue 2
Author(s): Amy L. Walz , Ariadne Ooms , Samantha Gadd , Daniela S. Gerhard , Malcolm A. Smith , Jaime M. Guidry Auvil , Daoud Meerzaman , Qing-Rong Chen , Chih Hao Hsu , Chunhua Yan , Cu Nguyen , Ying Hu , Reanne Bowlby , Denise Brooks , Yussanne Ma , Andrew J. Mungall , Richard A. Moore , Jacqueline Schein , Marco A. Marra , Vicki Huff , Jeffrey S. Dome , Yueh-Yun Chi , Charles G. Mullighan , Jing Ma , David A. Wheeler , Oliver A. Hampton , Nadereh Jafari , Nicole Ross , Julie M. Gastier-Foster , Elizabeth J. Perlman
We report the most common single-nucleotide substitution/deletion mutations in favorable histology Wilms tumors (FHWTs) to occur within SIX1/2 (7% of 534 tumors) and microRNA processing genes (miRNAPGs) DGCR8 and DROSHA (15% of 534 tumors). Comprehensive analysis of 77 FHWTs indicates that tumors with SIX1/2 and/or miRNAPG mutations show a pre-induction metanephric mesenchyme gene expression pattern and are significantly associated with both perilobar nephrogenic rests and 11p15 imprinting aberrations. Significantly decreased expression of mature Let-7a and the miR-200 family (responsible for mesenchymal-to-epithelial transition) in miRNAPG mutant tumors is associated with an undifferentiated blastemal histology. The combination of SIX and miRNAPG mutations in the same tumor is associated with evidence of RAS activation and a higher rate of relapse and death.

Teaser

Walz et al. identify recurrent SIX1, SIX2, DROSHA, and DGCR8 mutations in pre-therapy favorable histology Wilms tumors and find that mutant tumors more often have blastemal histology. Importantly, patients whose tumors have SIX1 or SIX2 mutations together with DROSHA or DGCR8 mutations have poorer survival.

Publication date: 9 February 2015
Source:Cancer Cell, Volume 27, Issue 2
Author(s): Jenny Wegert , Naveed Ishaque , Romina Vardapour , Christina Geörg , Zuguang Gu , Matthias Bieg , Barbara Ziegler , Sabrina Bausenwein , Nasenien Nourkami , Nicole Ludwig , Andreas Keller , Clemens Grimm , Susanne Kneitz , Richard D. Williams , Tas Chagtai , Kathy Pritchard-Jones , Peter van Sluis , Richard Volckmann , Jan Koster , Rogier Versteeg , Tomas Acha , Maureen J. O’Sullivan , Peter K. Bode , Felix Niggli , Godelieve A. Tytgat , Harm van Tinteren , Marry M. van den Heuvel-Eibrink , Eckart Meese , Christian Vokuhl , Ivo Leuschner , Norbert Graf , Roland Eils , Stefan M. Pfister , Marcel Kool , Manfred Gessler
Blastemal histology in chemotherapy-treated pediatric Wilms tumors (nephroblastoma) is associated with adverse prognosis. To uncover the underlying tumor biology and find therapeutic leads for this subgroup, we analyzed 58 blastemal type Wilms tumors by exome and transcriptome sequencing and validated our findings in a large replication cohort. Recurrent mutations included a hotspot mutation (Q177R) in the homeo-domain of SIX1 and SIX2 in tumors with high proliferative potential (18.1% of blastemal cases); mutations in the DROSHA/DGCR8 microprocessor genes (18.2% of blastemal cases); mutations in DICER1 and DIS3L2; and alterations in IGF2, MYCN, and TP53, the latter being strongly associated with dismal outcome. DROSHA and DGCR8 mutations strongly altered miRNA expression patterns in tumors, which was functionally validated in cell lines expressing mutant DROSHA.

Graphical abstract

Teaser

Blastemal type Wilms tumors are associated with poor prognosis. Wegert et al. identify recurrent mutations in SIX1 and SIX2 that correlate with high proliferation and in DROSHA and DGCR8 that affect miRNA biogenesis, as well as a high frequency of IGF2 overexpression in these tumors.

Evaluation of Commercially Available RNA Amplification Kits for RNA Sequencing Using Very Low Input Amounts of Total RNA.

J Biomol Tech. 2015 Jan 29;

Authors: Shanker S, Paulson A, Edenberg HJ, Peak A, Perera A, Alekseyev YO, Beckloff N, Bivens NJ, Donnelly R, Gillaspy AF, Grove D, Gu W, Jafari N, Kerley-Hamilton JS, Lyons RH, Tepper C, Nicolet CM

Abstract
This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.Multiple recent publications on RNA sequencing (RNA-seq) have demonstrated the power of next-generation sequencing technologies in whole-transcriptome analysis. Vendor-specific protocols used for RNA library construction often require at least 100 ng total RNA. However, under certain conditions, much less RNA is available for library construction. In these cases, effective transcriptome profiling requires amplification of subnanogram amounts of RNA. Several commercial RNA amplification kits are available for amplification prior to library construction for next-generation sequencing, but these kits have not been comprehensively field evaluated for accuracy and performance of RNA-seq for picogram amounts of RNA. To address this, 4 types of amplification kits were tested with 3 different concentrations, from 5 ng to 50 pg, of a commercially available RNA. Kits were tested at multiple sites to assess reproducibility and ease of use. The human total reference RNA used was spiked with a control pool of RNA molecules in order to further evaluate quantitative recovery of input material. Additional control data sets were generated from libraries constructed following polyA selection or ribosomal depletion using established kits and protocols. cDNA was collected from the different sites, and libraries were synthesized at a single site using established protocols. Sequencing runs were carried out on the Illumina platform. Numerous metrics were compared among the kits and dilutions used. Overall, no single kit appeared to meet all the challenges of small input material. However, it is encouraging that excellent data can be recovered with even the 50 pg input total RNA.

PMID: 25649271 [PubMed - as supplied by publisher]

Most undergraduates give high ratings to research experiences. Studies report that these experiences improve participation and persistence, often by strengthening students’ views of themselves as scientists. Yet, the evidence for these claims is weak. More than half the 60 studies reviewed rely on self-report surveys or interviews. Rather than introducing new images of science, research experiences may reinforce flawed images especially of research practices and conceptual understanding. The most convincing studies show benefits for mentoring and for communicating the nature of science, but the ideas that students learn are often isolated or fragmented rather than integrated and coherent. Rigorous research is needed to identify ways to design research experiences so that they promote integrated understanding. These studies need powerful and generalizable assessments that can document student progress, help distinguish effective and ineffective aspects of the experiences, and illustrate how students interpret the research experiences they encounter. To create research experiences that meet the needs of interested students and make effective use of scarce resources, we encourage systematic, iterative studies with multiple indicators of success. Authors: Marcia C. Linn, Erin Palmer, Anne Baranger, Elizabeth Gerard, Elisa Stone

CLIPdb: a CLIP-seq database for protein-RNA interactions.

BMC Genomics. 2015 Feb 5;16(1):51

Authors: Yang YC, Di C, Hu B, Zhou M, Liu Y, Song N, Li Y, Umetsu J, Lu Z

Abstract
BackgroundRNA-binding proteins (RBPs) play essential roles in gene expression regulation through their interactions with RNA transcripts, including coding, canonical non-coding and long non-coding RNAs. Large amounts of crosslinking immunoprecipitation (CLIP)-seq data (including HITS-CLIP, PAR-CLIP, and iCLIP) have been recently produced to reveal transcriptome-wide binding sites of RBPs at the single-nucleotide level.DescriptionHere, we constructed a database, CLIPdb, to describe RBP-RNA interactions based on 395 publicly available CLIP-seq data sets for 111 RBPs from four organisms: human, mouse, worm and yeast. We consistently annotated the CLIP-seq data sets and RBPs, and developed a user-friendly interface for rapid navigation of the CLIP-seq data. We applied a unified computational method to identify transcriptome-wide binding sites, making the binding sites directly comparable and the data available for integration across different CLIP-seq studies. The high-resolution binding sites of the RBPs can be visualized on the whole-genome scale using a browser. In addition, users can browse and download the identified binding sites of all profiled RBPs by querying genes of interest, including both protein coding genes and non-coding RNAs.ConclusionManually curated metadata and uniformly identified binding sites of publicly available CLIP-seq data sets will be a foundation for further integrative and comparative analyses. With maintained up-to-date data sets and improved functionality, CLIPdb (http://clipdb.ncrnalab.org) will be a valuable resource for improving the understanding of post-transcriptional regulatory networks.

PMID: 25652745 [PubMed - as supplied by publisher]

Related Articles

Characterization of the mammalian miRNA turnover landscape.

Nucleic Acids Res. 2015 Feb 27;43(4):2326-41

Authors: Guo Y, Liu J, Elfenbein SJ, Ma Y, Zhong M, Qiu C, Ding Y, Lu J

Abstract
Steady state cellular microRNA (miRNA) levels represent the balance between miRNA biogenesis and turnover. The kinetics and sequence determinants of mammalian miRNA turnover during and after miRNA maturation are not fully understood. Through a large-scale study on mammalian miRNA turnover, we report the co-existence of multiple cellular miRNA pools with distinct turnover kinetics and biogenesis properties and reveal previously unrecognized sequence features for fast turnover miRNAs. We measured miRNA turnover rates in eight mammalian cell types with a combination of expression profiling and deep sequencing. While most miRNAs are stable, a subset of miRNAs, mostly miRNA*s, turnovers quickly, many of which display a two-step turnover kinetics. Moreover, different sequence isoforms of the same miRNA can possess vastly different turnover rates. Fast turnover miRNA isoforms are enriched for 5' nucleotide bias against Argonaute-(AGO)-loading, but also additional 3' and central sequence features. Modeling based on two fast turnover miRNA*s miR-222-5p and miR-125b-1-3p, we unexpectedly found that while both miRNA*s are associated with AGO, they strongly differ in HSP90 association and sensitivity to HSP90 inhibition. Our data characterize the landscape of genome-wide miRNA turnover in cultured mammalian cells and reveal differential HSP90 requirements for different miRNA*s. Our findings also implicate rules for designing stable small RNAs, such as siRNAs.

PMID: 25653157 [PubMed - indexed for MEDLINE]

Nature Structural & Molecular Biology 22, 167 (2015). doi:10.1038/nsmb.2938

Authors: Keunwan Park, Betty W Shen, Fabio Parmeggiani, Po-Ssu Huang, Barry L Stoddard & David Baker