Hyeshik Chang

An atlas of chromatoid body components.

RNA. 2014 Feb 19;

Authors: Meikar O, Vagin VV, Chalmel F, Sõstar K, Lardenois A, Hammell M, Jin Y, Da Ros M, Wasik KA, Toppari J, Hannon GJ, Kotaja N

Abstract
The genome of male germ cells is actively transcribed during spermatogenesis to produce phase-specific protein-coding mRNAs and a considerable amount of different noncoding RNAs. Ribonucleoprotein (RNP) granule-mediated RNA regulation provides a powerful means to secure the quality and correct expression of the requisite transcripts. Haploid spermatids are characterized by a unique, unusually large cytoplasmic granule, the chromatoid body (CB), which emerges during the switch between the meiotic and post-meiotic phases of spermatogenesis. To better understand the role of the CB in male germ cell differentiation, we isolated CBs from mouse testes and revealed its full RNA and protein composition. We showed that the CB is mainly composed of RNA-binding proteins and other proteins involved RNA regulation. The CB was loaded with RNA, including pachytene piRNAs, a diverse set of mRNAs, and a number of uncharacterized long noncoding transcripts. The CB was demonstrated to accumulate nascent RNA during all the steps of round spermatid differentiation. Our results revealed the CB as a large germ cell-specific RNP platform that is involved in the control of the highly complex transcriptome of haploid male germ cells.

PMID: 24554440 [PubMed - as supplied by publisher]

Related Articles

Reproductive status alters transcriptomic response to infection in female Drosophila melanogaster.

G3 (Bethesda). 2013 May;3(5):827-40

Authors: Short SM, Lazzaro BP

Abstract
Mating and consequent reproduction significantly reduce the ability of female Drosophila melanogaster to defend against systemic bacterial infection. The goal of the present study was to identify genes likely to inform the mechanism of this post-mating immunosuppression. We used microarrays to contrast genome-wide transcript levels in virgin vs. mated females before and after infection. Because the immunosuppressive effect of mating is contingent on the presence of a germline in females, we repeated the entire experiment by using female mutants that do not form a germline. We found that multiple genes involved in egg production show reduced expression in response to infection, and that this reduction is stronger in virgins than it is in mated females. In germline-less females, expression of egg-production genes was predictably low and not differentially affected by infection. We also identified several immune responsive genes that are differentially induced after infection in virgins vs. mated females. Immune genes affected by mating status and egg production genes altered by infection are candidates to inform the mechanism of the trade-off between mating and immune defense.

PMID: 23550122 [PubMed - indexed for MEDLINE]

Long non-coding rnas in disease and development.

Pathology. 2014 Feb;46 Suppl 1:S26

Authors: Dinger ME

Abstract
Approximately 98% of the human genome comprises non-coding DNA, the function of which is largely unknown. Intriguingly, more than 85% of single nucleotide polymorphisms identified as disease-associated by genome-wide association studies (GWAS) occur in non-coding regions.The relatively recent discovery of widespread transcription of potentially functional long non-coding RNAs (lncRNAs) led us to investigate whether or not GWAS hits in non-coding regions could be reconciled by the transcription of regulatory RNAs. As lncRNAs typically show highly developmental-stage- and tissue-specific expression, they cannot be easily detected by RNA-Seq, which requires exponentially greater depth to detect increasingly rare transcripts. To overcome this problem, we developed a technique termed RNA-Capture-Seq, which targets RNA sequencing to specific areas of the genome.We have used this approach to target 300 chromosomal regions identified by GWAS. Using RNA from diverse human tissues, we identify thousands of novel differentially expressed transcripts. Although functional investigation of these transcripts is still underway, these experiments bring an intriguing new perspective into our understanding of how information in the genome is encoded and have considerable potential to identify novel regulators, which may prove valuable as biomarkers and therapeutic targets, involved in disease and development.

PMID: 24557238 [PubMed - in process]

Chromatin Immunoprecipitation Approaches to Determine Co-transcriptional Nature of Splicing.

Methods Mol Biol. 2014;1126:315-23

Authors: Bieberstein NI, Straube K, Neugebauer KM

Abstract
Chromatin immunoprecipitation (ChIP) is a common method used to determine the position along DNA where an antigen is found. The method was initially devised for protein antigens that come in direct contact with genomic DNA, such as components of the transcriptional machinery and histones. However, ChIP can also be extended to antigens that bind RNA, as demonstrated by the specific localization of spliceosomal components to particular gene regions that correlate with when and where introns and exons are transcribed. The activities of any RNA binding protein can in principle be monitored using ChIP, and RNA dependency of binding can also be assessed through RNase treatment. Combined with qPCR or high-throughput sequencing, this method allows the detection of RNA bound proteins at individual genes or genome-wide. Here, we present a detailed protocol for "splicing factor ChIP" in tissue culture cells.

PMID: 24549674 [PubMed - in process]

Rebecca M. Terns, Michael P. Terns.

Construction of Ligand-Responsive MicroRNAs that Operate Through Inhibition of Drosha Processing.

Methods Mol Biol. 2014;1111:259-67

Authors: Beisel CL, Bloom RJ, Smolke CD

Abstract
MicroRNAs (miRNAs) offer powerful tools for targeted gene silencing in almost all eukaryotes. These tools have received considerable attention for their utility in both fundamental genetic studies and as therapeutic agents. Rendering individual microRNAs responsive to endogenous or exogenously applied molecules (or ligands) can improve the stringency of silencing and can mediate autonomous control. This chapter describes the construction of ligand-responsive miRNAs that undergo reduced processing and subsequent gene silencing when bound by the recognized ligand. Following a simple set of rules, the engineered microRNAs can be readily modified to target different sequences and to bind different ligands. Individual miRNAs also can be incorporated into the same transcript for tunable, multi-gene silencing.

PMID: 24549626 [PubMed - in process]

Landscape of genomic alterations in cervical carcinomas

Nature 506, 7488 (2014). doi:10.1038/nature12881

Authors: Akinyemi I. Ojesina, Lee Lichtenstein, Samuel S. Freeman, Chandra Sekhar Pedamallu, Ivan Imaz-Rosshandler, Trevor J. Pugh, Andrew D. Cherniack, Lauren Ambrogio, Kristian Cibulskis, Bjørn Bertelsen, Sandra Romero-Cordoba, Victor Treviño, Karla Vazquez-Santillan, Alberto Salido Guadarrama, Alexi A. Wright, Mara W. Rosenberg, Fujiko Duke, Bethany Kaplan, Rui Wang, Elizabeth Nickerson, Heather M. Walline, Michael S. Lawrence, Chip Stewart, Scott L. Carter, Aaron McKenna, Iram P. Rodriguez-Sanchez, Magali Espinosa-Castilla, Kathrine Woie, Line Bjorge, Elisabeth Wik, Mari K. Halle, Erling A. Hoivik, Camilla Krakstad, Nayeli Belem Gabiño, Gabriela Sofia Gómez-Macías, Lezmes D. Valdez-Chapa, María Lourdes Garza-Rodríguez, German Maytorena, Jorge Vazquez, Carlos Rodea, Adrian Cravioto, Maria L. Cortes, Heidi Greulich, Christopher P. Crum, Donna S. Neuberg, Alfredo Hidalgo-Miranda, Claudia Rangel Escareno, Lars A. Akslen, Thomas E. Carey, Olav K. Vintermyr, Stacey B. Gabriel, Hugo A. Barrera-Saldaña, Jorge Melendez-Zajgla, Gad Getz, Helga B. Salvesen & Matthew Meyerson

Cervical cancer is responsible for 10–15% of cancer-related deaths in women worldwide. The aetiological role of infection with high-risk human papilloma viruses (HPVs) in cervical carcinomas is well established. Previous studies have also implicated somatic mutations in PIK3CA, PTEN, TP53, STK11 and KRAS as well as several copy-number alterations in the pathogenesis of cervical carcinomas. Here we report whole-exome sequencing analysis of 115 cervical carcinoma–normal paired samples, transcriptome sequencing of 79 cases and whole-genome sequencing of 14 tumour–normal pairs. Previously unknown somatic mutations in 79 primary squamous cell carcinomas include recurrent E322K substitutions in the MAPK1 gene (8%), inactivating mutations in the HLA-B gene (9%), and mutations in EP300 (16%), FBXW7 (15%), NFE2L2 (4%), TP53 (5%) and ERBB2 (6%). We also observe somatic ELF3 (13%) and CBFB (8%) mutations in 24 adenocarcinomas. Squamous cell carcinomas have higher frequencies of somatic nucleotide substitutions occurring at cytosines preceded by thymines (Tp*C sites) than adenocarcinomas. Gene expression levels at HPV integration sites were statistically significantly higher in tumours with HPV integration compared with expression of the same genes in tumours without viral integration at the same site. These data demonstrate several recurrent genomic alterations in cervical carcinomas that suggest new strategies to combat this disease.

Maturation of rRNA precursors requires an ordered series of endonucleolytic cleavages. One such cleavage site, designated A', upstream of mature 18S rRNA has been observed in metazoa. The studies in this paper show that cleavage at A' is not a prerequisite for downstream processing events but may be a quality control step for pre-rRNA transcripts.

Martin Dutertre, Sarah Lambert, Aura Carreira, Mounira Amor-Guéret, Stéphan Vagner.

Motivation: During the past decade, new classes of non-coding RNAs (ncRNAs) and their unexpected functions were discovered. Stable secondary structure is the key feature of many non-coding RNAs. Taking into account huge amounts of genomic data, development of computational methods to survey genomes for structured RNAs remains an actual problem, especially when homologous sequences are not available for comparative analysis. Existing programs scan genomes with a fixed window by efficiently constructing a matrix of RNA minimum free energies. A wide range of lengths of structured RNAs necessitates the use of many different window lengths that substantially increases the output size and computational efforts.

Results: In this article, we present an algorithm RNASurface to efficiently scan genomes by constructing a matrix of significance of RNA secondary structures and to identify all locally optimal structured RNA segments up to a predefined size. RNASurface significantly improves precision of identification of known ncRNA in Bacillus subtilis.

Availability and implementation: RNASurface C source code is available from http://bioinf.fbb.msu.ru/RNASurface/downloads.html.

Contact: ruslansoldatov@gmail.com

Supplementary Information: Supplementary data are available at Bioinformatics online.

Motivation: Prior biological knowledge greatly facilitates the meaningful interpretation of gene-expression data. Causal networks constructed from individual relationships curated from the literature are particularly suited for this task, since they create mechanistic hypotheses that explain the expression changes observed in datasets.

Results: We present and discuss a suite of algorithms and tools for inferring and scoring regulator networks upstream of gene-expression data based on a large-scale causal network derived from the Ingenuity Knowledge Base. We extend the method to predict downstream effects on biological functions and diseases and demonstrate the validity of our approach by applying it to example datasets.

Availability: The causal analytics tools ‘Upstream Regulator Analysis', ‘Mechanistic Networks', ‘Causal Network Analysis' and ‘Downstream Effects Analysis' are implemented and available within Ingenuity Pathway Analysis (IPA, http://www.ingenuity.com).

Supplementary information: Supplementary material is available at Bioinformatics online.

Related Articles

New universal rules of eukaryotic translation initiation fidelity.

PLoS Comput Biol. 2013;9(7):e1003136

Authors: Zur H, Tuller T

Abstract
The accepted model of eukaryotic translation initiation begins with the scanning of the transcript by the pre-initiation complex from the 5'end until an ATG codon with a specific nucleotide (nt) context surrounding it is recognized (Kozak rule). According to this model, ATG codons upstream to the beginning of the ORF should affect translation. We perform for the first time, a genome-wide statistical analysis, uncovering a new, more comprehensive and quantitative, set of initiation rules for improving the cost of translation and its efficiency. Analyzing dozens of eukaryotic genomes, we find that in all frames there is a universal trend of selection for low numbers of ATG codons; specifically, 16-27 codons upstream, but also 5-11 codons downstream of the START ATG, include less ATG codons than expected. We further suggest that there is selection for anti optimal ATG contexts in the vicinity of the START ATG. Thus, the efficiency and fidelity of translation initiation is encoded in the 5'UTR as required by the scanning model, but also at the beginning of the ORF. The observed nt patterns suggest that in all the analyzed organisms the pre-initiation complex often misses the START ATG of the ORF, and may start translation from an alternative initiation start-site. Thus, to prevent the translation of undesired proteins, there is selection for nucleotide sequences with low affinity to the pre-initiation complex near the beginning of the ORF. With the new suggested rules we were able to obtain a twice higher correlation with ribosomal density and protein levels in comparison to the Kozak rule alone (e.g. for protein levels r=0.7 vs. r=0.31; p<10(-12)).

PMID: 23874179 [PubMed - indexed for MEDLINE]

The expression level of small non-coding RNAs derived from the first exon of protein-coding genes is predictive of cancer status.

EMBO Rep. 2014 Feb 17;

Authors: Zovoilis A, Mungall AJ, Moore R, Varhol R, Chu A, Wong T, Marra M, Jones SJ

Abstract
Small non-coding RNAs (smRNAs) are known to be significantly enriched near the transcriptional start sites of genes. However, the functional relevance of these smRNAs remains unclear, and they have not been associated with human disease. Within the cancer genome atlas project (TCGA), we have generated small RNA datasets for many tumor types. In prior cancer studies, these RNAs have been regarded as transcriptional "noise," due to their apparent chaotic distribution. In contrast, we demonstrate their striking potential to distinguish efficiently between cancer and normal tissues and classify patients with cancer to subgroups of distinct survival outcomes. This potential to predict cancer status is restricted to a subset of these smRNAs, which is encoded within the first exon of genes, highly enriched within CpG islands and negatively correlated with DNA methylation levels. Thus, our data show that genome-wide changes in the expression levels of small non-coding RNAs within first exons are associated with cancer.

PMID: 24534129 [PubMed - as supplied by publisher]

Molecular biology: Protein binding cannot subdue a lively RNA

Nature 506, 7488 (2014). doi:10.1038/nature13055

Authors: Kathleen B. Hall

Ribosomes, the cell's protein-synthesis machines, are assembled from their components in a defined order. It emerges that the first assembly step must overcome dynamic structural rearrangements. See Article p.334

Protein-guided RNA dynamics during early ribosome assembly

Nature 506, 7488 (2014). doi:10.1038/nature13039

Authors: Hajin Kim, Sanjaya C. Abeysirigunawarden, Ke Chen, Megan Mayerle, Kaushik Ragunathan, Zaida Luthey-Schulten, Taekjip Ha & Sarah A. Woodson

The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer

The transcription of rRNA is critical to all living cells and is tightly controlled at the level of chromatin structure. Although the widespread adoption of genomic technologies including chromatin immunoprecipitation with massively parallel short-read sequencing (ChIP-seq) has allowed for the interrogation of chromatin structure on a genome-wide scale, until recently rDNA has not been analyzed by this technique. We extended genomic analysis of rDNA to mouse (Mus musculus), in which rDNA is similar in structure but highly divergent in sequence compared with human rDNA. Comparison of rDNA histone marks between mouse embryonic stem cells (mESCs) and more differentiated mouse cell types revealed differences between pluripotent and differentiated states. We also observed substantial divergence in rDNA histone modification patterns between mESCs and human embryonic stem cells (hESCs). Surprisingly, we found that the pluripotency factor OCT4 was bound to rDNA in similar patterns in mESCs and hESCs. Extending this analysis, we found that an additional 17 pluripotency-associated factors were bound to rDNA in mESCs, suggesting novel modes of rDNA regulation in pluripotent cells. Taken together, our results provide a detailed view of rDNA chromatin structure in an important model system and enable high-resolution comparison of rDNA regulation between mouse and human.

The large number of RNA-binding proteins and translation factors encoded in the Drosophila and other metazoan genomes predicts widespread use of post-transcriptional regulation in cellular and developmental processes. Previous studies identified roles for several RNA-binding proteins in dendrite branching morphogenesis of Drosophila larval sensory neurons. To determine the larger contribution of post-transcriptional gene regulation to neuronal morphogenesis, we conducted an RNA interference screen to identify additional Drosophila proteins annotated as either RNA-binding proteins or translation factors that function in producing the complex dendritic trees of larval class IV dendritic arborization neurons. We identified 88 genes encoding such proteins whose knockdown resulted in aberrant dendritic morphology, including alterations in dendritic branch number, branch length, field size, and patterning of the dendritic tree. In particular, splicing and translation initiation factors were associated with distinct and characteristic phenotypes, suggesting that different morphogenetic events are best controlled at specific steps in post-transcriptional messenger RNA metabolism. Many of the factors identified in the screen have been implicated in controlling the subcellular distributions and translation of maternal messenger RNAs; thus, common post-transcriptional regulatory strategies may be used in neurogenesis and in the generation of asymmetry in the female germline and embryo.

Nature Reviews Genetics. doi:10.1038/nrg3700

Author: Isabel Lokody

Osteoclastogenesis is under the control of posttranscriptional and transcriptional events. However, posttranscriptional regulation of osteoclastogenesis is incompletely understood. CNOT3 is a component of the CCR4 family that regulates mRNA stability, but its function in bone is not known. Here, we show that Cnot3 deficiency by deletion of a single allele...

Endogenous RNA interference is driven by copy number.

Elife. 2014;3:e01581

Authors: Cruz C, Houseley J

Abstract
A plethora of non-protein coding RNAs are produced throughout eukaryotic genomes, many of which are transcribed antisense to protein-coding genes and could potentially instigate RNA interference (RNAi) responses. Here we have used a synthetic RNAi system to show that gene copy number is a key factor controlling RNAi for transcripts from endogenous loci, since transcripts from multi-copy loci form double stranded RNA more efficiently than transcripts from equivalently expressed single-copy loci. Selectivity towards transcripts from high-copy DNA is therefore an emergent property of a minimal RNAi system. The ability of RNAi to selectively degrade transcripts from high-copy loci would allow suppression of newly emerging transposable elements, but such a surveillance system requires transcription. We show that low-level genome-wide pervasive transcription is sufficient to instigate RNAi, and propose that pervasive transcription is part of a defense mechanism capable of directing a sequence-independent RNAi response against transposable elements amplifying within the genome. DOI: http://dx.doi.org/10.7554/eLife.01581.001.

PMID: 24520161 [PubMed]

Nature Protocols 9, 597 (2014). doi:10.1038/nprot.2014.036

Authors: Ruixue Duan, Xiaolei Zuo, Shutao Wang, Xiyun Quan, Dongliang Chen, Zhifei Chen, Lei Jiang, Chunhai Fan & Fan Xia

This protocol describes an isothermal amplification approach for ultrasensitive detection of specific microRNAs (miRNAs). It achieves this level of sensitivity through quadratic amplification of the target oligonucleotide by using a Bst DNA polymerase–induced strand-displacement reaction and a lambda exonuclease–aided recycling reaction. First, the

Alphaviruses use secondary structural elements in their genomic RNA to avoid host detection. Authors: Jennifer L. Hyde, Christina L. Gardner, Taishi Kimura, James P. White, Gai Liu, Derek W. Trobaugh, Cheng Huang, Marco Tonelli, Slobodan Paessler, Kiyoshi Takeda, William B. Klimstra, Gaya K. Amarasinghe, Michael S. Diamond

Sequencing of RNA from thousands of individual immune cells allows unbiased identification of cellular subtypes. Authors: Diego Adhemar Jaitin, Ephraim Kenigsberg, Hadas Keren-Shaul, Naama Elefant, Franziska Paul, Irina Zaretsky, Alexander Mildner, Nadav Cohen, Steffen Jung, Amos Tanay, Ido Amit

Kevin S. Bonham, Megan H. Orzalli, Kachiko Hayashi, Amaya I. Wolf, Christoph Glanemann, Wolfgang Weninger, Akiko Iwasaki, David M. Knipe, Jonathan C. Kagan. The Toll-like receptors (TLRs) of the innate immune system are unusual in that individual family members are located on different organelles, yet most activate a common signaling pathway important....

Manira Rayamajhi, Edward A. Miao, Nathaniel J. Moorman. The functional significance of protein diversification through translational regulation in mammals is largely unexplored. Brubaker et al. now describe the generation of two functionally distinct m....

Don Benjamin, Michael N. Hall. mTORC1 is activated primarily on the lysosome. Menon et al. and Demetriades et al. show that mTORC1 deactivation on the lysosome is determined by recruitment of its negative regulator, the tumor s....

Joseph V. Geisberg, Zarmik Moqtaderi, Xiaochun Fan, Fatih Ozsolak, Kevin Struhl. We measured half-lives of 21,248 mRNA 3′ isoforms in yeast by rapidly depleting RNA polymerase II from the nucleus and performing direct RNA sequencing throughout the decay process. Interestingly,....

Hiroshi Nishimasu, F. Ann Ran, Patrick D. Hsu, Silvana Konermann, Soraya I. Shehata, Naoshi Dohmae, Ryuichiro Ishitani, Feng Zhang, Osamu Nureki. The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in compl....

The mammalian target of rapamycin (mTOR) promotes cell growth and proliferation by promoting mRNA translation and increasing the protein synthetic capacity of the cell. Although mTOR globally promotes translation by regulating the mRNA 5' cap-binding protein eIF4E (eukaryotic initiation factor 4E), it also preferentially regulates the translation of certain classes of mRNA via unclear mechanisms. To help fill this gap in knowledge, we performed a quantitative proteomic screen to identify proteins that associate with the mRNA 5' cap in an mTOR-dependent manner. Using this approach, we identified many potential regulatory factors, including the putative RNA-binding protein LARP1 (La-related protein 1). Our results indicate that LARP1 associates with actively translating ribosomes via PABP and that LARP1 stimulates the translation of mRNAs containing a 5' terminal oligopyrimidine (TOP) motif, encoding for components of the translational machinery. We found that LARP1 associates with the mTOR complex 1 (mTORC1) and is required for global protein synthesis as well as cell growth and proliferation. Together, these data reveal important molecular mechanisms involved in TOP mRNA translation and implicate LARP1 as an important regulator of cell growth and proliferation.

Nature advance online publication 16 February 2014. doi:10.1038/nature12971

Authors: Daan C. Swarts, Matthijs M. Jore, Edze R. Westra, Yifan Zhu, Jorijn H. Janssen, Ambrosius P. Snijders, Yanli Wang, Dinshaw J. Patel, José Berenguer, Stan J. J. Brouns & John van der Oost

RNA interference is widely distributed in eukaryotes and has a variety of functions, including antiviral defence and gene regulation. All RNA interference pathways use small single-stranded RNA (ssRNA) molecules that guide proteins of the Argonaute (Ago) family to complementary ssRNA targets: RNA-guided RNA interference. The role of prokaryotic Ago variants has remained elusive, although bioinformatics analysis has suggested their involvement in host defence. Here we demonstrate that Ago of the bacterium Thermus thermophilus (TtAgo) acts as a barrier for the uptake and propagation of foreign DNA. In vivo, TtAgo is loaded with 5′-phosphorylated DNA guides, 13–25 nucleotides in length, that are mostly plasmid derived and have a strong bias for a 5′-end deoxycytidine. These small interfering DNAs guide TtAgo to cleave complementary DNA strands. Hence, despite structural homology to its eukaryotic counterparts, TtAgo functions in host defence by DNA-guided DNA interference.