Hyeshik Chang

Related Articles

Small RNAs derived from structural non-coding RNAs.

Methods. 2013 Sep 1;63(1):76-84

Authors: Chen CJ, Heard E

Abstract
It has been shown in small RNA sequencing-based studies that some small RNA fragments are specifically processed from known structural non-coding RNAs, either through Dicer-dependent or Dicer-independent pathways. Although these small RNAs are often less abundant compared to microRNAs in normal mammalian tissues, they are always present in all sequenced libraries. In this paper, we use the ncPRO-seq pipeline, to describe different profiles of these small RNA fragments, and to discuss their potential processing pathways and functions. To assess whether more small RNA fragments can be detected in small RNA sequencing datasets, we decided to focus on small nuclear RNAs, abbreviated as snRNAs, which are associated with Sm ribonucleoproteins to form functional RNA-protein complexes. Here, we describe a group of small RNA fragments derived from snRNAs, which are typically highly enriched in regions bound by Sm proteins. Based on this, we propose the existence of a potential novel small RNA family associated with Sm proteins.

PMID: 23684746 [PubMed - indexed for MEDLINE]

Structure of a Eukaryotic RNase III Postcleavage Complex Reveals a Double-Ruler Mechanism for Substrate Selection.

Mol Cell. 2014 Apr 2;

Authors: Liang YH, Lavoie M, Comeau MA, Abou Elela S, Ji X

Abstract
Ribonuclease III (RNase III) enzymes are a family of double-stranded RNA (dsRNA)-specific endoribonucleases required for RNA maturation and gene regulation. Prokaryotic RNase III enzymes have been well characterized, but how eukaryotic RNase IIIs work is less clear. Here, we describe the structure of the Saccharomyces cerevisiae RNase III (Rnt1p) postcleavage complex and explain why Rnt1p binds to RNA stems capped with an NGNN tetraloop. The structure shows specific interactions between a structural motif located at the end of the Rnt1p dsRNA-binding domain (dsRBD) and the guanine nucleotide in the second position of the loop. Strikingly, structural and biochemical analyses indicate that the dsRBD and N-terminal domains (NTDs) of Rnt1p function as two rulers that measure the distance between the tetraloop and the cleavage site. These findings provide a framework for understanding eukaryotic RNase IIIs.

PMID: 24703949 [PubMed - as supplied by publisher]

Nature Biotechnology 32, 387 (2014). doi:10.1038/nbt.2851

Authors: Wenxue Zhao, Joshua L Pollack, Denitza P Blagev, Noah Zaitlen, Michael T McManus & David J Erle

Functional characterization of noncoding sequences is crucial for understanding the human genome and learning how genetic variation contributes to disease. 3′ untranslated regions (UTRs) are an important class of noncoding sequences, but their functions remain largely uncharacterized. We developed a method for massively parallel functional annotation of sequences from 3′ UTRs (fast-UTR) and used this approach to measure the effects of a total of >450 kilobases of 3′ UTR sequences from >2,000 human genes on steady-state mRNA abundance, mRNA stability and protein production. We found widespread regulatory effects on mRNA that were coupled to effects on mRNA stability and protein production. Furthermore, we discovered 87 novel cis-regulatory elements and measured the effects of genetic variation within known and novel 3′ UTR motifs. This work shows how massively parallel approaches can improve the functional annotation of noncoding sequences, advance our understanding of cis-regulatory mechanisms and quantify the effects of human genetic variation.

Nature Biotechnology 32, 341 (2014). doi:10.1038/nbt.2850

Authors: Nathan Boley, Marcus H Stoiber, Benjamin W Booth, Kenneth H Wan, Roger A Hoskins, Peter J Bickel, Susan E Celniker & James B Brown

LaSSO, a strategy for genome-wide mapping of intronic lariats and branch-points using RNA-seq.

Genome Res. 2014 Apr 7;

Authors: Bitton DA, Rallis C, Jeffares DC, Smith GC, Chen YY, Codlin S, Marguerat S, Bahler J

Abstract
Both canonical and alternative splicing of RNAs is governed by intronic sequence elements and produces transient lariat structures fastened by branch-points within introns. To map precisely the location of branch-points on a genomic scale, we developed LaSSO (Lariat Sequence Site Origin), a data-driven algorithm which utilizes RNA-seq data. Using fission yeast cells lacking the debranching enzyme Dbr1, LaSSO not only accurately identified canonical splicing events, but also pinpointed novel, but rare, exon-skipping events, which may reflect aberrantly spliced transcripts. Compromised intron turnover perturbed gene regulation at multiple levels, including splicing and protein translation. Notably, Dbr1 function was also critical for the expression of mitochondrial genes, and for the processing of self-spliced mitochondrial introns. LaSSO showed better sensitivity and accuracy than algorithms used for computational branch-point prediction or for empirical branch-point determination. Even when applied to a human data set acquired in the presence of debranching activity, LaSSO identified both canonical and exon skipping branch-points. LaSSO thus provides an effective approach for defining high-resolution maps of branch-site sequences and intronic elements on a genomic scale. LaSSO should be useful to validate introns and uncover branch-point sequences in any eukaryote, and it could be integrated to RNA-seq pipelines.

PMID: 24709818 [PubMed - as supplied by publisher]

There is growing scientific and industry interest, and also substantial NIH Common Fund investment, in extracellular RNA (exRNA) as a form of intercellular communication. Many uncertainties remain about what constitutes bona fide exRNA, how it is released to bodily fluids, and what function it may have. In the mix of provocative studies are some that suggest an influence of diet on exRNA content and function. This letter recommends caution in conclusions about diet-derived exRNA based on bioinformatic analysis.

Nature advance online publication 13 April 2014. doi:10.1038/nature13173

Authors: Barbara Treutlein, Doug G. Brownfield, Angela R. Wu, Norma F. Neff, Gary L. Mantalas, F. Hernan Espinoza, Tushar J. Desai, Mark A. Krasnow & Stephen R. Quake

The mammalian lung is a highly branched network in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that mediate gas exchange. Although this transformation has been studied by marker expression analysis and fate-mapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types are still incompletely known, in part because of the limited number of lineage markers and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations. Here we show that single-cell transcriptome analysis circumvents these problems and enables direct measurement of the various cell types and hierarchies in the developing lung. We used microfluidic single-cell RNA sequencing (RNA-seq) on 198 individual cells at four different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups by using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or the previous purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell-type diversity in the distal lung and led to the discovery of many previously unknown cell-type markers, including transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full life cycle of the alveolar type 2 cell lineage. This single-cell genomics approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.

The RNA world hypothesis describes a stage in the early evolution of life in which RNA served as genome and as the only genome-encoded catalyst. To test whether RNA world organisms could have used cyclic trimetaphosphate as an energy source, we developed an in vitro selection strategy for isolating ribozymes that catalyze the triphosphorylation of RNA 5'-hydroxyl groups with trimetaphosphate. Several active sequences were isolated, and one ribozyme was analyzed in more detail. The ribozyme was truncated to 96 nt, while retaining full activity. It was converted to a trans-format and reacted with rates of 0.16 min^–1 under optimal conditions. The secondary structure appears to contain a four-helical junction motif. This study showed that ribozymes can use trimetaphosphate to triphosphorylate RNA 5'-hydroxyl groups and suggested that RNA world organisms could have used trimetaphosphate as their energy source.

Protein-RNA interactions play important roles in many biological processes. Given the high cost and technique difficulties in experimental methods, computationally predicting the binding complexes from individual protein and RNA structures is pressingly needed, in which a reliable scoring function is one of the critical components. Here, we have developed a knowledge-based scoring function, referred to as ITScore-PR, for protein-RNA binding mode prediction by using a statistical mechanics-based iterative method. The pairwise distance-dependent atomic interaction potentials of ITScore-PR were derived from experimentally determined protein–RNA complex structures. For validation, we have compared ITScore-PR with 10 other scoring methods on four diverse test sets. For bound docking, ITScore-PR achieved a success rate of up to 86% if the top prediction was considered and up to 94% if the top 10 predictions were considered, respectively. For truly unbound docking, the respective success rates of ITScore-PR were up to 24 and 46%. ITScore-PR can be used stand-alone or easily implemented in other docking programs for protein–RNA recognition.

It has previously been assumed that the generally high stability of microRNAs (miRNAs) reflects their tight association with Argonaute (Ago) proteins, essential components of the RNA-induced silencing complex (RISC). However, recent data have suggested that the majority of mature miRNAs are not, in fact, Ago associated. Here, we demonstrate that endogenous human miRNAs vary widely, by >100-fold, in their level of RISC association and show that the level of Ago binding is a better indicator of inhibitory potential than is the total level of miRNA expression. While miRNAs of closely similar sequence showed comparable levels of RISC association in the same cell line, these varied between different cell types. Moreover, the level of RISC association could be modulated by overexpression of complementary target mRNAs. Together, these data indicate that the level of RISC association of a given endogenous miRNA is regulated by the available RNA targetome and predicts miRNA function.

Molecular biology: DNA regulator acts on RNA too

Nature 508, 7495 (2014). doi:10.1038/508152d

A DNA-binding protein that regulates several genes also attaches to RNA, revealing another way in which the protein acts as a 'master weaver' of the genome.Félix Recillas-Targa of the National Autonomous University of Mexico in Mexico City, Danny Reinberg of New York University's Langone

Nature advance online publication 09 April 2014. doi:10.1038/nature13238

Authors: Arthur Prindle, Jangir Selimkhanov, Howard Li, Ivan Razinkov, Lev S. Tsimring & Jeff Hasty

One promise of synthetic biology is the creation of genetic circuitry that enables the execution of logical programming in living cells. Such ‘wet programming’ is positioned to transform a wide and diverse swathe of biotechnology ranging from therapeutics and diagnostics to water treatment strategies. Although progress in the development of a library of genetic modules continues apace, a major challenge for their integration into larger circuits is the generation of sufficiently fast and precise communication between modules. An attractive approach is to integrate engineered circuits with host processes that facilitate robust cellular signalling. In this context, recent studies have demonstrated that bacterial protein degradation can trigger a precise response to stress by overloading a limited supply of intracellular proteases. Here we use protease competition to engineer rapid and tunable coupling of genetic circuits across multiple spatial and temporal scales. We characterize coupling delay times that are more than an order of magnitude faster than standard transcription-factor-based coupling methods (less than 1 min compared with ∼20–40 min) and demonstrate tunability through manipulation of the linker between the protein and its degradation tag. We use this mechanism as a platform to couple genetic clocks at the intracellular and colony level, then synchronize the multi-colony dynamics to reduce variability in both clocks. We show how the coupled clock network can be used to encode independent environmental inputs into a single time series output, thus enabling frequency multiplexing (information transmitted on a common channel by distinct frequencies) in a genetic circuit context. Our results establish a general framework for the rapid and tunable coupling of genetic circuits through the use of native ‘queueing’ processes such as competitive protein degradation.

Nature advance online publication 09 April 2014. doi:10.1038/nature13166

Authors: Yuexin Zhou, Shiyou Zhu, Changzu Cai, Pengfei Yuan, Chunmei Li, Yanyi Huang & Wensheng Wei

Targeted genome editing technologies are powerful tools for studying biology and disease, and have a broad range of research applications. In contrast to the rapid development of toolkits to manipulate individual genes, large-scale screening methods based on the complete loss of gene expression are only now beginning to be developed. Here we report the development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis. Using knockout library screens, we successfully identified the host genes essential for the intoxication of cells by anthrax and diphtheria toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity but will also enable the discovery of genes that participate in other biological processes.

Motivation: High-throughput sequencing has been used to probe RNA structures, by treating RNAs with reagents that preferentially cleave or mark certain nucleotides according to their local structures, followed by sequencing of the resulting fragments. The data produced contain valuable information for studying various RNA properties.

Results: We developed methods for statistically modeling these structure-probing data and extracting structural features from them. We show that the extracted features can be used to predict RNA ‘zipcodes’ in yeast, regions bound by the She complex in asymmetric localization. The prediction accuracy was better than using raw RNA probing data or sequence features. We further demonstrate the use of the extracted features in identifying binding sites of RNA binding proteins from whole-transcriptome global photoactivatable-ribonucleoside-enhanced cross-linking and immunopurification (gPAR-CLIP) data.

Availability: The source code of our implemented methods is available at http://yiplab.cse.cuhk.edu.hk/probrna/.

Contact: kevinyip@cse.cuhk.edu.hk

Supplementary information: Supplementary data are available at Bioinformatics online.

Motivation: Identifying subclonal mutations and their implications requires accurate estimation of mutant allele fractions from possibly duplicated sequencing reads. Removing duplicate reads assumes that polymerase chain reaction amplification from library constructions is the primary source. The alternative—sampling coincidence from DNA fragmentation—has not been systematically investigated.

Results: With sufficiently high-sequencing depth, sampling-induced read duplication is non-negligible, and removing duplicate reads can overcorrect read counts, causing systemic biases in variant allele fraction and copy number variation estimations. Minimal overcorrection occurs when duplicate reads are identified accounting for their mate reads, inserts are of a variety of lengths and samples are sequenced in separate batches. We investigate sampling-induced read duplication in deep sequencing data with 500x to 2000x duplicates-removed sequence coverage. We provide a quantitative solution to overcorrection and guidance for effective designs of deep sequencing platforms that facilitate accurate estimation of variant allele fraction and copy number variation.

Availability and implementation: A Python implementation is freely available at https://bitbucket.org/wanding/duprecover/overview.

Contact: wzhou1@mdanderson.org, kchen3@mdanderson.org

Supplementary information: Supplementary data are available at Bioinformatics online.

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A Model-Based Approach to Identify Binding Sites in CLIP-Seq Data.

PLoS One. 2014;9(4):e93248

Authors: Wang T, Chen B, Kim M, Xie Y, Xiao G

Abstract
Cross-linking immunoprecipitation coupled with high-throughput sequencing (CLIP-Seq) has made it possible to identify the targeting sites of RNA-binding proteins in various cell culture systems and tissue types on a genome-wide scale. Here we present a novel model-based approach (MiClip) to identify high-confidence protein-RNA binding sites from CLIP-seq datasets. This approach assigns a probability score for each potential binding site to help prioritize subsequent validation experiments. The MiClip algorithm has been tested in both HITS-CLIP and PAR-CLIP datasets. In the HITS-CLIP dataset, the signal/noise ratios of miRNA seed motif enrichment produced by the MiClip approach are between 17% and 301% higher than those by the ad hoc method for the top 10 most enriched miRNAs. In the PAR-CLIP dataset, the MiClip approach can identify ∼50% more validated binding targets than the original ad hoc method and two recently published methods. To facilitate the application of the algorithm, we have released an R package, MiClip (http://cran.r-project.org/web/packages/MiClip/index.html), and a public web-based graphical user interface software (http://galaxy.qbrc.org/tool_runner?tool_id=mi_clip) for customized analysis.

PMID: 24714572 [PubMed - in process]

This method paper presents a novel strategy based on the use of transcription activator-like effector nucleases (TALENs) together with homologous recombination (HR) to disrupt miRNA loci in cell lines. As such, it introduces a new tool that will be useful in many studies of miRNA function.

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Long noncoding RNAs in spermatogenesis: insights from recent high-throughput transcriptome studies.

Reproduction. 2014;147(5):R131-41

Authors: Luk AC, Chan WY, Rennert OM, Lee TL

Abstract
Spermatogenesis is a complex developmental process in which undifferentiated spermatogonia are differentiated into spermatocytes and spermatids through two rounds of meiotic division and finally giving rise to mature spermatozoa (sperm). These processes involve many testis- or male germ cell-specific gene products that undergo strict developmental regulations. As a result, identifying critical, regulatory genes controlling spermatogenesis provide the clues not only to the regulatory mechanism of spermatogenesis at the molecular level, but also to the identification of candidate genes for infertility or contraceptives development. Despite the biological importance in male germ cell development, the underlying mechanisms of stage-specific gene regulation and cellular transition during spermatogenesis remain largely elusive. Previous genomic studies on transcriptome profiling were largely limited to protein-coding genes. Importantly, protein-coding genes only account for a small percentage of transcriptome; the majority are noncoding transcripts that do not translate into proteins. Although small noncoding RNAs (ncRNAs) such as microRNAs, siRNAs, and Piwi-interacting RNAs are extensively investigated in male germ cell development, the role of long ncRNAs (lncRNAs), commonly defined as ncRNAs longer than 200 bp, is relatively unexplored. Herein, we summarize recent transcriptome studies on spermatogenesis and show examples that a subset of noncoding transcript population, known as lncRNAs, constitutes a novel regulatory target in spermatogenesis.

PMID: 24713396 [PubMed - in process]

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The dynamic epitranscriptome: N(6)-methyladenosine and gene expression control.

Nat Rev Mol Cell Biol. 2014 Apr 9;

Authors: Meyer KD, Jaffrey SR

Abstract
N(6)-methyladenosine (m(6)A) is a modified base that has long been known to be present in non-coding RNAs, ribosomal RNA, polyadenylated RNA and at least one mammalian mRNA. However, our understanding of the prevalence of this modification has been fundamentally redefined by transcriptome-wide m(6)A mapping studies, which have shown that m(6)A is present in a large subset of the transcriptome in specific regions of mRNA. This suggests that mRNA may undergo post-transcriptional methylation to regulate its fate and function, which is analogous to methyl modifications in DNA. Thus, the pattern of methylation constitutes an mRNA 'epitranscriptome'. The identification of adenosine methyltransferases ('writers'), m(6)A demethylating enzymes ('erasers') and m(6)A-binding proteins ('readers') is helping to define cellular pathways for the post-transcriptional regulation of mRNAs.

PMID: 24713629 [PubMed - as supplied by publisher]

Nature Reviews Genetics. doi:10.1038/nrg3707

Authors: Jeffrey Rogers & Richard A. Gibbs

by Sergio Ruiz-Carmona, Daniel Alvarez-Garcia, Nicolas Foloppe, A. Beatriz Garmendia-Doval, Szilveszter Juhos, Peter Schmidtke, Xavier Barril, Roderick E. Hubbard, S. David Morley

Identification of chemical compounds with specific biological activities is an important step in both chemical biology and drug discovery. When the structure of the intended target is available, one approach is to use molecular docking programs to assess the chemical complementarity of small molecules with the target; such calculations provide a qualitative measure of affinity that can be used in virtual screening (VS) to rank order a list of compounds according to their potential to be active. rDock is a molecular docking program developed at Vernalis for high-throughput VS (HTVS) applications. Evolved from RiboDock, the program can be used against proteins and nucleic acids, is designed to be computationally very efficient and allows the user to incorporate additional constraints and information as a bias to guide docking. This article provides an overview of the program structure and features and compares rDock to two reference programs, AutoDock Vina (open source) and Schrödinger's Glide (commercial). In terms of computational speed for VS, rDock is faster than Vina and comparable to Glide. For binding mode prediction, rDock and Vina are superior to Glide. The VS performance of rDock is significantly better than Vina, but inferior to Glide for most systems unless pharmacophore constraints are used; in that case rDock and Glide are of equal performance. The program is released under the Lesser General Public License and is freely available for download, together with the manuals, example files and the complete test sets, at http://rdock.sourceforge.net/

Macros in microRNA target identification: A comparative analysis of in silico, in vitro, and in vivo approaches to microRNA target identification.

RNA Biol. 2014 Apr 2;11(4)

Authors: Tarang S, Weston MD

Abstract
MicroRNAs (miRNAs) are short RNA molecules that modulate post-transcriptional gene expression by partial or incomplete base-pairing to the complementary sequences on their target genes. Sequence-based miRNA target gene recognition enables the utilization of computational methods, which are highly informative in identifying a subset of putative miRNA targets from the genome. Subsequently, single miRNA-target gene binding is evaluated experimentally by in vitro assays to validate and quantify the transcriptional or post-transcriptional effects of miRNA-target gene interaction. Although ex vivo approaches are instructive in providing a basis for further analyses, in vivo genetic studies are critical to determine the occurrence and biological relevance of miRNA targets under physiological conditions. In the present review, we summarize the important features of each of the experimental approaches, their technical and biological limitations, and future challenges in light of the complexity of miRNA target gene recognition.

PMID: 24717361 [PubMed - as supplied by publisher]

Background: MicroRNAs (miRNAs) are established regulators of development, cell identity and disease. Although nearly two thousand human miRNA genes are known and new ones are continuously discovered, no attempt has been made to gauge the total miRNA content of the human genome. Results: Employing an innovative computational method on massively pooled small RNA sequencing data, we report 2,469 novel human miRNA candidates of which 1,098 are validated by in-house and published experiments. Almost 300 candidates are robustly expressed in a neuronal cell system and are regulated during differentiation or when biogenesis factors Dicer, Drosha, DGCR8 or Ago2 are silenced. To improve expression profiling we devised a quantitative miRNA capture system. In a kidney cell system 400 candidates interact with DGCR8 at transcript positions that suggest miRNA hairpin recognition, and 1,000 of the new miRNA candidates interact with Ago1 or Ago2, indicating that they are directly bound by miRNA effector proteins. From kidney cell CLASH experiments, in which miRNA-target pairs are ligated and sequenced, we observe hundreds of interactions between novel miRNAs and mRNA targets. The novel miRNA candidates are specifically but lowly expressed, raising the possibility that not all may be functional. Interestingly, the majority are evolutionarily young and overrepresented in the human brain. Conclusions: In summary, we present evidence that the complement of human miRNA genes is substantially larger than anticipated, and that more are likely to be discovered in the future as more tissues and experimental conditions are sequenced to greater depth.

If there is a "secret of Stradivarius," top players can't detect it

Structures of part of the signal recognition complex help explain how newly synthesized proteins are inserted into membranes. Authors: Jan Timo Grotwinkel, Klemens Wild, Bernd Segnitz, Irmgard Sinning

The Period protein homolog LIN-42 negatively regulates microRNA biogenesis in C. elegans.

Dev Biol. 2014 Mar 31;

Authors: Van Wynsberghe PM, Finnegan EF, Stark T, Angelus EP, Homan KE, Yeo GW, Pasquinelli AE

Abstract
MicroRNAs (miRNAs) are small RNAs that post-transcriptionally regulate gene expression in many multicellular organisms. They are encoded in the genome and transcribed into primary (pri-) miRNAs before two processing steps that ultimately produce the mature miRNA. In order to generate the appropriate amount of a particular miRNA in the correct location at the correct time, proper regulation of miRNA biogenesis is essential. Here we identify the Period protein homolog LIN-42 as a new regulator of miRNA biogenesis in Caenorhabditis elegans. We mapped a spontaneous suppressor of the normally lethal let-7(n2853) allele to the lin-42 gene. Mutations in this allele (ap201) or a second lin-42 allele (n1089) caused increased mature let-7 miRNA levels at most time points when mature let-7 miRNA is normally expressed. Levels of pri-let-7 and a let-7 transcriptional reporter were also increased in lin-42(n1089) worms. These results indicate that LIN-42 normally represses pri-let-7 transcription and thus the accumulation of let-7 miRNA. This inhibition is not specific to let-7, as pri- and mature levels of lin-4 and miR-35 were also increased in lin-42 mutants. Furthermore, small RNA-seq analysis showed widespread increases in the levels of mature miRNAs in lin-42 mutants. Thus, we propose that the period protein homolog LIN-42 is a global regulator of miRNA biogenesis.

PMID: 24699545 [PubMed - as supplied by publisher]

Nature Protocols 9, 989 (2014). doi:10.1038/nprot.2014.058

Authors: Tim R Mercer, Michael B Clark, Joanna Crawford, Marion E Brunck, Daniel J Gerhardt, Ryan J Taft, Lars K Nielsen, Marcel E Dinger & John S Mattick

RNA sequencing (RNAseq) samples the majority of expressed genes infrequently, owing to the large size, complex splicing and wide dynamic range of eukaryotic transcriptomes. This results in sparse sequencing coverage that can hinder robust isoform assembly and quantification. RNA capture sequencing (CaptureSeq) addresses this challenge

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Transcriptional profiling of cells sorted by RNA abundance.

Nat Methods. 2014 Mar 30;

Authors: Klemm S, Semrau S, Wiebrands K, Mooijman D, Faddah DA, Jaenisch R, van Oudenaarden A

Abstract
We have developed a quantitative technique for sorting cells on the basis of endogenous RNA abundance, with a molecular resolution of 10-20 transcripts. We demonstrate efficient and unbiased RNA extraction from transcriptionally sorted cells and report a high-fidelity transcriptome measurement of mouse induced pluripotent stem cells (iPSCs) isolated from a heterogeneous reprogramming culture. This method is broadly applicable to profiling transcriptionally distinct cellular states without requiring antibodies or transgenic fluorescent proteins.

PMID: 24681693 [PubMed - as supplied by publisher]

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The applications of single-cell genomics.

Hum Mol Genet. 2013 Oct 15;22(R1):R22-6

Authors: Lovett M

Abstract
We all start out as a single totipotent cell that is programmed to produce a multicellular organism. How do individual cells make those complex developmental switches? How do single cells within a tissue or organ differ, how do they coordinate their actions or go astray in a disease process? These are long-standing and fundamental questions in biology that are now becoming tractable because of advances in microfluidics, DNA amplification and DNA sequencing. Methods for studying single-cell transcriptomes (or at least the polyadenylated mRNA fraction of it) are by far the furthest ahead and reveal remarkable heterogeneity between morphologically identical cells. The analysis of genomic DNA variation is not far behind. The other 'omics' of single cells pose greater technological obstacles, but they are progressing and promise to yield highly integrated large data sets in the near future.

PMID: 23922233 [PubMed - indexed for MEDLINE]

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The human transcriptome is enriched for miRNA-binding sites located in cooperativity-permitting distance.

RNA Biol. 2013 Jul;10(7):1125-35

Authors: Rinck A, Preusse M, Laggerbauer B, Lickert H, Engelhardt S, Theis FJ

Abstract
MiRNAs are short, non-coding RNAs that regulate gene expression post-transcriptionally through specific binding to mRNA. Deregulation of miRNAs is associated with various diseases and interference with miRNA function has proven therapeutic potential. Most mRNAs are thought to be regulated by multiple miRNAs and there is some evidence that such joint activity is enhanced if a short distance between sites allows for cooperative binding. Until now, however, the concept of cooperativity among miRNAs has not been addressed in a transcriptome-wide approach. Here, we computationally screened human mRNAs for distances between miRNA binding sites that are expected to promote cooperativity. We find that sites with a maximal spacing of 26 nucleotides are enriched for naturally occurring miRNAs compared with control sequences. Furthermore, miRNAs with similar characteristics as indicated by either co-expression within a specific tissue or co-regulation in a disease context are predicted to target a higher number of mRNAs cooperatively than unrelated miRNAs. These bioinformatic data were compared with genome-wide sets of biochemically validated miRNA targets derived by Argonaute crosslinking and immunoprecipitation (HITS-CLIP and PAR-CLIP). To ease further research into combined and cooperative miRNA function, we developed miRco, a database connecting miRNAs and respective targets involved in distance-defined cooperative regulation (mips.helmholtz-muenchen.de/mirco). In conclusion, our findings suggest that cooperativity of miRNA-target interaction is a widespread phenomenon that may play an important role in miRNA-mediated gene regulation.

PMID: 23696004 [PubMed - indexed for MEDLINE]