Hyeshik Chang

Nature advance online publication 29 January 2014. doi:10.1038/nature13011

Authors: Samuel H. Sternberg, Sy Redding, Martin Jinek, Eric C. Greene & Jennifer A. Doudna

Nature Methods 11, 163 (2014). doi:10.1038/nmeth.2772

Authors: Saiful Islam, Amit Zeisel, Simon Joost, Gioele La Manno, Pawel Zajac, Maria Kasper, Peter Lönnerberg & Sten Linnarsson

Single-cell RNA sequencing (RNA-seq) is a powerful tool to reveal cellular heterogeneity, discover new cell types and characterize tumor microevolution. However, losses in cDNA synthesis and bias in cDNA amplification lead to severe quantitative errors. We show that molecular labels—random sequences that label individual molecules—can nearly eliminate amplification noise, and that microfluidic sample preparation and optimized reagents produce a fivefold improvement in mRNA capture efficiency.

Nature Methods 11, 113 (2014). doi:10.1038/nmeth.2837

Bar charts are too frequently used to communicate data that they cannot represent well. We strongly encourage the use of more appropriate plots to display statistical samples.

Nature Methods 11, 129 (2014). doi:10.1038/nmeth.2820

Nature Methods 11, 117 (2014). doi:10.1038/nmeth.2807

Authors: Marc Streit & Nils Gehlenborg

Creating a simple yet effective plot requires an understanding of data and tasks.

Nature Genetics 46, 99 (2014). doi:10.1038/ng.2887

Author: Brooke LaFlamme

The importance of RNA-protein interactions in controlling mRNA regulation and non-coding RNA function is increasingly appreciated. A variety of methods exist to comprehensively define RNA-protein interactions. We describe these methods and the considerations required for designing and interpreting these experiments.

Combining the prediction of interactions between mRNAs and RNA-binding proteins with experimental expression profiles uncovers novel regulatory paradigms concerning proliferation and differentiation processes.See related research, http://genomebiology.com/2014/15/1/R13

Genome Biology's editorial team discuss the highlights of our RBPome issue, which includes research, method, review and comment articles

Guest Editors John Rinn and Jernej Ule: why Genome Biology's RBPome issue coincides with an exciting time for RNA-protein interactions

Analysis of single-cell gene expression promises a more precise understanding of molecular mechanisms of a living system. Most techniques only allow studies of the expressions for limited numbers of gene species. When amplification of cDNA was carried out for analysing more genes, amplification biases were frequently reported. A non-biased and efficient global-amplification method, which uses a single-cell cDNA library immobilized on beads, was developed for analysing entire gene expressions for single cells. Every step in this analysis from reverse transcription to cDNA amplification was optimized. By removing degrading excess primers, the bias due to the digestion of cDNA was prevented. Since the residual reagents, which affect the efficiency of each subsequent reaction, could be removed by washing beads, the conditions for uniform and maximized amplification of cDNAs were achieved. The differences in the amplification rates for randomly selected eight genes were within 1.5-folds, which could be negligible for most of the applications of single-cell analysis. The global amplification gives a large amount of amplified cDNA (>100 μg) from a single cell (2-pg mRNA), and that amount is enough for downstream analysis. The proposed global-amplification method was used to analyse transcript ratios of multiple cDNA targets (from several copies to several thousand copies) quantitatively.

In many bacteria and archaea, small RNAs derived from clustered regularly interspaced short palindromic repeats (CRISPRs) associate with CRISPR-associated (Cas) proteins to target foreign DNA for destruction. In Type I and III CRISPR/Cas systems, the Cas6 family of endoribonucleases generates functional CRISPR-derived RNAs by site-specific cleavage of repeat sequences in precursor transcripts. CRISPR repeats differ widely in both sequence and structure, with varying propensity to form hairpin folds immediately preceding the cleavage site. To investigate the evolution of distinct mechanisms for the recognition of diverse CRISPR repeats by Cas6 enzymes, we determined crystal structures of two Thermus thermophilus Cas6 enzymes both alone and bound to substrate and product RNAs. These structures show how the scaffold common to all Cas6 endonucleases has evolved two binding sites with distinct modes of RNA recognition: one specific for a hairpin fold and the other for a single-stranded 5'-terminal segment preceding the hairpin. These findings explain how divergent Cas6 enzymes have emerged to mediate highly selective pre-CRISPR-derived RNA processing across diverse CRISPR systems.

Catalytic RNAs are attractive objects for studying molecular evolution. To understand how RNA libraries can evolve from randomness toward highly active catalysts, we analyze the original samples that led to the discovery of Diels–Alderase ribozymes by next-generation sequencing. Known structure-activity relationships are used to correlate abundance with catalytic performance. We find that efficient catalysts arose not just from selection for reactivity among the members of the starting library, but from improvement of less potent precursors by mutations. We observe changes in the ribozyme population in response to increasing selection pressure. Surprisingly, even after many rounds of enrichment, the libraries are highly diverse, suggesting that potential catalysts are more abundant in random space than generally thought. To highlight the use of next-generation sequencing as a tool for in vitro selections, we also apply this technique to a recent, less characterized ribozyme selection. Making use of the correlation between sequence evolution and catalytic activity, we predict mutations that improve ribozyme activity and validate them biochemically. Our study reveals principles underlying ribozyme in vitro selections and provides guidelines to render future selections more efficient, as well as to predict the conservation of key structural elements, allowing the rational improvement of catalysts.

Motivation: RNA-seq is replacing microarrays as the primary tool for gene expression studies. Many RNA-seq studies have used insufficient biological replicates, resulting in low statistical power and inefficient use of sequencing resources.

Results: We show the explicit trade-off between more biological replicates and deeper sequencing in increasing power to detect differentially expressed (DE) genes. In the human cell line MCF7, adding more sequencing depth after 10 M reads gives diminishing returns on power to detect DE genes, whereas adding biological replicates improves power significantly regardless of sequencing depth. We also propose a cost-effectiveness metric for guiding the design of large-scale RNA-seq DE studies. Our analysis showed that sequencing less reads and performing more biological replication is an effective strategy to increase power and accuracy in large-scale differential expression RNA-seq studies, and provided new insights into efficient experiment design of RNA-seq studies.

Availability and implementation: The code used in this paper is provided on: http://home.uchicago.edu/~jiezhou/replication/. The expression data is deposited in the Gene Expression Omnibus under the accession ID GSE51403.

Contact: kpwhite@uchicago.edu

Supplementary information: Supplementary data are available at Bioinformatics online.

Stepwise assembly of multiple Lin28 proteins on the terminal loop of let-7 miRNA precursors.

Nucleic Acids Res. 2014 Jan 21;

Authors: Desjardins A, Bouvette J, Legault P

Abstract
Lin28 inhibits the biogenesis of let-7 miRNAs through direct interactions with let-7 precursors. Previous studies have described seemingly inconsistent Lin28 binding sites on pre-let-7 RNAs. Here, we reconcile these data by examining the binding mechanism of Lin28 to the terminal loop of pre-let-7g (TL-let-7g) using biochemical and biophysical methods. First, we investigate Lin28 binding to TL-let-7g variants and short RNA fragments and identify three independent binding sites for Lin28 on TL-let-7g. We then determine that Lin28 assembles in a stepwise manner on TL-let-7g to form a stable 1:3 complex. We show that the cold-shock domain (CSD) of Lin28 is responsible for remodelling the terminal loop of TL-let-7g, whereas the NCp7-like domain facilitates the initial binding of Lin28 to TL-let-7g. This stable binding of multiple Lin28 molecules to the terminal loop of pre-let-7g extends to other precursors of the let-7 family, but not to other pre-miRNAs tested. We propose a model for stepwise assembly of the 1:1, 1:2 and 1:3 pre-let-7g/Lin28 complexes. Stepwise multimerization of Lin28 on pre-let-7 is required for maximum inhibition of Dicer cleavage for a least one member of the let-7 family and may be important for orchestrating the activity of the several factors that regulate let-7 biogenesis.

PMID: 24452802 [PubMed - as supplied by publisher]

Related Articles

PIPE-CLIP: a comprehensive online tool for CLIP-seq data analysis.

Genome Biol. 2014 Jan 22;15(1):R18

Authors: Chen B, Yun J, Kim MS, Mendell JT, Xie Y

Abstract
CLIP-seq is widely used to study genome-wide interactions between RNA-binding proteins and RNAs. However, there are few tools available to analyze CLIP-seq data, thus creating a bottleneck to the implementation of this methodology. Here, we present PIPE-CLIP, a Galaxy framework-based comprehensive online pipeline for reliable analysis of data generated by three types of CLIP-seq protocol: HITS-CLIP, PAR-CLIP and iCLIP. PIPE-CLIP provides both data processing and statistical analysis to determine candidate cross-linking regions, which are comparable to those regions identified from the original studies or using existing computational tools. PIPE-CLIP is available at http://pipeclip.qbrc.org/.

PMID: 24451213 [PubMed - as supplied by publisher]

X-inactivation: xist RNA uses chromosome contacts to coat the x.

Curr Biol. 2014 Jan 20;24(2):R80-2

Authors: Leung KN, Panning B

Abstract
The mechanisms by which Xist RNA associates with the X chromosome to mediate alterations in chromatin structure remain mysterious. Recent genome-wide Xist RNA distribution studies suggest that this long noncoding RNA uses 3-dimensional chromosome contacts to move to its sites of action.

PMID: 24456982 [PubMed - in process]

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: 24457730 [PubMed - in process]

Ribosome profiling: new views of translation, from single codons to genome scale.

Nat Rev Genet. 2014 Jan 28;

Authors: Ingolia NT

Abstract
Genome-wide analyses of gene expression have so far focused on the abundance of mRNA species as measured either by microarray or, more recently, by RNA sequencing. However, neither approach provides information on protein synthesis, which is the true end point of gene expression. Ribosome profiling is an emerging technique that uses deep sequencing to monitor in vivo translation. Studies using ribosome profiling have already provided new insights into the identity and the amount of proteins that are produced by cells, as well as detailed views into the mechanism of protein synthesis itself.

PMID: 24468696 [PubMed - as supplied by publisher]

Related Articles

Evaluating cell lines as tumour models by comparison of genomic profiles.

Nat Commun. 2013;4:2126

Authors: Domcke S, Sinha R, Levine DA, Sander C, Schultz N

Abstract
Cancer cell lines are frequently used as in vitro tumour models. Recent molecular profiles of hundreds of cell lines from The Cancer Cell Line Encyclopedia and thousands of tumour samples from the Cancer Genome Atlas now allow a systematic genomic comparison of cell lines and tumours. Here we analyse a panel of 47 ovarian cancer cell lines and identify those that have the highest genetic similarity to ovarian tumours. Our comparison of copy-number changes, mutations and mRNA expression profiles reveals pronounced differences in molecular profiles between commonly used ovarian cancer cell lines and high-grade serous ovarian cancer tumour samples. We identify several rarely used cell lines that more closely resemble cognate tumour profiles than commonly used cell lines, and we propose these lines as the most suitable models of ovarian cancer. Our results indicate that the gap between cell lines and tumours can be bridged by genomically informed choices of cell line models for all tumour types.

PMID: 23839242 [PubMed - indexed for MEDLINE]

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Landscape and variation of RNA secondary structure across the human transcriptome.

Nature. 2014 Jan 30;505(7485):706-9

Authors: Wan Y, Qu K, Zhang QC, Flynn RA, Manor O, Ouyang Z, Zhang J, Spitale RC, Snyder MP, Segal E, Chang HY

Abstract
In parallel to the genetic code for protein synthesis, a second layer of information is embedded in all RNA transcripts in the form of RNA structure. RNA structure influences practically every step in the gene expression program. However, the nature of most RNA structures or effects of sequence variation on structure are not known. Here we report the initial landscape and variation of RNA secondary structures (RSSs) in a human family trio (mother, father and their child). This provides a comprehensive RSS map of human coding and non-coding RNAs. We identify unique RSS signatures that demarcate open reading frames and splicing junctions, and define authentic microRNA-binding sites. Comparison of native deproteinized RNA isolated from cells versus refolded purified RNA suggests that the majority of the RSS information is encoded within RNA sequence. Over 1,900 transcribed single nucleotide variants (approximately 15% of all transcribed single nucleotide variants) alter local RNA structure. We discover simple sequence and spacing rules that determine the ability of point mutations to impact RSSs. Selective depletion of 'riboSNitches' versus structurally synonymous variants at precise locations suggests selection for specific RNA shapes at thousands of sites, including 3' untranslated regions, binding sites of microRNAs and RNA-binding proteins genome-wide. These results highlight the potentially broad contribution of RNA structure and its variation to gene regulation.

PMID: 24476892 [PubMed - in process]

Drosha-independent DGCR8/Pasha pathway regulates neuronal morphogenesis.

Proc Natl Acad Sci U S A. 2014 Jan 28;111(4):1421-6

Authors: Luhur A, Chawla G, Wu YC, Li J, Sokol NS

Abstract
Cleavage of microRNAs and mRNAs by Drosha and its cofactor Pasha/DGCR8 is required for animal development, but whether these proteins also have independent roles in development has been unclear. Known phenotypes associated with loss of either one of these two proteins are very similar and consistent with their joint function, even though both cofactors are involved with additional distinct RNA biogenesis pathways. Here, we report clear phenotypic differences between drosha and pasha/dgcr8 null alleles in two postembryonic lineages in the Drosophila brain: elimination of pasha/dgcr8 leads to defects that are not shared by drosha null mutations in the morphology of gamma neurons in the mushroom body lineage, as well as many neurons in the anterodorsal projection neuron lineage. These morphological defects are not detected in neurons that are genetically depleted of two additional microRNA pathway components, dicer-1 and argonaute1, indicating that they are not due to loss of microRNA activity. They are, however, phenocopied by a newly identified recessive gain-of-function allele in drosha that probably interferes with the microRNA independent functions of Pasha/DGCR8. These data therefore identify a general Drosha-independent DGCR8/Pasha pathway that promotes proper morphology in multiple neuronal lineages. Given that reduction of human DGCR8/Pasha may contribute to the cognitive and behavioral characteristics of DiGeorge syndrome patients, disruption of this newly described pathway could underlie human neurological disease.

PMID: 24474768 [PubMed - in process]

Related Articles

Poly(A)-tail profiling reveals an embryonic switch in translational control.

Nature. 2014 Jan 29;

Authors: Subtelny AO, Eichhorn SW, Chen GR, Sive H, Bartel DP

Abstract
Poly(A) tails enhance the stability and translation of most eukaryotic messenger RNAs, but difficulties in globally measuring poly(A)-tail lengths have impeded greater understanding of poly(A)-tail function. Here we describe poly(A)-tail length profiling by sequencing (PAL-seq) and apply it to measure tail lengths of millions of individual RNAs isolated from yeasts, cell lines, Arabidopsis thaliana leaves, mouse liver, and zebrafish and frog embryos. Poly(A)-tail lengths were conserved between orthologous mRNAs, with mRNAs encoding ribosomal proteins and other 'housekeeping' proteins tending to have shorter tails. As expected, tail lengths were coupled to translational efficiencies in early zebrafish and frog embryos. However, this strong coupling diminished at gastrulation and was absent in non-embryonic samples, indicating a rapid developmental switch in the nature of translational control. This switch complements an earlier switch to zygotic transcriptional control and explains why the predominant effect of microRNA-mediated deadenylation concurrently shifts from translational repression to mRNA destabilization.

PMID: 24476825 [PubMed - as supplied by publisher]

by Chengtian Zhao, Viktoria Andreeva, Yann Gibert, Melissa LaBonty, Victoria Lattanzi, Shubhangi Prabhudesai, Yi Zhou, Leonard Zon, Kathleen L. McCann, Susan Baserga, Pamela C. Yelick

During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome.

by Weixiong Zhang

Takashi S. Miki, Hannes Richter, Stefan Rüegger, Helge Großhans. XRN2 is an essential eukaryotic exoribonuclease that processes and degrades various substrates. Here we identify the previously uncharacterized protein R05D11.6/PAXT-1 as a subunit of an XRN2 comp....

Jun Liang, Laurent Lacroix, Adrien Gamot, Suresh Cuddapah, Sophie Queille, Priscillia Lhoumaud, Pierre Lepetit, Pascal G.P. Martin, Jutta Vogelmann, Franck Court, Magali Hennion, Gaël Micas, Serge Urbach, Olivier Bouchez, Marcelo Nöllmann, Keji Zhao, Eldon Emberly, Olivier Cuvier. Eukaryotic chromosomes are partitioned into topologically associating domains (TADs) that are demarcated by distinct insulator-binding proteins (IBPs) in Drosophila. Whether IBPs regulate s....

Yuan Tian, Dhirendra K. Simanshu, Jin-Biao Ma, Jong-Eun Park, Inha Heo, V. Narry Kim, Dinshaw J. Patel. We have solved two families of crystal structures of the human Dicer “platform-PAZ-connector helix” cassette in complex with small interfering RNAs (siRNAs). The structures possess two adjacently ....

G. Ekin Atilla-Gokcumen, Eleonora Muro, Josep Relat-Goberna, Sofia Sasse, Anne Bedigian, Margaret L. Coughlin, Sergi Garcia-Manyes, Ulrike S. Eggert. Although massive membrane rearrangements occur during cell division, little is known about specific roles that lipids might play in this process. We report that the lipidome changes with the cell ....

Yuyu Niu, Bin Shen, Yiqiang Cui, Yongchang Chen, Jianying Wang, Lei Wang, Yu Kang, Xiaoyang Zhao, Wei Si, Wei Li, Andy Peng Xiang, Jiankui Zhou, Xuejiang Guo, Ye Bi, Chenyang Si, Bian Hu, Guoying Dong, Hong Wang, Zuomin Zhou, Tianqing Li, Tao Tan, Xiuqiong Pu, Fang Wang, Shaohui Ji, Qi Zhou, Xingxu Huang, Weizhi Ji, Jiahao Sha. Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of monkeys in biomedical researches has been significantly hindered ....