Hyeshik Chang

The initiation of mammalian protein synthesis and mRNA scanning mechanism

Nature 500, 7462 (2013). doi:10.1038/nature12355

Authors: Ivan B. Lomakin & Thomas A. Steitz

During translation initiation in eukaryotes, the small ribosomal subunit binds messenger RNA at the 5′ end and scans in the 5′ to 3′ direction to locate the initiation codon, form the 80S initiation complex and start protein synthesis. This simple, yet intricate, process is guided

Nature Structural & Molecular Biology 20, 994 (2013). doi:10.1038/nsmb.2619

Authors: Claudia Keller, Raghavendran Kulasegaran-Shylini, Yukiko Shimada, Hans-Rudolf Hotz & Marc Bühler

Nilesh Chitnis, Dariusz Pytel, J. Alan Diehl.

Biology: Cell sex matters

Nature 500, 7460 (2013). doi:10.1038/500023a

Author: Elizabeth Pollitzer

Male and female cells can behave differently — it is time that researchers, journals and funders took this seriously, says Elizabeth Pollitzer.

The EMBO Journal. doi:10.1038/emboj.2013.164

Authors: Eva Hörmanseder, Thomas Tischer & Thomas U Mayer

Nature Genetics 45, 851 (2013). doi:10.1038/ng.2722

Author: Emily Niemitz

PARma is a complete data analysis software for AGO-PAR-CLIP experiments to identify target sites of microRNAs as well as the microRNA binding to these sites. It integrates specific characteristics of the experiments into a generative model. The model and a novel pattern discovery tool are iteratively applied to data to estimate seed activity probabilities, cluster confidence scores and to assign the most probable microRNA. Based on differential PAR-CLIP analysis and comparison to RIP-Chip data, we show that PARma is more accurate than existing approaches. PARma is available from http://www.bio.ifi.lmu.de/PARma.

A select group of microRNAs identified in blood samples can differentiate between Alzheimer's disease, other neurological disorders and age-matched healthy controls with high accuracy.Please see related Research article, http://genomebiology.com/2013/14/7/R78

Background: Hanwoo (Korean cattle), which originated from natural crossbreeding between taurine and zebu cattle, migrated to the Korean peninsula through North China. Hanwoo were raised as draft animals until the 1970s without the introduction of foreign germplasm. Since 1979, Hanwoo has been bred as beef cattle. Genetic variation was analyzed by whole-genome deep resequencing of a Hanwoo bull. The Hanwoo genome was compared to that of two other breeds, Black Angus and Holstein, and genes within regions of homozygosity were investigated to elucidate the genetic and genomic characteristics of Hanwoo. Results: The Hanwoo bull genome was sequenced to 45.6-fold coverage using the ABI SOLiD system. In total, 4.7 million single-nucleotide polymorphisms and 0.4 million small indels were identified by comparison with the Btau4.0 reference assembly. Of the total number of SNPs and indels, 58% and 87%, respectively, were novel. The overall genotype concordance between the SNPs and BovineSNP50 BeadChip data was 96.4%. Of 1.6 million genetic differences in Hanwoo, approximately 25,000 non-synonymous SNPs, splice-site variants, and coding indels (NS/SS/Is) were detected in 8,360 genes. Among 1,045 genes containing reliable specific NS/SS/Is in Hanwoo, 109 genes contained more than one novel damaging NS/SS/I. Of the genes containing NS/SS/Is, 610 genes were assigned as trait-associated genes. Moreover, 16, 78, and 51 regions of homozygosity (ROHs) were detected in Hanwoo, Black Angus, and Holstein, respectively. 'Regulation of actin filament length' was revealed as a significant gene ontology term and 25 trait-associated genes for meat quality and disease resistance were found in 753 genes that resided in the ROHs of Hanwoo. In Hanwoo, 43 genes were located in common ROHs between whole-genome resequencing and SNP chips in BTA2, 10, and 13 coincided with quantitative trait loci for meat fat traits. In addition, the common ROHs in BTA2 and 16 were in agreement between Hanwoo and Black Angus. Conclusions: We identified 4.7 million SNPs and 0.4 million small indels by whole-genome resequencing of a Hanwoo bull. Approximately 25,000 non-synonymous SNPs, splice-site variants, and coding indels (NS/SS/Is) were detected in 8,360 genes. Additionally, we found 25 trait-associated genes for meat quality and disease resistance among 753 genes that resided in the ROHs of Hanwoo. These findings will provide useful genomic information for identifying genes or casual mutations associated with economically important traits in cattle.

Nature Methods 10, 709 (2013). doi:10.1038/nmeth.2567

Authors: Christopher J Rowlands, Elijah Y S Yew & Peter T C So

Massive parallelization of scanning-based super-resolution imaging allows fast imaging of large fields of view.

Nature Reviews Genetics. doi:10.1038/nrg3542

Authors: Ehud Shapiro, Tamir Biezuner & Sten Linnarsson

Nature Reviews Molecular Cell Biology 14, 464 (2013). doi:10.1038/nrm3633

Author: Alison Schuldt

After mRNA transcription, ribosomes undergo controlled release and recycling of their components. In some cases, however, ribosomes can reinitiate translation at nearby open reading frames. Here, Pestova and colleagues use an in vitro translation system to determine the specific combinations of eukaryotic initiation factors

The 5′-untranslated region (5′-UTR) of mRNAs contains elements that affect expression, yet the rules by which these regions exert their effect are poorly understood. Here, we studied the impact of 5′-UTR sequences on protein levels in yeast, by constructing a large-scale library of mutants that differ only in the 10...

In mammalian cells, the cytoplasmic function of Dicer is well established, but evidence for its roles in the nucleus is fragmentary. This work demonstrates that the C-terminal double-stranded RNA binding domain of human Dicer (hDicer) includes a nuclear localization signal that depends on the importin-β family of import factors. The N-terminal helicase domain also contributes to hDicer localization. The authors conclude that hDicer is a shuttling protein that exhibits cytoplasmic localization at steady state.

Nature Protocols 8, 1620 (2013). doi:10.1038/nprot.2013.096

Authors: Chayasith Uttamapinant, Mateo I Sanchez, Daniel S Liu, Jennifer Z Yao & Alice Y Ting

This protocol describes an efficient method to site-specifically label cell-surface or purified proteins with chemical probes in two steps: probe incorporation mediated by enzymes (PRIME) followed by chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC). In the PRIME step, Escherichia colilipoic acid ligase (LplA)

by C. J. Zopf, Katie Quinn, Joshua Zeidman, Narendra Maheshri

The large variability in mRNA and protein levels found from both static and dynamic measurements in single cells has been largely attributed to random periods of transcription, often occurring in bursts. The cell cycle has a pronounced global role in affecting transcriptional and translational output, but how this influences transcriptional statistics from noisy promoters is unknown and generally ignored by current stochastic models. Here we show that variable transcription from the synthetic tetO promoter in S. cerevisiae is dominated by its dependence on the cell cycle. Real-time measurements of fluorescent protein at high expression levels indicate tetO promoters increase transcription rate ∼2-fold in S/G2/M similar to constitutive genes. At low expression levels, where tetO promoters are thought to generate infrequent bursts of transcription, we observe random pulses of expression restricted to S/G2/M, which are correlated between homologous promoters present in the same cell. The analysis of static, single-cell mRNA measurements at different points along the cell cycle corroborates these findings. Our results demonstrate that highly variable mRNA distributions in yeast are not solely the result of randomly switching between periods of active and inactive gene expression, but instead largely driven by differences in transcriptional activity between G1 and S/G2/M.

by Justin Ritz, Joshua S. Martin, Alain Laederach

Sequence conservation and co-variation of base pairs are hallmarks of structured RNAs. For certain RNAs (e.g. riboswitches), a single sequence must adopt at least two alternative secondary structures to effectively regulate the message. If alternative secondary structures are important to the function of an RNA, we expect to observe evolutionary co-variation supporting multiple conformations. We set out to characterize the evolutionary co-variation supporting alternative conformations in riboswitches to determine the extent to which alternative secondary structures are conserved. We found strong co-variation support for the terminator, P1, and anti-terminator stems in the purine riboswitch by extending alignments to include terminator sequences. When we performed Boltzmann suboptimal sampling on purine riboswitch sequences with terminators we found that these sequences appear to have evolved to favor specific alternative conformations. We extended our analysis of co-variation to classic alignments of group I/II introns, tRNA, and other classes of riboswitches. In a majority of these RNAs, we found evolutionary evidence for alternative conformations that are compatible with the Boltzmann suboptimal ensemble. Our analyses suggest that alternative conformations are selected for and thus likely play functional roles in even the most structured of RNAs.

Associations can be artificially created through reactivation of memory engrams in mouse dentate gyrus granule cells.

Authors: Steve Ramirez, Xu Liu, Pei-Ann Lin, Junghyup Suh, Michele Pignatelli, Roger L. Redondo, Tomás J. Ryan, Susumu Tonegawa

Guillermo Rodrigo, Thomas E. Landrain, Shensi Shen, Alfonso Jaramillo.

microRNAs (miRNAs) are post-transcriptional regulators of gene expression which may act as buffering agents to stabilize gene regulatory networks. Here, we identify two miRNAs which are maternally required for normal embryonic primordial germ cell development in Drosophila melanogaster. Embryos derived from miR-969 and miR-9c mutant mothers had on average reduced germ cell numbers. Intriguingly, this reduction correlated with an increase in the variance of this quantitative phenotypic trait. Analysis of an independent set of maternal mutant genotypes suggests that reduction of germ cell number need not lead to increased variance. Our observations are consistent with the hypothesis that miR-969 and miR-9c contribute to stabilizing the processes that control germ number, supporting phenotypic robustness.

Nature advance online publication 28 July 2013. doi:10.1038/nature12364

Authors: Zhigang Xue, Kevin Huang, Chaochao Cai, Lingbo Cai, Chun-yan Jiang, Yun Feng, Zhenshan Liu, Qiao Zeng, Liming Cheng, Yi E. Sun, Jia-yin Liu, Steve Horvath & Guoping Fan

Mammalian pre-implantation development is a complex process involving dramatic changes in the transcriptional architecture. We report here a comprehensive analysis of transcriptome dynamics from oocyte to morula in both human and mouse embryos, using single-cell RNA sequencing. Based on single-nucleotide variants in human blastomere messenger RNAs and paternal-specific single-nucleotide polymorphisms, we identify novel stage-specific monoallelic expression patterns for a significant portion of polymorphic gene transcripts (25 to 53%). By weighted gene co-expression network analysis, we find that each developmental stage can be delineated concisely by a small number of functional modules of co-expressed genes. This result indicates a sequential order of transcriptional changes in pathways of cell cycle, gene regulation, translation and metabolism, acting in a step-wise fashion from cleavage to morula. Cross-species comparisons with mouse pre-implantation embryos reveal that the majority of human stage-specific modules (7 out of 9) are notably preserved, but developmental specificity and timing differ between human and mouse. Furthermore, we identify conserved key members (or hub genes) of the human and mouse networks. These genes represent novel candidates that are likely to be key in driving mammalian pre-implantation development. Together, the results provide a valuable resource to dissect gene regulatory mechanisms underlying progressive development of early mammalian embryos.

Related Articles

Detecting and Comparing Non-Coding RNAs in the High-Throughput Era.

Int J Mol Sci. 2013;14(8):15423-58

Authors: Bussotti G, Notredame C, Enright AJ

Abstract
In recent years there has been a growing interest in the field of non-coding RNA. This surge is a direct consequence of the discovery of a huge number of new non-coding genes and of the finding that many of these transcripts are involved in key cellular functions. In this context, accurately detecting and comparing RNA sequences has become important. Aligning nucleotide sequences is a key requisite when searching for homologous genes. Accurate alignments reveal evolutionary relationships, conserved regions and more generally any biologically relevant pattern. Comparing RNA molecules is, however, a challenging task. The nucleotide alphabet is simpler and therefore less informative than that of amino-acids. Moreover for many non-coding RNAs, evolution is likely to be mostly constrained at the structural level and not at the sequence level. This results in very poor sequence conservation impeding comparison of these molecules. These difficulties define a context where new methods are urgently needed in order to exploit experimental results to their full potential. This review focuses on the comparative genomics of non-coding RNAs in the context of new sequencing technologies and especially dealing with two extremely important and timely research aspects: the development of new methods to align RNAs and the analysis of high-throughput data.

PMID: 23887659 [PubMed - in process]

Nature advance online publication 28 July 2013. doi:10.1038/nature12460

Authors: Bhaskar Chetnani & Alfonso Mondragón

A crystal structure of two bound RNA molecules not only provides insight into how regulatory riboswitch sequences affect messenger RNA expression, but also expands our understanding of RNA structure and architecture.

Inferring Potential microRNA-microRNA Associations Based on Targeting Propensity and Connectivity in the Context of Protein Interaction Network.

PLoS One. 2013;8(7):e69719

Authors: Sun J, Zhou M, Yang H, Deng J, Wang L, Wang Q

Abstract
MicroRNAs (miRNAs) are a group of small non-coding RNAs that play important regulatory roles at the post-transcriptional level. Although several computational methods have been developed to compare miRNAs, it is still a challenging and a badly needed task with the availability of various biological data resources. In this study, we proposed a novel graph theoretic property based computational framework and method, called miRFunSim, for quantifying the associations between miRNAs based on miRNAs targeting propensity and proteins connectivity in the integrated protein-protein interaction network. To evaluate the performance of our method, we applied the miRFunSim method to compute functional similarity scores of miRNA pairs between 100 miRNAs whose target genes have been experimentally supported and found that the functional similarity scores of miRNAs in the same family or in the same cluster are significantly higher compared with other miRNAs which are consistent with prior knowledge. Further validation analysis on experimentally verified miRNA-disease associations suggested that miRFunSim can effectively recover the known miRNA pairs associated with the same disease and achieve a higher AUC of 83.1%. In comparison with similar methods, our miRFunSim method can achieve more effective and more reliable performance for measuring the associations of miRNAs. We also conducted the case study examining liver cancer based on our method, and succeeded in uncovering the candidate liver cancer related miRNAs such as miR-34 which also has been proven in the latest study.

PMID: 23874989 [PubMed - in process]

The impact of age, biogenesis, and genomic clustering on Drosophila microRNA evolution.

RNA. 2013 Jul 23;

Authors: Mohammed J, Flynt AS, Siepel A, Lai EC

Abstract
The molecular evolutionary signatures of miRNAs inform our understanding of their emergence, biogenesis, and function. The known signatures of miRNA evolution have derived mostly from the analysis of deeply conserved, canonical loci. In this study, we examine the impact of age, biogenesis pathway, and genomic arrangement on the evolutionary properties of Drosophila miRNAs. Crucial to the accuracy of our results was our curation of high-quality miRNA alignments, which included nearly 150 corrections to ortholog calls and nucleotide sequences of the global 12-way Drosophilid alignments currently available. Using these data, we studied primary sequence conservation, normalized free-energy values, and types of structure-preserving substitutions. We expand upon common miRNA evolutionary patterns that reflect fundamental features of miRNAs that are under functional selection. We observe that melanogaster-subgroup-specific miRNAs, although recently emerged and rapidly evolving, nonetheless exhibit evolutionary signatures that are similar to well-conserved miRNAs and distinct from other structured noncoding RNAs and bulk conserved non-miRNA hairpins. This provides evidence that even young miRNAs may be selected for regulatory activities. More strikingly, we observe that mirtrons and clustered miRNAs both exhibit distinct evolutionary properties relative to solo, well-conserved miRNAs, even after controlling for sequence depth. These studies highlight the previously unappreciated impact of biogenesis strategy and genomic location on the evolutionary dynamics of miRNAs, and affirm that miRNAs do not evolve as a unitary class.

PMID: 23882112 [PubMed - as supplied by publisher]

Optical control of mammalian endogenous transcription and epigenetic states.

Nature. 2013 Jul 23;

Authors: Konermann S, Brigham MD, Trevino A, Hsu PD, Heidenreich M, Le Cong, Platt RJ, Scott DA, Church GM, Zhang F

Abstract
The dynamic nature of gene expression enables cellular programming, homeostasis and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high-precision spatiotemporal control of many cellular functions. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here we describe the development of light-inducible transcriptional effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical cofactors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of freely behaving mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states.

PMID: 23877069 [PubMed - as supplied by publisher]

NSun2-Mediated Cytosine-5 Methylation of Vault Noncoding RNA Determines Its Processing into Regulatory Small RNAs.

Cell Rep. 2013 Jul 17;

Authors: Hussain S, Sajini AA, Blanco S, Dietmann S, Lombard P, Sugimoto Y, Paramor M, Gleeson JG, Odom DT, Ule J, Frye M

Abstract
Autosomal-recessive loss of the NSUN2 gene has been identified as a causative link to intellectual disability disorders in humans. NSun2 is an RNA methyltransferase modifying cytosine-5 in transfer RNAs (tRNAs), yet the identification of cytosine methylation in other RNA species has been hampered by the lack of sensitive and reliable molecular techniques. Here, we describe miCLIP as an additional approach for identifying RNA methylation sites in transcriptomes. miCLIP is a customized version of the individual-nucleotide-resolution crosslinking and immunoprecipitation (iCLIP) method. We confirm site-specific methylation in tRNAs and additional messenger and noncoding RNAs (ncRNAs). Among these, vault ncRNAs contained six NSun2-methylated cytosines, three of which were confirmed by RNA bisulfite sequencing. Using patient cells lacking the NSun2 protein, we further show that loss of cytosine-5 methylation in vault RNAs causes aberrant processing into Argonaute-associated small RNA fragments that can function as microRNAs. Thus, impaired processing of vault ncRNA may contribute to the etiology of NSun2-deficiency human disorders.

PMID: 23871666 [PubMed - as supplied by publisher]

Nature advance online publication 21 July 2013. doi:10.1038/nature12355

Authors: Ivan B. Lomakin & Thomas A. Steitz

Related Articles

A competitive regulatory mechanism discriminates between juxtaposed splice sites and pri-miRNA structures.

Nucleic Acids Res. 2013 Jul 17;

Authors: Mattioli C, Pianigiani G, Pagani F

Abstract
We have explored the functional relationships between spliceosome and Microprocessor complex activities in a novel class of microRNAs (miRNAs), named Splice site Overlapping (SO) miRNAs, whose pri-miRNA hairpins overlap splice sites. We focused on the evolutionarily conserved SO miR-34b, and we identified two indispensable elements for recognition of its 3' splice site: a branch point located in the hairpin and a downstream purine-rich exonic splicing enhancer. In minigene systems, splicing inhibition owing to exonic splicing enhancer deletion or AG 3'ss mutation increases miR-34b levels. Moreover, small interfering-mediated silencing of Drosha and/or DGCR8 improves splicing efficiency and abolishes miR-34b production. Thus, the processing of this 3' SO miRNA is regulated in an antagonistic manner by the Microprocessor and the spliceosome owing to competition between these two machineries for the nascent transcript. We propose that this novel mechanism is commonly used to regulate the relative amount of SO miRNA and messenger RNA produced from primary transcripts.

PMID: 23863840 [PubMed - as supplied by publisher]

Related Articles

Genome-wide identification of genes regulated in trans by transposable element small interfering RNAs.

RNA Biol. 2013 Aug 1;10(8):1379-95

Authors: McCue AD, Nuthikattu S, Slotkin RK

Abstract
Transposable elements (TEs) are known to influence the regulation of neighboring genes through a variety of mechanisms. Additionally, it was recently discovered that TEs can regulate non-neighboring genes through the trans-acting nature of small interfering RNAs (siRNAs). When the epigenetic repression of TEs is lost, TEs become transcriptionally active, and the host cell acts to repress mutagenic transposition by degrading TE mRNAs into siRNAs. In this study, we have performed a genome-wide analysis in the model plant Arabidopsis thaliana and found that TE siRNA-based regulation of genic mRNAs is more pervasive than the two formerly characterized proof-of-principle examples. We identified 27 candidate genic mRNAs that do not contain a TE fragment but are regulated through partial complementarity by the accumulation of TE siRNAs and are therefore influenced by TE epigenetic activation. We have experimentally confirmed several gene targets and demonstrated that they respond to the accumulation of specific 21 nucleotide TE siRNAs that are incorporated into the Arabidopsis Argonaute1 protein. Additionally, we found that one TE siRNA specifically targets and inhibits the formation of a host protein that acts to repress TE activity, suggesting that TEs harbor and potentially evolutionarily select short sequences to act as suppressors of host TE repression.

PMID: 23863322 [PubMed - indexed for MEDLINE]