Hyeshik Chang

New therapeutic strategies are needed to treat infections caused by drug-resistant bacteria, which constitute a major growing threat to human health. Here, we use a high-throughput technology to identify combinatorial genetic perturbations that can enhance the killing of drug-resistant bacteria with antibiotic treatment. This strategy, Combinatorial Genetics En Masse (CombiGEM),...

A proteogenomics approach integrating proteomics and ribosome profiling increases the efficiency of protein identification and enables the discovery of alternative translation start sites.

Proteomics. 2014 Aug 22;

Authors: Koch A, Gawron D, Steyaert S, Ndah E, Crappé J, Keulenaer SD, Meester ED, Ma M, Shen B, Gevaert K, Criekinge WV, Damme PV, Menschaert G

Abstract
Next-generation transcriptome sequencing is increasingly integrated with mass spectrometry to enhance MS-based protein and peptide identification. Recently, a breakthrough in transcriptome analysis was achieved with the development of ribosome profiling (ribo-seq). This technology is based on the deep sequencing of ribosome-protected mRNA fragments, thereby enabling the direct observation of in vivo protein synthesis at the transcript level. In order to explore the impact of a ribo-seq-derived protein sequence search space on MS/MS spectrum identification, we performed a comprehensive proteome study on a human cancer cell line, using both shotgun and N-terminal proteomics, next to ribosome profiling, which was used to delineate (alternative) translational reading-frames. By including protein-level evidence of sample-specific genetic variation and alternative translation, this strategy improved the identification score of 69 proteins and identified 22 new proteins in the shotgun experiment. Furthermore, we discovered 18 new alternative translation start sites in the N-terminal proteomics data and observed a correlation between the quantitative measures of ribo-seq and shotgun proteomics with a Pearson correlation coefficient ranging from 0.483 to 0.664. Overall, this study demonstrated the benefits of ribosome profiling for MS-based protein and peptide identification and we believe this approach could develop into a common practice for next-generation proteomics. This article is protected by copyright. All rights reserved.

PMID: 25156699 [PubMed - as supplied by publisher]

Nature Reviews Genetics. doi:10.1038/nrg3772

Authors: Nongluk Plongthongkum, Dinh H. Diep & Kun Zhang

Comparative analysis of regulatory information and circuits across distant species

Nature 512, 7515 (2014). doi:10.1038/nature13668

Authors: Alan P. Boyle, Carlos L. Araya, Cathleen Brdlik, Philip Cayting, Chao Cheng, Yong Cheng, Kathryn Gardner, LaDeana W. Hillier, Judith Janette, Lixia Jiang, Dionna Kasper, Trupti Kawli, Pouya Kheradpour, Anshul Kundaje, Jingyi Jessica Li, Lijia Ma, Wei Niu, E. Jay Rehm, Joel Rozowsky, Matthew Slattery, Rebecca Spokony, Robert Terrell, Dionne Vafeados, Daifeng Wang, Peter Weisdepp, Yi-Chieh Wu, Dan Xie, Koon-Kiu Yan, Elise A. Feingold, Peter J. Good, Michael J. Pazin, Haiyan Huang, Peter J. Bickel, Steven E. Brenner, Valerie Reinke, Robert H. Waterston, Mark Gerstein, Kevin P. White, Manolis Kellis & Michael Snyder

Despite the large evolutionary distances between metazoan species, they can show remarkable commonalities in their biology, and this has helped to establish fly and worm as model organisms for human biology. Although studies of individual elements and factors have explored similarities in gene regulation, a large-scale comparative analysis of basic principles of transcriptional regulatory features is lacking. Here we map the genome-wide binding locations of 165 human, 93 worm and 52 fly transcription regulatory factors, generating a total of 1,019 data sets from diverse cell types, developmental stages, or conditions in the three species, of which 498 (48.9%) are presented here for the first time. We find that structural properties of regulatory networks are remarkably conserved and that orthologous regulatory factor families recognize similar binding motifs in vivo and show some similar co-associations. Our results suggest that gene-regulatory properties previously observed for individual factors are general principles of metazoan regulation that are remarkably well-preserved despite extensive functional divergence of individual network connections. The comparative maps of regulatory circuitry provided here will drive an improved understanding of the regulatory underpinnings of model organism biology and how these relate to human biology, development and disease.

Publication date: September 2014
Source:Trends in Genetics, Volume 30, Issue 9
Author(s): Ralf-Peter Jansen , Dierk Niessing , Sebastian Baumann , Michael Feldbrügge
Active transport and local translation of mRNAs ensure the appropriate spatial organization of proteins within cells. Recent work has shown that this process is intricately connected to membrane trafficking. Here, we focus on new findings obtained in fungal model systems. Important highlights are that RNA-binding proteins recognize cargo mRNA synergistically and that mRNAs are co-transported with membranous compartments such as the endoplasmic reticulum (ER) and endosomes. We further discuss a novel concept of endosome-coupled translation that loads shuttling endosomes with septin cargo, a process important for correct septin filamentation. Interestingly, evidence is accumulating that RNA and membrane trafficking are also tightly interwoven in higher eukaryotes, suggesting that this phenomenon is a common theme and not an exception restricted to fungi.

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Determination of in vivo RNA kinetics using RATE-seq.

RNA. 2014 Aug 26;

Authors: Neymotin B, Athanasiadou R, Gresham D

Abstract
The abundance of a transcript is determined by its rate of synthesis and its rate of degradation; however, global methods for quantifying RNA abundance cannot distinguish variation in these two processes. Here, we introduce RNA approach to equilibrium sequencing (RATE-seq), which uses in vivo metabolic labeling of RNA and approach to equilibrium kinetics, to determine absolute RNA degradation and synthesis rates. RATE-seq does not disturb cellular physiology, uses straightforward normalization with exogenous spike-ins, and can be readily adapted for studies in most organisms. We demonstrate the use of RATE-seq to estimate genome-wide kinetic parameters for coding and noncoding transcripts in Saccharomyces cerevisiae.

PMID: 25161313 [PubMed - as supplied by publisher]

Nature Methods 11, 875 (2014). doi:10.1038/nmeth.3096

A clear idea of the performance—the strengths but also the limits—of biological research methods is critical for generating reliable data that others are able to reproduce.

Synthetic analogs of the 5' end of mRNA (cap structure) are widely used in molecular studies on mechanisms of cellular processes such as translation, intracellular transport, splicing, and turnover. The best-characterized cap binding protein is translation initiation factor 4E (eIF4E). Recognition of the mRNA cap by eIF4E is a critical, rate-limiting step for efficient translation initiation and is considered a major target for anticancer therapy. Here, we report a facile methodology for the preparation of N2-triazole-containing monophosphate cap analogs and present their biological evaluation as inhibitors of protein synthesis. Five analogs possessing this unique hetero-cyclic ring spaced from the m7-guanine of the cap structure at a distance of one or three carbon atoms and/or additionally substituted by various groups containing the benzene ring were synthesized. All obtained compounds turned out to be effective translation inhibitors with IC₅₀ similar to dinucleotide triphosphate m⁷GpppG. As these compounds possess a reduced number of phosphate groups and, thereby, a negative charge, which may support their cell penetration, this type of cap analog might be promising in terms of designing new potential therapeutic molecules. In addition, an exemplary dinucleotide from a corresponding mononucleotide containing benzyl substituted 1,2,3-triazole was prepared and examined. The superior inhibitory properties of this analog (10-fold vs. m⁷GpppG) suggest the usefulness of such compounds for the preparation of mRNA transcripts with high translational activity.

Nonsense-mediated mRNA decay (NMD) eliminates different classes of mRNA substrates including transcripts with long 3' UTRs. Current models of NMD suggest that the long physical distance between the poly(A) tail and the termination codon reduces the interaction between cytoplasmic poly(A)-binding protein (PABPC1) and the eukaryotic release factor 3a (eRF3a) during translation termination. In the absence of PABPC1 binding, eRF3a recruits the NMD factor UPF1 to the terminating ribosome, triggering mRNA degradation. Here, we have used the MS2 tethering system to investigate the suppression of NMD by PABPC1. We show that tethering of PABPC1 between the termination codon and a long 3' UTR specifically inhibits NMD-mediated mRNA degradation. Contrary to the current model, tethered PABPC1 mutants unable to interact with eRF3a still efficiently suppress NMD. We find that the interaction of PABPC1 with eukaryotic initiation factor 4G (eIF4G), which mediates the circularization of mRNAs, is essential for NMD inhibition by tethered PABPC1. Furthermore, recruiting either eRF3a or eIF4G in proximity to an upstream termination codon antagonizes NMD. While tethering of an eRF3a mutant unable to interact with PABPC1 fails to suppress NMD, tethered eIF4G inhibits NMD in a PABPC1-independent manner, indicating a sequential arrangement of NMD antagonizing factors. In conclusion, our results establish a previously unrecognized link between translation termination, mRNA circularization, and NMD suppression, thereby suggesting a revised model for the activation of NMD at termination codons upstream of long 3' UTR.

Ribo-Seq maps the location of translating ribosomes on mature mRNA transcripts. While during normal translation, ribosome density is constant along the length of the mRNA coding region, this can be altered in response to translational regulatory events. In the present study, we developed a method to detect translational regulation of individual mRNAs from their ribosome profiles, utilizing changes in ribosome density. We used mathematical modeling to show that changes in ribosome density should occur along the mRNA at the point of regulation. We analyzed a Ribo-Seq data set obtained for mouse embryonic stem cells and showed that normalization by corresponding RNA-Seq can be used to improve the Ribo-Seq quality by removing bias introduced by deep-sequencing and alignment artifacts. After normalization, we applied a change point algorithm to detect changes in ribosome density present in individual mRNA ribosome profiles. Additional sequence and gene isoform information obtained from the UCSC Genome Browser allowed us to further categorize the detected changes into different mechanisms of regulation. In particular, we detected several mRNAs with known post-transcriptional regulation, e.g., premature termination for selenoprotein mRNAs and translational control of Atf4, but also several more mRNAs with hitherto unknown translational regulation. Additionally, our approach proved useful for identification of new transcript isoforms.

Nature Reviews Molecular Cell Biology 15, 563 (2014). doi:10.1038/nrm3865

Cells respond to stress by transcribing survival genes. Zid and O'shea found that, in response to glucose starvation in budding yeast, the translation of a subset of transcriptionally activated mRNAs was increased, and that of another subset was decreased. Transcripts of the first subset were

Related Articles

Discovery, identification and sequence analysis of RNAs selected for very short or long poly A tail in immature bovine oocytes.

Mol Hum Reprod. 2014 Feb;20(2):127-38

Authors: Gohin M, Fournier E, Dufort I, Sirard MA

Abstract
A major challenge in applying genomics to oocyte physiology is that many RNAs are present but will not be translated into proteins, making it difficult to draw conclusions from RNAseq and array data. Oocyte maturation and early embryo development rely on maternal storage of specific RNAs with a short poly(A) tail, which must be elongated for translation. To resolve the role of key genes during that period, we aimed to characterize both extremes of mRNA: deadenylated RNA and long polyA tails mRNA population in immature bovine oocytes. Using magnetic beads coupled to oligodT, we isolated deadenylated (A-, 20-50 adenosines) from polyadenylated (A+, up to 200 adenosines) RNAs. After transcriptomic analysis, we observed that A+ candidates are associated with short-term processes required for immediate cell survival (translation or protein transport) or meiotic resumption, while several A- candidates are involved in processes (chromatin modification, gene transcription and post-transcriptional modifications) that will be extremely important in the development of the early embryo. In addition to a list of candidates probably translated early or late, sequence analysis revealed that cytoplasmic polyadenylation element (CPE) and U(3)GU(3) were enriched in A- sequences. Moreover, a motif associated with polyadenylation signals (MAPS, U(5)CU(2)) appeared to be enriched in 3'untranslated regions (UTR) with CPE or U(3)GU(3) sequences in bovine but also in zebrafish and Xenopus tropicalis. To further validate our methodology, we measured specific tail length of known candidates (AURKA, PTTG1, H2A1) but also determined the poly(A) tail length of other candidate RNAs (H3F3A, H1FOO, DAZAP2, ATF1, ATF2, KAT5, DAZL, ELAVL2). In conclusion, we have reported a methodology to isolate deadenylated from polyadenylated RNAs in samples with small total RNA quantities such as mammals. Moreover, we identified deadenylated RNAs in bovine oocytes that may be stored for the long-term process of early embryo development and described a conserved motif enriched in the 3'UTR of deadenylated RNAs.

PMID: 24233545 [PubMed - indexed for MEDLINE]

The specialized protein synthesis functions of the cytosol and endoplasmic reticulum compartments are conferred by the signal recognition particle (SRP) pathway, which directs the cotranslational trafficking of signal sequence-encoding mRNAs from the cytosol to the endoplasmic reticulum (ER). Although subcellular mRNA distributions largely mirror the binary pattern predicted by the SRP pathway model, studies in mammalian cells, yeast, and Drosophila have also demonstrated that cytosolic protein-encoding mRNAs are broadly represented on ER-bound ribosomes. A mechanism for such noncanonical mRNA localization remains, however, to be identified. Here, we examine the hypothesis that de novo translation initiation on ER-bound ribosomes serves as a mechanism for localizing cytosolic protein-encoding mRNAs to the ER. As a test of this hypothesis, we performed single molecule RNA fluorescence in situ hybridization studies of subcellular mRNA distributions and report that a substantial fraction of mRNAs encoding the cytosolic protein GAPDH resides in close proximity to the ER. Consistent with these data, analyses of subcellular mRNA and ribosome distributions in multiple cell lines demonstrated that cytosolic protein mRNA-ribosome distributions were strongly correlated, whereas signal sequence-encoding mRNA-ribosome distributions were divergent. Ribosome footprinting studies of ER-bound polysomes revealed a substantial initiation codon read density enrichment for cytosolic protein-encoding mRNAs. We also demonstrate that eukaryotic initiation factor 2α is bound to the ER via a salt-sensitive, ribosome-independent mechanism. Combined, these data support ER-localized translation initiation as a mechanism for mRNA recruitment to the ER.

IRBIS is a computational pipeline for detecting conserved complementary regions in unaligned orthologous sequences. Unlike other methods, it follows the "first-fold-then-align" principle in which all possible combinations of complementary k-mers are searched for simultaneous conservation. The novel trimming procedure reduces the size of the search space and improves the performance to the point where large-scale analyses of intra- and intermolecular RNA–RNA interactions become possible. In this article, I provide a rigorous description of the method, benchmarking on simulated and real data, and a set of stringent predictions of intramolecular RNA structure in placental mammals, drosophilids, and nematodes. I discuss two particular cases of long-range RNA structures that are likely to have a causal effect on single- and multiple-exon skipping, one in the mammalian gene Dystonin and the other in the insect gene Ca-α₁D. In Dystonin, one of the two complementary boxes contains a binding site of Rbfox splicing factor similar to the RNA bridge that was recently described in Enah gene. I also report that snoRNAs and long noncoding RNAs (lncRNAs) have a high capacity of base-pairing to introns of protein-coding genes, suggesting possible involvement of these transcripts in splicing regulation. I also find that conserved sequences that occur equally likely on both strands of DNA (e.g., transcription factor binding sites) contribute strongly to the false-discovery rate and, therefore, would confound every such analysis. IRBIS is an open-source software that is available at http://genome.crg.es/~dmitri/irbis.

Related Articles

RNA-Based Regulation: Dynamics and Response to Perturbations of Competing RNAs.

Biophys J. 2014 Aug 19;107(4):1011-1022

Authors: Figliuzzi M, De Martino A, Marinari E

Abstract
The observation that, through a titration mechanism, microRNAs (miRNAs) can act as mediators of effective interactions among their common targets (competing endogenous RNAs or ceRNAs) has brought forward the idea (i.e., the ceRNA hypothesis) that RNAs can regulate each other in extended cross-talk networks. Such an ability might play a major role in posttranscriptional regulation to shape a cell's protein repertoire. Recent work focusing on the emergent properties of the cross-talk networks has emphasized the high flexibility and selectivity that may be achieved at stationarity. On the other hand, dynamical aspects, possibly crucial on the relevant timescales, are far less clear. We have carried out a dynamical study of the ceRNA hypothesis on a model of posttranscriptional regulation. Sensitivity analysis shows that ceRNA cross-talk is dynamically extended, i.e., it may take place on timescales shorter than those required to achieve stationarity even in cases where no cross-talk occurs in the steady state, and is possibly amplified. In addition, in the case of large, transfection-like perturbations, the system may develop a strongly nonlinear, threshold response. Finally, we show that the ceRNA effect provides a very efficient way for a cell to achieve fast positive shifts in the level of a ceRNA when necessary. These results indicate that competition for miRNAs may indeed provide an elementary mechanism to achieve system-level regulatory effects on the transcriptome over physiologically relevant timescales.

PMID: 25140437 [PubMed - as supplied by publisher]

Publication date: 4 September 2014
Source:Molecular Cell, Volume 55, Issue 5
Author(s): Jason A. West , Christopher P. Davis , Hongjae Sunwoo , Matthew D. Simon , Ruslan I. Sadreyev , Peggy I. Wang , Michael Y. Tolstorukov , Robert E. Kingston
Mechanistic roles for many lncRNAs are poorly understood, in part because their direct interactions with genomic loci and proteins are difficult to assess. Using a method to purify endogenous RNAs and their associated factors, we mapped the genomic binding sites for two highly expressed human lncRNAs, NEAT1 and MALAT1. We show that NEAT1 and MALAT1 localize to hundreds of genomic sites in human cells, primarily over active genes. NEAT1 and MALAT1 exhibit colocalization to many of these loci, but display distinct gene body binding patterns at these sites, suggesting independent but complementary functions for these RNAs. We also identified numerous proteins enriched by both lncRNAs, supporting complementary binding and function, in addition to unique associated proteins. Transcriptional inhibition or stimulation alters localization of NEAT1 on active chromatin sites, implying that underlying DNA sequence does not target NEAT1 to chromatin, and that localization responds to cues involved in the transcription process.

Graphical abstract

Teaser

NEAT1 and MALAT1 are two of the most abundant mammalian lncRNAs. West et al. map the genomic binding of NEAT1 and MALAT1 and identify interacting proteins via RNA pull-down followed by mass spectrometry, showing these RNAs play a synergistic role in nuclear organization linking active genes and nuclear subdomains.

A riboswitch is both part of, and regulates the activity of, a small noncoding regulatory RNA. [Also see Perspective by Chen and Gottesman] Authors: J. R. Mellin, Mikael Koutero, Daniel Dar, Marie-Anne Nahori, Rotem Sorek, Pascale Cossart

A riboswitch is both part of, and regulates the activity of, a small noncoding regulatory RNA. [Also see Perspective by Chen and Gottesman] Authors: Sruti DebRoy, Margo Gebbie, Arati Ramesh, Jonathan R. Goodson, Melissa R. Cruz, Ambro van Hoof, Wade C. Winkler, Danielle A. Garsin

Nutrient metabolism in bacteria is controlled by a circuit in which RNA is regulated by RNA [Also see Reports by DebRoy et al. and Mellin et al.] Authors: Jiandong Chen, Susan Gottesman

Publication date: 11 September 2014
Source:Cell Reports, Volume 8, Issue 5
Author(s): Mario Leonardo Squadrito , Caroline Baer , Frédéric Burdet , Claudio Maderna , Gregor D. Gilfillan , Robert Lyle , Mark Ibberson , Michele De Palma
MicroRNA (miRNA) transfer via exosomes may mediate cell-to-cell communication. Interestingly, specific miRNAs are enriched in exosomes in a cell-type-dependent fashion. However, the mechanisms whereby miRNAs are sorted to exosomes and the significance of miRNA transfer to acceptor cells are unclear. We used macrophages and endothelial cells (ECs) as a model of heterotypic cell communication in order to investigate both processes. RNA profiling of macrophages and their exosomes shows that miRNA sorting to exosomes is modulated by cell-activation-dependent changes of miRNA target levels in the producer cells. Genetically perturbing the expression of individual miRNAs or their targeted transcripts promotes bidirectional miRNA relocation from the cell cytoplasm/P bodies (sites of miRNA activity) to multivesicular bodies (sites of exosome biogenesis) and controls miRNA sorting to exosomes. Furthermore, the use of Dicer-deficient cells and reporter lentiviral vectors (LVs) for miRNA activity shows that exosomal miRNAs are transferred from macrophages to ECs to detectably repress targeted sequences.

Graphical abstract

Teaser

Cell-derived microvesicles called exosomes mediate the exchange of microRNAs among cells. However, the mechanisms controlling microRNA sorting to exosomes are poorly understood. Squadrito et al. employ multiple techniques to show that the sorting of microRNAs to exosomes is partly regulated by the cellular levels of their target gene transcripts, which fluctuate in response to the cell activation state. These findings identify a general, motif-independent mechanism of microRNA sorting to exosomes that may be relevant to intercellular communication.

Publication date: 11 September 2014
Source:Cell Reports, Volume 8, Issue 5
Author(s): Nicholas T. Ingolia , Gloria A. Brar , Noam Stern-Ginossar , Michael S. Harris , Gaëlle J.S. Talhouarne , Sarah E. Jackson , Mark R. Wills , Jonathan S. Weissman
Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be noncoding, including 5′ UTRs and long noncoding RNAs (lncRNAs). Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs). Here, we show hallmarks of translation in these footprints: copurification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including the induction of immune responses following human cytomegalovirus (HCMV) infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts at understanding how cells manage and exploit its consequences.

Graphical abstract

Teaser

Ribosome profiling, an experimental approach for detecting and quantifying translation, detected apparent ribosome footprints outside of annotated protein-coding genes, including many 5′ UTRs and some lncRNAs. Here, Ingolia et al. verify the presence of assembled 80S ribosomes on these RNAs through computational and experimental approaches. Furthermore, they show that peptides are translated from these regions in human cytomegalovirus (HCMV) and serve as HCMV-specific antigens, suggesting that endogenous human 5′ UTRs and lncRNAs may encode self-antigens and cancer neoantigens as well.

Genomic analysis of RNA localization.

RNA Biol. 2014 Aug 19;11(8)

Authors: Taliaferro JM, Wang ET, Burge CB

Abstract
The localization of mRNAs to specific subcellular sites is widespread, allowing cells to spatially restrict and regulate protein production, and playing important roles in development and cellular physiology. This process has been studied in mechanistic detail for several RNAs. However, the generality or specificity of RNA localization systems and mechanisms that impact the many thousands of localized mRNAs has been difficult to assess. In this review, we discuss the current state of the field in determining which RNAs localize, which RNA sequences mediate localization, the protein factors involved, and the biological implications of localization. For each question, we examine prominent systems and techniques that are used to study individual messages, highlight recent genome-wide studies of RNA localization, and discuss the potential for adapting other high-throughput approaches to the study of localization.

PMID: 25136858 [PubMed - as supplied by publisher]

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Efficient in vivo deletion of a large imprinted lncRNA by CRISPR/Cas9.

RNA Biol. 2014;11(7):829-35

Authors: Han J, Zhang J, Chen L, Shen B, Zhou J, Hu B, Du Y, Tate PH, Huang X, Zhang W

Abstract
Recent genome-wide studies have revealed that the majority of the mouse genome is transcribed as non-coding RNAs (ncRNAs) and growing evidence supports the importance of ncRNAs in regulating gene expression and epigenetic processes. However, the low efficiency of conventional gene targeting strategies has hindered the functional study of ncRNAs in vivo, particularly in generating large fragment deletions of long non-coding RNAs (lncRNAs) with multiple expression variants. The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has recently been applied as an efficient tool for engineering site-specific mutations of protein-coding genes in the genome. In this study, we explored the potential of using the CRISPR/Cas9 system to generate large genomic deletions of lncRNAs in mice. We developed an efficient one-step strategy to target the maternally expressed lncRNA, Rian, on chromosome 12 in mice. We showed that paired sgRNAs can precisely generate large deletions up to 23kb and the deletion efficiency can be further improved up to 33% by combining multiple sgRNAs. The deletion successfully abolished the expression of Rian from the maternally inherited allele, validating the biological relevance of the mutations in studying an imprinted locus. Mutation of Rian has differential effects on expression of nearby genes in different somatic tissues. Taken together, we have established a robust one-step method to engineer large deletions to knockout lncRNA genes with the CRISPR/Cas9 system. Our work will facilitate future functional studies of other lncRNAs in vivo.

PMID: 25137067 [PubMed - indexed for MEDLINE]

Background: Variability in protein levels is generated through intricate control of the different gene decoding phases. Presently little is known about the links between the various gene expression stages. Here we investigated the relationship between transcription and translation regulatory properties encoded in mammalian genes. Results: We found that the TATA-box, a core promoter element known to enhance transcriptional output, is associated not only with higher mRNA levels but also with positive translation regulatory features and elevated translation efficiency. Further investigation revealed general association between transcription and translation regulatory trends. Specifically, translation inhibitory features such as the presence of upstream AUG (uAUG) and increased lengths of the 5[prime]UTR, the coding sequence and the 3[prime]UTR, are strongly associated with lower translation as well as lower transcriptional rate. Conclusions: Our findings reveal that co-occurrence of several gene-encoded transcription and translation regulatory features with the same trend substantially contributes to the final mRNA and protein expression levels and enables their coordination.

Related Articles

Temporally and spatially restricted gene expression profiling.

Curr Genomics. 2014 Aug;15(4):278-92

Authors: Tallafuss A, Washbourne P, Postlethwait J

Abstract
Identifying gene function in specific cells is critical for understanding the processes that make cells unique. Several different methods are available to isolate actively transcribed RNA or actively translated RNA in specific cells at a chosen time point. Cell-specific mRNA isolation can be accomplished by the expression of transgenes in cells of interest, either directly from a specific promoter or using a modular system such as Gal4/UAS or Cre/lox. All of the methods described in this review, namely thiol-labeling of RNA (TU-tagging or RABT), TRAP (translating ribosome affinity purification) and INTACT (isolation of nuclei tagged in specific cell types), allow next generation sequencing, permitting the identification of enriched gene transcripts within the specific cell-type. We describe here the general concept of each method, include examples, evaluate possible problems related to each technique, and suggest the types of questions for which each method is best suited.

PMID: 25132798 [PubMed]

Related Articles

RNA structural analysis by evolving SHAPE chemistry.

Wiley Interdiscip Rev RNA. 2014 Aug 15;

Authors: Spitale RC, Flynn RA, Torre EA, Kool ET, Chang HY

Abstract
RNA is central to the flow of biological information. From transcription to splicing, RNA localization, translation, and decay, RNA is intimately involved in regulating every step of the gene expression program, and is thus essential for health and understanding disease. RNA has the unique ability to base-pair with itself and other nucleic acids to form complex structures. Hence the information content in RNA is not simply its linear sequence of bases, but is also encoded in complex folding of RNA molecules. A general chemical functionality that all RNAs have is a 2'-hydroxyl group in the ribose ring, and the reactivity of the 2'-hydroxyl in RNA is gated by local nucleotide flexibility. In other words, the 2'-hydroxyl is reactive at single-stranded and conformationally flexible positions but is unreactive at nucleotides constrained by base-pairing. Recent efforts have been focused on developing reagents that modify RNA as a function of RNA 2' hydroxyl group reactivity. Such RNA structure probing techniques can be read out by primer extension in experiments termed RNA SHAPE (selective 2'- hydroxyl acylation and primer extension). Herein, we describe the efforts devoted to the design and utilization of SHAPE probes for characterizing RNA structure. We also describe current technological advances that are being applied to utilize SHAPE chemistry with deep sequencing to probe many RNAs in parallel. The merging of chemistry with genomics is sure to open the door to genome-wide exploration of RNA structure and function. For further resources related to this article, please visit the WIREs website. Conflict of interest: The authors have declared no conflicts of interest for this article.

PMID: 25132067 [PubMed - as supplied by publisher]

Publication date: 18 August 2014
Source:Current Biology, Volume 24, Issue 16
Author(s): Bo W. Han , Phillip D. Zamore
PIWI-interacting RNAs (piRNAs) are single-stranded, 23–36 nucleotide (nt) RNAs that act as guides for an animal-specific class of Argonaute proteins, the PIWI proteins. The first piRNAs — derived from the Suppressor of Stellate locus in Drosophila melanogaster testes — were discovered in 2001. Although the authors noted the larger size of those ‘rasiRNAs’ (repeat-associated siRNAs), piRNAs were not recognized until 2006 as a distinct class of small silencing RNAs that derive from single-stranded, rather than double-stranded RNA (dsRNA), precursors. It was their association with PIWI, but not AGO, proteins and their independence from Dicer — the RNase III enzyme that cleaves dsRNAs to release small interfering RNAs (siRNAs) — that finally distinguished piRNAs from siRNAs. Moreover, while siRNAs are expressed ubiquitously, piRNAs are predominantly found in animal gonads and are thought to be indispensable for fertility.

Teaser

Han and Zamore provide an over-view of the piRNA class of small RNAs, explaining how piRNAs were first discovered, how they are produced, and how they function in gene silencing.

One of the most challenging unanswered questions regarding the structural biology of biomolecular machines such as the two-subunit ribosome is whether and how these machines coordinate seemingly independent and random conformational fluctuations to maximize and regulate their functional efficiencies. To address this question, we have used ribosome mutagenesis or a...

The exoribonuclease Xrn1 participates in mRNA homeostasis downstream of the energy and nutrient-sensing kinase Snf1.

Nature Chemical Biology 10, 695 (2014). doi:10.1038/nchembio.1619

Author: Joshua M. Finkelstein