Hyeshik Chang

The genetic code is an abstraction of how mRNA codons and tRNA anticodons molecularly interact during protein synthesis; the stability and regulation of this interaction remains largely unexplored. Here, we characterized the expression of mRNA and tRNA genes quantitatively at multiple time points in two developing mouse tissues. We discovered that mRNA codon pools are highly stable over development and simply reflect the genomic background; in contrast, precise regulation of tRNA gene families is required to create the corresponding tRNA transcriptomes. The dynamic regulation of tRNA genes during development is controlled in order to generate an anticodon pool that closely corresponds to messenger RNAs. Thus, across development, the pools of mRNA codons and tRNA anticodons are invariant and highly correlated, revealing a stable molecular interaction interlocking transcription and translation.

It remains an open question when and how the first cell fate decision is made in mammals. Using deep single-cell RNA-seq of matched sister blastomeres, we report highly reproducible inter-blastomere differences among 10 2-cell and five 4-cell mouse embryos. Inter-blastomere gene expression differences dominated between-embryo differences and noise, and were sufficient to cluster sister blastomeres into distinct groups. Dozens of protein-coding genes exhibited reproducible bimodal expression in sister blastomeres, which cannot be explained by random fluctuations. The protein expression of one gene out of four of these bimodal genes tested, Gadd45a, exhibited clear inter-blastomeric contrasts. We traced some of the bimodal mRNA expressions to embryonic genome activation, and others to blastomere-specific RNA depletion. Inter-blastomere differences created coexpression gene networks that were much stronger and larger than those that can possibly be created by random noise. The highly correlated gene pairs at the 4-cell stage overlapped with those showing the same directions of differential expression between inner cell mass (ICM) and trophectoderm (TE). These data substantiate the hypothesis of inter-blastomere differences in 2- and 4-cell mouse embryos, and associate these differences with ICM/TE differences.

High-throughput sequencing (HTS) methods for analyzing RNA populations (RNA-Seq) are gaining rapid application to many experimental situations. The steps in an RNA-Seq experiment require thought and planning, especially because the expense in time and materials is currently higher and the protocols are far less routine than those used for other high-throughput methods, such as microarrays. As always, good experimental design will make analysis and interpretation easier. Having a clear biological question, an idea about the best way to do the experiment, and an understanding of the number of replicates needed will make the entire process more satisfying. Whether the goal is capturing transcriptome complexity from a tissue or identifying small fragments of RNA cross-linked to a protein of interest, conversion of the RNA to cDNA followed by direct sequencing using the latest methods is a developing practice, with new technical modifications and applications appearing every day. Even more rapid are the development and improvement of methods for analysis of the very large amounts of data that arrive at the end of an RNA-Seq experiment, making considerations regarding reproducibility, validation, visualization, and interpretation increasingly important. This introduction is designed to review and emphasize a pathway of analysis from experimental design through data presentation that is likely to be successful, with the recognition that better methods are right around the corner.

The onset of development is marked by two major, posttranscriptionally controlled, events: oocyte maturation (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest). Using quantitative mass spectrometry, we previously described proteome remodeling during Drosophila egg activation. Here, we describe our quantitative mass spectrometry-based...

The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein–RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3' overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells.

The synthesis of adequate amounts of ribosomes is an essential task for the cell. It is therefore not surprising that regulatory circuits exist to organize the synthesis of ribosomal components. It has been shown that defect in ribosome biogenesis (ribosomal stress) induces apoptosis or cell cycle arrest through activation of the tumor suppressor p53. This mechanism is thought to be implicated in the pathophysiology of a group of genetic diseases such as Diamond Blackfan Anemia which are called ribosomopathies. We have identified an additional response to ribosomal stress that includes the activation of eukaryotic translation elongation factor 2 kinase with a consequent inhibition of translation elongation. This leads to a translational reprogramming in the cell that involves the structurally defined group of messengers called terminal oligopyrimidine (TOP) mRNAs which encode ribosomal proteins and translation factors. In fact, while general protein synthesis is decreased by the impairment of elongation, TOP mRNAs are recruited on polysomes causing a relative increase in the synthesis of TOP mRNA-encoded proteins compared to other proteins. Therefore, in response to ribosomal stress, there is a change in the translation pattern of the cell which may help restore a sufficient level of ribosomes.

Related Articles

Construction of Trypanosoma brucei Illumina RNA-Seq Libraries Enriched for Transcript Ends.

Methods Mol Biol. 2015;1201:165-75

Authors: Kolev NG, Ullu E, Tschudi C

Abstract
High-throughput RNA sequencing (RNA-Seq) has quickly occupied center stage in the repertoire of available tools for transcriptomics. Among many advantages, the single-nucleotide resolution of this powerful approach allows mapping on a genome-wide scale of splice junctions and polyadenylation sites, and thus, the precise definition of mature transcript boundaries. This greatly facilitated the transcriptome annotation of the human pathogen Trypanosoma brucei, a protozoan organism in which all mRNA molecules are matured by spliced leader (SL) trans-splicing from longer polycistronic precursors. The protocols described here for the generation of three types of libraries for Illumina RNA-Seq, 5'-SL enriched, 5'-triphosphate-end enriched, and 3'-poly(A) enriched, enabled the discovery of an unprecedented heterogeneity of pre-mRNA-processing sites, a large number of novel coding and noncoding transcripts from previously unannotated genes, and quantify the cellular abundance of RNA molecules. The method for producing 5'-triphosphate-end-enriched libraries was instrumental for obtaining evidence that transcription initiation by RNA polymerase II in trypanosomes is bidirectional and biosynthesis of mRNA precursors is primed not only at the beginning of unidirectional gene clusters, but also at specific internal sites.

PMID: 25388113 [PubMed - in process]

Short loop-targeting oligoribonucleotides antagonize Lin28 and enable pre-let-7 processing and suppression of cell growth in let-7-deficient cancer cells.

Nucleic Acids Res. 2014 Nov 6;

Authors: Roos M, Rebhan MA, Lucic M, Pavlicek D, Pradere U, Towbin H, Civenni G, Catapano CV, Hall J

Abstract
MicroRNAs (miRNAs) originate from stem-loop-containing precursors (pre-miRNAs, pri-miRNAs) and mature by means of the Drosha and Dicer endonucleases and their associated factors. The let-7 miRNAs have prominent roles in developmental differentiation and in regulating cell proliferation. In cancer, the tumor suppressor function of let-7 is abrogated by overexpression of Lin28, one of several RNA-binding proteins that regulate let-7 biogenesis by interacting with conserved motifs in let-7 precursors close to the Dicer cleavage site. Using in vitro assays, we have identified a binding site for short modified oligoribonucleotides ('looptomirs') overlapping that of Lin28 in pre-let-7a-2. These looptomirs selectively antagonize the docking of Lin28, but still permit processing of pre-let-7a-2 by Dicer. Looptomirs restored synthesis of mature let-7 and inhibited growth and clonogenic potential in Lin28 overexpressing hepatocarcinoma cells, thereby demonstrating a promising new means to rescue defective miRNA biogenesis in Lin28-dependent cancers.

PMID: 25378324 [PubMed - as supplied by publisher]

Article

During translation, several ribosomes assemble on an mRNA strand to form polyribosomes. Here the authors describe—using cryo electron tomography—a compact state of the eukaryotic polysome where individual ribosomes contact each other and form a tight helical assembly that creates a continuous inner channel for the mRNA.

Nature Communications doi: 10.1038/ncomms6294

Authors: Alexander G. Myasnikov, Zhanna A. Afonina, Jean-François Ménétret, Vladimir A. Shirokov, Alexander S. Spirin, Bruno P. Klaholz

Article

Despite our growing understanding of their complexity, different types of RNA are still classified using technical rather than functional criteria. Andersson et al. show that categorization of RNAs based on stability and direction of transcription is an effective means of functional classification.

Nature Communications doi: 10.1038/ncomms6336

Authors: Robin Andersson, Peter Refsing Andersen, Eivind Valen, Leighton J. Core, Jette Bornholdt, Mette Boyd, Torben Heick Jensen, Albin Sandelin

Nature advance online publication 12 November 2014. doi:10.1038/nature13976

Authors: Caroline Dias, Jian Feng, Haosheng Sun, Ning yi Shao, Michelle S. Mazei-Robison, Diane Damez-Werno, Kimberly Scobie, Rosemary Bagot, Benoit LaBonté, Efrain Ribeiro, XiaoChuan Liu, Pamela Kennedy, Vincent Vialou, Deveroux Ferguson, Catherine Peña, Erin S. Calipari, Ja Wook Koo, Ezekiell Mouzon, Subroto Ghose, Carol Tamminga, Rachael Neve, Li Shen & Eric J. Nestler

Nature Protocols 9, 2823 (2014). doi:10.1038/nprot.2014.187

Authors: Thomas O Auer, Karine Duroure, Jean-Paul Concordet & Filippo Del Bene

Here we present a protocol for the conversion of eGFP-transgenic zebrafish lines into lines expressing Gal4 from the same locus. This conversion allows the in-depth analysis of the former eGFP-expressing cell population; with the Gal4-upstream activating sequence (UAS) system, diverse UAS

Nature Biotechnology 32, 1141 (2014). doi:10.1038/nbt.3011

Authors: Robert J Citorik, Mark Mimee & Timothy K Lu

Current antibiotics tend to be broad spectrum, leading to indiscriminate killing of commensal bacteria and accelerated evolution of drug resistance. Here, we use CRISPR-Cas technology to create antimicrobials whose spectrum of activity is chosen by design. RNA-guided nucleases (RGNs) targeting specific DNA sequences are delivered efficiently to microbial populations using bacteriophage or bacteria carrying plasmids transmissible by conjugation. The DNA targets of RGNs can be undesirable genes or polymorphisms, including antibiotic resistance and virulence determinants in carbapenem-resistant Enterobacteriaceae and enterohemorrhagic Escherichia coli. Delivery of RGNs significantly improves survival in a Galleria mellonella infection model. We also show that RGNs enable modulation of complex bacterial populations by selective knockdown of targeted strains based on genetic signatures. RGNs constitute a class of highly discriminatory, customizable antimicrobials that enact selective pressure at the DNA level to reduce the prevalence of undesired genes, minimize off-target effects and enable programmable remodeling of microbiota.

Nature Biotechnology 32, 1102 (2014). doi:10.1038/nbt.3060

Author: Lynn L Silver

RNA-guided nucleases provide a strategy for killing specific bacterial species in complex populations.

Systematic Analysis of the Role of RNA-Binding Proteins in the Regulation of RNA Stability.

PLoS Genet. 2014 Nov;10(11):e1004684

Authors: Hasan A, Cotobal C, Duncan CD, Mata J

Abstract
mRNA half-lives are transcript-specific and vary over a range of more than 100-fold in eukaryotic cells. mRNA stabilities can be regulated by sequence-specific RNA-binding proteins (RBPs), which bind to regulatory sequence elements and modulate the interaction of the mRNA with the cellular RNA degradation machinery. However, it is unclear if this kind of regulation is sufficient to explain the large range of mRNA stabilities. To address this question, we examined the transcriptome of 74 Schizosaccharomyces pombe strains carrying deletions in non-essential genes encoding predicted RBPs (86% of all such genes). We identified 25 strains that displayed changes in the levels of between 4 and 104 mRNAs. The putative targets of these RBPs formed biologically coherent groups, defining regulons involved in cell separation, ribosome biogenesis, meiotic progression, stress responses and mitochondrial function. Moreover, mRNAs in these groups were enriched in specific sequence motifs in their coding sequences and untranslated regions, suggesting that they are coregulated at the posttranscriptional level. We performed genome-wide RNA stability measurements for several RBP mutants, and confirmed that the altered mRNA levels were caused by changes in their stabilities. Although RBPs regulate the decay rates of multiple regulons, only 16% of all S. pombe mRNAs were affected in any of the 74 deletion strains. This suggests that other players or mechanisms are required to generate the observed range of RNA half-lives of a eukaryotic transcriptome.

PMID: 25375137 [PubMed - as supplied by publisher]

Related Articles

Fingerprints of a message: integrating positional information on the transcriptome.

Front Cell Dev Biol. 2014;2:39

Authors: Dassi E, Quattrone A

Abstract
The recent explosion of high-throughput sequencing methods applied to RNA molecules is allowing us to go beyond the description of sequence variants and their relative abundances, as measured by RNA-seq. We can now probe for RNA engagement in polysomes, for ribosomes, RNA binding proteins and microRNAs binding sites, for RNA secondary structure and for RNA methylation. These descriptors produce a steadily growing multidimensional array of positional information on RNA sequences, whose effective integration only would bring to decipher the regulatory interplay occurring between proteins, RNAs and their modifications on the transcriptome. This interplay ultimately dictates the degree of mRNA availability to translation, and thus the occurrence of cell phenotypes. However, several issues in data presentation are slowing down effective integration. A standardization effort for new dataset types produced should be urgently undertaken to solve these issues. Providing uniformed experimental details along with datasets processed to be directly usable and employing shared formats would greatly simplify integration efforts, strengthening hypotheses stemming from correlative observations and eventually bringing to mechanistic understanding.

PMID: 25364746 [PubMed]

Related Articles

Genome-Wide Distribution of RNA-DNA Hybrids Identifies RNase H Targets in tRNA Genes, Retrotransposons and Mitochondria.

PLoS Genet. 2014 Oct;10(10):e1004716

Authors: El Hage A, Webb S, Kerr A, Tollervey D

Abstract
During transcription, the nascent RNA can invade the DNA template, forming extended RNA-DNA duplexes (R-loops). Here we employ ChIP-seq in strains expressing or lacking RNase H to map targets of RNase H activity throughout the budding yeast genome. In wild-type strains, R-loops were readily detected over the 35S rDNA region, transcribed by Pol I, and over the 5S rDNA, transcribed by Pol III. In strains lacking RNase H activity, R-loops were elevated over other Pol III genes, notably tRNAs, SCR1 and U6 snRNA, and were also associated with the cDNAs of endogenous TY1 retrotransposons, which showed increased rates of mobility to the 5'-flanking regions of tRNA genes. Unexpectedly, R-loops were also associated with mitochondrial genes in the absence of RNase H1, but not of RNase H2. Finally, R-loops were detected on actively transcribed protein-coding genes in the wild-type, particularly over the second exon of spliced ribosomal protein genes.

PMID: 25357144 [PubMed - as supplied by publisher]

The Y chromosome in date plum encodes a small RNA involved in male sex determination. Authors: Takashi Akagi, Isabelle M. Henry, Ryutaro Tao, Luca Comai

Structures of microRNA bound to the Ago2 repressor protein and messenger RNA elucidate target specificity. [Also see Perspective by Patel] Authors: Nicole T. Schirle, Jessica Sheu-Gruttadauria, Ian J. MacRae

Structures of microRNA-mRNA–human Argonaute reveal where and how intermolecular interactions are specified [Also see Research Article by Schirle et al.] Author: Dinshaw J. Patel

A new method reveals exactly which proteins are synthesized in the neighborhood of the endoplasmic reticulum and mitochondria. [Also see Perspective by Shao and Hegde] Authors: Christopher C. Williams, Calvin H. Jan, Jonathan S. Weissman

A new method reveals exactly which proteins are synthesized in the neighborhood of the endoplasmic reticulum and mitochondria. [Also see Perspective by Shao and Hegde] Authors: Calvin H. Jan, Christopher C. Williams, Jonathan S. Weissman

New experimental strategies reveal spatial and temporal features of protein synthesis and degradation in cells [Also see Research Article by Jan et al. and Reports by Williams et al. and Foresti et al.] Authors: Sichen Shao, Ramanujan S. Hegde

Multiple microRNAs converge to regulate calcium homeostasis in the endoplasmic reticulum.

Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells

Nature 515, 7525 (2014). doi:10.1038/nature13802

Authors: Thomas M. Carlile, Maria F. Rojas-Duran, Boris Zinshteyn, Hakyung Shin, Kristen M. Bartoli & Wendy V. Gilbert

Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure. Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function—it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1–4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease.

Article

Rapid and stable manipulation of gene expression in the malarial parasite Plasmodium falciparum remains a significant challenge. Goldfless et al . adapt a system for the inducible control of mRNA translation for use in Plasmodium and demonstrate its use to validate targets of antimalarial drugs.

Nature Communications doi: 10.1038/ncomms6329

Authors: Stephen J. Goldfless, Jeffrey C. Wagner, Jacquin C. Niles

Nature Structural & Molecular Biology 21, 945 (2014). doi:10.1038/nsmb.2911

Author: Samie R Jaffrey

The fate of an mRNA is regulated by internal base modifications that generate the modified nucleotides N6-methyladenosine, 5-methylcytosine and inosine. Three new studies show that yeast and human mRNAs also contain pseudouridine residues and that pseudouridylation is induced in various stress states, hinting at a new pathway for post-transcriptional control of mRNA.

Nature Reviews Genetics. doi:10.1038/nrg3813

Authors: Stefanie Gerstberger, Markus Hafner & Thomas Tuschl

Publication date: February 2015
Source:Trends in Cell Biology, Volume 25, Issue 2
Author(s): John M. Lucocq , Terry M. Mayhew , Yannick Schwab , Anna M. Steyer , Christian Hacker
Systems-based understanding of living organisms depends on acquiring huge datasets from arrays of genes, transcripts, proteins, and lipids. These data, referred to as ‘omes’, are assembled using ‘omics’ methodologies. Currently a comprehensive, quantitative view of cellular and organellar systems in 3D space at nanoscale/molecular resolution is missing. We introduce here the term ‘morphome’ for the distribution of living matter within a 3D biological system, and ‘morphomics’ for methods of collecting 3D data systematically and quantitatively. A sampling-based approach termed stereology currently provides rapid, precise, and minimally biased morphomics. We propose that stereology solves the ‘big data’ problem posed by emerging wide-scale electron microscopy (EM) and can establish quantitative links between the newer nanoimaging platforms such as electron tomography, cryo-EM, and correlative microscopy.

Nature Reviews Drug Discovery 13, 812 (2014). doi:10.1038/nrd4464

Author: Sarah Crunkhorn

Harnessing synthetic lethality — which occurs when the inhibition of two non-essential genes is lethal — is attracting significant attention as a strategy to selectively treat cancer. Here, the authors use the data mining synthetic lethality identification pipeline (DAISY) to analyse the accumulating cancer genomic