Joha

Nature Biotechnology. doi:10.1038/nbt.3289

Authors: Vijay Ramani, Ruolan Qiu & Jay Shendure

We present an unbiased method to globally resolve RNA structures through pairwise contact measurements between interacting regions. RNA proximity ligation (RPL) uses proximity ligation of native RNA followed by deep sequencing to yield chimeric reads with ligation junctions in the vicinity of structurally proximate bases. We apply RPL in both baker's yeast (Saccharomyces cerevisiae) and human cells and generate contact probability maps for ribosomal and other abundant RNAs, including yeast snoRNAs, the RNA subunit of the signal recognition particle and the yeast U2 spliceosomal RNA homolog. RPL measurements correlate with established secondary structures for these RNA molecules, including stem-loop structures and long-range pseudoknots. We anticipate that RPL will complement the current repertoire of computational and experimental approaches in enabling the high-throughput determination of secondary and tertiary RNA structures.

Quantitative evolutionary dynamics using high-resolution lineage tracking

Nature 519, 7542 (2015). doi:10.1038/nature14279

Authors: Sasha F. Levy, Jamie R. Blundell, Sandeep Venkataram, Dmitri A. Petrov, Daniel S. Fisher & Gavin Sherlock

Evolution of large asexual cell populations underlies ∼30% of deaths worldwide, including those caused by bacteria, fungi, parasites, and cancer. However, the dynamics underlying these evolutionary processes remain poorly understood because they involve many competing beneficial lineages, most of which never rise above extremely low

Nature advance online publication 24 November 2014. doi:10.1038/nature13923

Authors: Eliezer Calo, Ryan A. Flynn, Lance Martin, Robert C. Spitale, Howard Y. Chang & Joanna Wysocka

DEAD-box RNA helicases are vital for the regulation of various aspects of the RNA life cycle, but the molecular underpinnings of their involvement, particularly in mammalian cells, remain poorly understood. Here we show that the DEAD-box RNA helicase DDX21 can sense the transcriptional status of both RNA polymerase (Pol) I and II to control multiple steps of ribosome biogenesis in human cells. We demonstrate that DDX21 widely associates with Pol I- and Pol II-transcribed genes and with diverse species of RNA, most prominently with non-coding RNAs involved in the formation of ribonucleoprotein complexes, including ribosomal RNA, small nucleolar RNAs (snoRNAs) and 7SK RNA. Although broad, these molecular interactions, both at the chromatin and RNA level, exhibit remarkable specificity for the regulation of ribosomal genes. In the nucleolus, DDX21 occupies the transcribed rDNA locus, directly contacts both rRNA and snoRNAs, and promotes rRNA transcription, processing and modification. In the nucleoplasm, DDX21 binds 7SK RNA and, as a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, is recruited to the promoters of Pol II-transcribed genes encoding ribosomal proteins and snoRNAs. Promoter-bound DDX21 facilitates the release of the positive transcription elongation factor b (P-TEFb) from the 7SK snRNP in a manner that is dependent on its helicase activity, thereby promoting transcription of its target genes. Our results uncover the multifaceted role of DDX21 in multiple steps of ribosome biogenesis, and provide evidence implicating a mammalian RNA helicase in RNA modification and Pol II elongation control.

RNA editing increases transcriptome diversity through post-transcriptional modifications of RNA. Adenosine deaminases that act on RNA (ADARs) catalyze the adenosine-to-inosine (A-to-I) conversion, the most common type of RNA editing in higher eukaryotes. Caenorhabditis elegans has two ADARs, ADR-1 and ADR-2, but their functions remain unclear. Here, we profiled the RNA editomes of C. elegans at different developmental stages of wild-type and ADAR mutants. We developed a new computational pipeline with a "bisulfite-seq-mapping-like" step and achieved a threefold increase in identification sensitivity. A total of 99.5% of the 47,660 A-to-I editing sites were found in clusters. Of the 3080 editing clusters, 65.7% overlapped with DNA transposons in noncoding regions and 73.7% could form hairpin structures. The numbers of editing sites and clusters were highest at the L1 and embryonic stages. The editing frequency of a cluster positively correlated with the number of editing sites within it. Intriguingly, for 80% of the clusters with 10 or more editing sites, almost all expressed transcripts were edited. Deletion of adr-1 reduced the editing frequency but not the number of editing clusters, whereas deletion of adr-2 nearly abolished RNA editing, indicating a modulating role of ADR-1 and an essential role of ADR-2 in A-to-I editing. Quantitative proteomics analysis showed that adr-2 mutant worms altered the abundance of proteins involved in aging and lifespan regulation. Consistent with this finding, we observed that worms lacking RNA editing were short-lived. Taken together, our results reveal a sophisticated landscape of RNA editing and distinct modes of action of different ADARs.

Related Articles

Eri1: a conserved enzyme at the crossroads of multiple RNA-processing pathways.

Trends Genet. 2014 Jul;30(7):298-307

Authors: Thomas MF, L'Etoile ND, Ansel KM

Abstract
Eri1 is an evolutionarily conserved 3'-5' exoribonuclease that participates in 5.8S rRNA 3' end processing and turnover of replication-dependent histone mRNAs. Over the course of evolution, Eri1 has also been recruited into a variety of conserved and species-specific regulatory small RNA pathways that include endogenous small interfering (si)RNAs and miRNAs. Recent advances in Eri1 biology illustrate the importance of RNA metabolism in epigenetic gene regulation and illuminate common principles and players in RNA biogenesis and turnover. In this review, we highlight Eri1 as a member of a growing class of ribosome- and histone mRNA-associated proteins that have been recruited into divergent RNA metabolic pathways. We summarize recent advances in the understanding of Eri1 function in these pathways and discuss how Eri1 impacts gene expression and physiology in a variety of eukaryotic species. This emerging view highlights the possibility for crosstalk and coregulation of diverse cellular processes regulated by RNA.

PMID: 24929628 [PubMed - in process]

Related Articles

CstF-64 supports pluripotency and regulates cell cycle progression in embryonic stem cells through histone 3' end processing.

Nucleic Acids Res. 2014;42(13):8330-42

Authors: Youngblood BA, Grozdanov PN, MacDonald CC

Abstract
Embryonic stem cells (ESCs) exhibit a unique cell cycle with a shortened G1 phase that supports their pluripotency, while apparently buffering them against pro-differentiation stimuli. In ESCs, expression of replication-dependent histones is a main component of this abbreviated G1 phase, although the details of this mechanism are not well understood. Similarly, the role of 3' end processing in regulation of ESC pluripotency and cell cycle is poorly understood. To better understand these processes, we examined mouse ESCs that lack the 3' end-processing factor CstF-64. These ESCs display slower growth, loss of pluripotency and a lengthened G1 phase, correlating with increased polyadenylation of histone mRNAs. Interestingly, these ESCs also express the τCstF-64 paralog of CstF-64. However, τCstF-64 only partially compensates for lost CstF-64 function, despite being recruited to the histone mRNA 3' end-processing complex. Reduction of τCstF-64 in CstF-64-deficient ESCs results in even greater levels of histone mRNA polyadenylation, suggesting that both CstF-64 and τCstF-64 function to inhibit polyadenylation of histone mRNAs. These results suggest that CstF-64 plays a key role in modulating the cell cycle in ESCs while simultaneously controlling histone mRNA 3' end processing.

PMID: 24957598 [PubMed - indexed for MEDLINE]

Related Articles

Molecular mechanisms for the regulation of histone mRNA stem-loop-binding protein by phosphorylation.

Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):E2937-46

Authors: Zhang J, Tan D, DeRose EF, Perera L, Dominski Z, Marzluff WF, Tong L, Hall TM

Abstract
Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop-binding protein (SLBP). The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. We show that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and we use structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA.

PMID: 25002523 [PubMed - indexed for MEDLINE]

Related Articles

Human DDX6 effects miRNA-mediated gene silencing via direct binding to CNOT1.

RNA. 2014 Sep;20(9):1398-409

Authors: Rouya C, Siddiqui N, Morita M, Duchaine TF, Fabian MR, Sonenberg N

Abstract
MicroRNAs (miRNAs) play critical roles in a variety of biological processes through widespread effects on protein synthesis. Upon association with the miRNA-induced silencing complex (miRISC), miRNAs repress target mRNA translation and accelerate mRNA decay. Degradation of the mRNA is initiated by shortening of the poly(A) tail by the CCR4-NOT deadenylase complex followed by the removal of the 5' cap structure and exonucleolytic decay of the mRNA. Here, we report a direct interaction between the large scaffolding subunit of CCR4-NOT, CNOT1, with the translational repressor and decapping activator protein, DDX6. DDX6 binds to a conserved CNOT1 subdomain in a manner resembling the interaction of the translation initiation factor eIF4A with eIF4G. Importantly, mutations that disrupt the DDX6-CNOT1 interaction impair miRISC-mediated gene silencing in human cells. Thus, CNOT1 facilitates recruitment of DDX6 to miRNA-targeted mRNAs, placing DDX6 as a downstream effector in the miRNA silencing pathway.

PMID: 25035296 [PubMed - indexed for MEDLINE]

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

Nature Reviews Molecular Cell Biology 15, 632 (2014). doi:10.1038/nrm3880

Author: Eytan Zlotorynski

Following translational stress, mRNAs colocalize with mRNA decay factors in cytoplasmic processing (P) bodies. Tracking fluorescently tagged mRNAs in human cells and using fluorescence in situ hybridization, the authors found that following amino acid starvation, mRNAs that lack poly(A)-tails accumulated in the P bodies;

Publication date: 9 October 2014
Source:Cell, Volume 159, Issue 2
Author(s): Randall J. Platt , Sidi Chen , Yang Zhou , Michael J. Yim , Lukasz Swiech , Hannah R. Kempton , James E. Dahlman , Oren Parnas , Thomas M. Eisenhaure , Marko Jovanovic , Daniel B. Graham , Siddharth Jhunjhunwala , Matthias Heidenreich , Ramnik J. Xavier , Robert Langer , Daniel G. Anderson , Nir Hacohen , Aviv Regev , Guoping Feng , Phillip A. Sharp , Feng Zhang
CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras ^G12D mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.

Graphical abstract

Teaser

Viral and nonviral delivery of sgRNAs in CRISPR-Cas9 knockin mice enables diverse genome engineering applications in biology and disease modeling.

Publication date: 25 September 2014
Source:Cell, Volume 159, Issue 1
Author(s): Jesse M. Engreitz , Klara Sirokman , Patrick McDonel , Alexander A. Shishkin , Christine Surka , Pamela Russell , Sharon R. Grossman , Amy Y. Chow , Mitchell Guttman , Eric S. Lander
Intermolecular RNA-RNA interactions are used by many noncoding RNAs (ncRNAs) to achieve their diverse functions. To identify these contacts, we developed a method based on RNA antisense purification to systematically map RNA-RNA interactions (RAP-RNA) and applied it to investigate two ncRNAs implicated in RNA processing: U1 small nuclear RNA, a component of the spliceosome, and Malat1, a large ncRNA that localizes to nuclear speckles. U1 and Malat1 interact with nascent transcripts through distinct targeting mechanisms. Using differential crosslinking, we confirmed that U1 directly hybridizes to 5′ splice sites and 5′ splice site motifs throughout introns and found that Malat1 interacts with pre-mRNAs indirectly through protein intermediates. Interactions with nascent pre-mRNAs cause U1 and Malat1 to localize proximally to chromatin at active genes, demonstrating that ncRNAs can use RNA-RNA interactions to target specific pre-mRNAs and genomic sites. RAP-RNA is sensitive to lower abundance RNAs as well, making it generally applicable for investigating ncRNAs.

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Comprehensive mapping of intermolecular RNA-RNA interactions for U1 snRNA and Malat1 lncRNA reveals mechanisms for targeting noncoding RNAs to chromatin at active gene loci.

Publication date: Available online 16 October 2014
Source:Cell
Author(s): Stephane E. Castel , Jie Ren , Sonali Bhattacharjee , An-Yun Chang , Mar Sánchez , Alberto Valbuena , Francisco Antequera , Robert A. Martienssen
Nuclear RNAi is an important regulator of transcription and epigenetic modification, but the underlying mechanisms remain elusive. Using a genome-wide approach in the fission yeast S. pombe, we have found that Dcr1, but not other components of the canonical RNAi pathway, promotes the release of Pol II from the 3′ end of highly transcribed genes, and, surprisingly, from antisense transcription of rRNA and tRNA genes, which are normally transcribed by Pol I and Pol III. These Dcr1-terminated loci correspond to sites of replication stress and DNA damage, likely resulting from transcription-replication collisions. At the rDNA loci, release of Pol II facilitates DNA replication and prevents homologous recombination, which would otherwise lead to loss of rDNA repeats especially during meiosis. Our results reveal a novel role for Dcr1-mediated transcription termination in genome maintenance and may account for widespread regulation of genome stability by nuclear RNAi in higher eukaryotes.

Graphical abstract

Teaser

Dicer regulates genomic stability by preventing replication stress and DNA damage resulting from transcription-replication collisions. Dicer-mediated release of Pol II during transcription termination facilitates DNA replication and prevents homologous replication.

by Alex J. Cornish, Florian Markowetz

Linking networks of molecular interactions to cellular functions and phenotypes is a key goal in systems biology. Here, we adapt concepts of spatial statistics to assess the functional content of molecular networks. Based on the guilt-by-association principle, our approach (called SANTA) quantifies the strength of association between a gene set and a network, and functionally annotates molecular networks like other enrichment methods annotate lists of genes. As a general association measure, SANTA can (i) functionally annotate experimentally derived networks using a collection of curated gene sets and (ii) annotate experimentally derived gene sets using a collection of curated networks, as well as (iii) prioritize genes for follow-up analyses. We exemplify the efficacy of SANTA in several case studies using the S. cerevisiae genetic interaction network and genome-wide RNAi screens in cancer cell lines. Our theory, simulations, and applications show that SANTA provides a principled statistical way to quantify the association between molecular networks and cellular functions and phenotypes. SANTA is available from http://bioconductor.org/packages/release/bioc/html/SANTA.html.

Motivation: Gene set analysis is the analysis of a set of genes that collectively contribute to a biological process. Most popular gene set analysis methods are based on empirical P-value that requires large number of permutations. Despite numerous gene set analysis methods developed in the past decade, the most popular methods still suffer from serious limitations.

Results: We present a gene set analysis method (mGSZ) based on Gene Set Z-scoring function (GSZ) and asymptotic P-values. Asymptotic P-value calculation requires fewer permutations, and thus speeds up the gene set analysis process. We compare the GSZ-scoring function with seven popular gene set scoring functions and show that GSZ stands out as the best scoring function. In addition, we show improved performance of the GSA method when the max-mean statistics is replaced by the GSZ scoring function. We demonstrate the importance of both gene and sample permutations by showing the consequences in the absence of one or the other. A comparison of asymptotic and empirical methods of P-value estimation demonstrates a clear advantage of asymptotic P-value over empirical P-value. We show that mGSZ outperforms the state-of-the-art methods based on two different evaluations. We compared mGSZ results with permutation and rotation tests and show that rotation does not improve our asymptotic P-values. We also propose well-known asymptotic distribution models for three of the compared methods.

Availability and implementation: mGSZ is available as R package from cran.r-project.org.

Contact: pashupati.mishra@helsinki.fi

Supplementary information: Available at http://ekhidna.biocenter.helsinki.fi/downloads/pashupati/mGSZ.html

Nature Chemical Biology. doi:10.1038/nchembio.1654

Authors: Chao Xu, Xiao Wang, Ke Liu, Ian A Roundtree, Wolfram Tempel, Yanjun Li, Zhike Lu, Chuan He & Jinrong Min

N6-methyladenosine (m6A) is the most abundant internal modification of nearly all eukaryotic mRNAs and has recently been reported to be recognized by the YTH domain family proteins. Here we present the crystal structures of the YTH domain of YTHDC1, a member of the YTH domain family, and its complex with an m6A-containing RNA. Our structural studies, together with transcriptome-wide identification of YTHDC1-binding sites and biochemical experiments, not only reveal the specific mode of m6A-YTH binding but also explain the preferential recognition of the GG(m6A)C sequences by YTHDC1.

Publication date: 14 August 2014
Source:Cell, Volume 158, Issue 4
Author(s): Patrick Cahan , Hu Li , Samantha A. Morris , Edroaldo Lummertz da Rocha , George Q. Daley , James J. Collins
Somatic cell reprogramming, directed differentiation of pluripotent stem cells, and direct conversions between differentiated cell lineages represent powerful approaches to engineer cells for research and regenerative medicine. We have developed CellNet, a network biology platform that more accurately assesses the fidelity of cellular engineering than existing methodologies and generates hypotheses for improving cell derivations. Analyzing expression data from 56 published reports, we found that cells derived via directed differentiation more closely resemble their in vivo counterparts than products of direct conversion, as reflected by the establishment of target cell-type gene regulatory networks (GRNs). Furthermore, we discovered that directly converted cells fail to adequately silence expression programs of the starting population and that the establishment of unintended GRNs is common to virtually every cellular engineering paradigm. CellNet provides a platform for quantifying how closely engineered cell populations resemble their target cell type and a rational strategy to guide enhanced cellular engineering.

Graphical abstract

Teaser

A network biology platform, CellNet, enables a more accurate assessment of the fidelity of cellular engineering (e.g., directed differentiation and directed conversion) and provides strategies for improving cell derivations.

Nature Biotechnology 32, 933 (2014). doi:10.1038/nbt.2943

Authors: Jeffrey J Quinn, Ibrahim A Ilik, Kun Qu, Plamen Georgiev, Ci Chu, Asifa Akhtar & Howard Y Chang

Publication date: 28 August 2014
Source:Cell, Volume 158, Issue 5
Author(s): Jan P. Erzberger , Florian Stengel , Riccardo Pellarin , Suyang Zhang , Tanja Schaefer , Christopher H.S. Aylett , Peter Cimermančič , Daniel Boehringer , Andrej Sali , Ruedi Aebersold , Nenad Ban
Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. Yeast eIF3 engages 40S in a clamp-like manner, fully encircling 40S to position key initiation factors on opposite ends of the mRNA channel, providing a platform for the recruitment, assembly, and regulation of the translation initiation machinery. The structures of eIF3 components reported here also have implications for understanding the architecture of the mammalian 43S preinitiation complex and the complex of eIF3, 40S, and the hepatitis C internal ribosomal entry site RNA.

Graphical abstract

Teaser

A hybrid approach drawing on X-ray structures, crosslinking coupled to mass spectrometry, electron microscopy, and integrative modeling yields mechanistic insights into how eIF3 coordinates translation initiation.

Publication date: 11 September 2014
Source:Cell, Volume 158, Issue 6
Author(s): Edward M. De Robertis

Publication date: 11 September 2014
Source:Cell, Volume 158, Issue 6
Author(s): Ivan Topisirovic , Nahum Sonenberg
Transfer RNAs (tRNAs) deliver amino acids to the ribosome during mRNA translation. Gingold et al. now provide evidence that alterations in the cellular tRNA repertoire are tightly coordinated with changes in mRNA expression. These changes in the tRNA repertoire dictate translational programs that distinguish differentiating from proliferating cells.

Teaser

Transfer RNAs (tRNAs) deliver amino acids to the ribosome during mRNA translation. Gingold et al. now provide evidence that alterations in the cellular tRNA repertoire are tightly coordinated with changes in mRNA expression. These changes in the tRNA repertoire dictate translational programs that distinguish differentiating from proliferating cells.

Publication date: 11 September 2014
Source:Cell, Volume 158, Issue 6
Author(s): Yasuhiro Takashima , Ge Guo , Remco Loos , Jennifer Nichols , Gabriella Ficz , Felix Krueger , David Oxley , Fatima Santos , James Clarke , William Mansfield , Wolf Reik , Paul Bertone , Austin Smith
Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.

Graphical abstract

Teaser

Invoking the transcription factor circuitry that defines mouse embryonic stem cell identity converts human pluripotent cells to a more naive state, characterized by altered transcriptional and metabolic activity, absence of lineage priming, and global DNA hypomethylation.

Publication date: 11 September 2014
Source:Cell, Volume 158, Issue 6
Author(s): Hila Gingold , Disa Tehler , Nanna R. Christoffersen , Morten M. Nielsen , Fazila Asmar , Susanne M. Kooistra , Nicolaj S. Christophersen , Lise Lotte Christensen , Michael Borre , Karina D. Sørensen , Lars D. Andersen , Claus L. Andersen , Esther Hulleman , Tom Wurdinger , Elisabeth Ralfkiær , Kristian Helin , Kirsten Grønbæk , Torben Ørntoft , Sebastian M. Waszak , Orna Dahan , Jakob Skou Pedersen , Anders H. Lund , Yitzhak Pilpel
A dichotomous choice for metazoan cells is between proliferation and differentiation. Measuring tRNA pools in various cell types, we found two distinct subsets, one that is induced in proliferating cells, and repressed otherwise, and another with the opposite signature. Correspondingly, we found that genes serving cell-autonomous functions and genes involved in multicellularity obey distinct codon usage. Proliferation-induced and differentiation-induced tRNAs often carry anticodons that correspond to the codons enriched among the cell-autonomous and the multicellularity genes, respectively. Because mRNAs of cell-autonomous genes are induced in proliferation and cancer in particular, the concomitant induction of their codon-enriched tRNAs suggests coordination between transcription and translation. Histone modifications indeed change similarly in the vicinity of cell-autonomous genes and their corresponding tRNAs, and in multicellularity genes and their tRNAs, suggesting the existence of transcriptional programs coordinating tRNA supply and demand. Hence, we describe the existence of two distinct translation programs that operate during proliferation and differentiation.

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Teaser

Genes associated with proliferation and differentiation are preferentially encoded by different codons, and the tRNA pool shifts accordingly in transitions between these two states and in cancer.

Publication date: 11 September 2014
Source:Cell, Volume 158, Issue 6
Author(s): Matthew T. Weirauch , Ally Yang , Mihai Albu , Atina G. Cote , Alejandro Montenegro-Montero , Philipp Drewe , Hamed S. Najafabadi , Samuel A. Lambert , Ishminder Mann , Kate Cook , Hong Zheng , Alejandra Goity , Harm van Bakel , Jean-Claude Lozano , Mary Galli , Mathew G. Lewsey , Eryong Huang , Tuhin Mukherjee , Xiaoting Chen , John S. Reece-Hoyes , Sridhar Govindarajan , Gad Shaulsky , Albertha J.M. Walhout , François-Yves Bouget , Gunnar Ratsch , Luis F. Larrondo , Joseph R. Ecker , Timothy R. Hughes
Transcription factor (TF) DNA sequence preferences direct their regulatory activity, but are currently known for only ∼1% of eukaryotic TFs. Broadly sampling DNA-binding domain (DBD) types from multiple eukaryotic clades, we determined DNA sequence preferences for >1,000 TFs encompassing 54 different DBD classes from 131 diverse eukaryotes. We find that closely related DBDs almost always have very similar DNA sequence preferences, enabling inference of motifs for ∼34% of the ∼170,000 known or predicted eukaryotic TFs. Sequences matching both measured and inferred motifs are enriched in chromatin immunoprecipitation sequencing (ChIP-seq) peaks and upstream of transcription start sites in diverse eukaryotic lineages. SNPs defining expression quantitative trait loci in Arabidopsis promoters are also enriched for predicted TF binding sites. Importantly, our motif “library” can be used to identify specific TFs whose binding may be altered by human disease risk alleles. These data present a powerful resource for mapping transcriptional networks across eukaryotes.

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Teaser

The binding sites for more than one-third of known transcription factors across eukaryotes can be inferred following direct characterization of a representative subset of DNA-binding domains. This Resource provides an avenue for assessing the transcriptional impact of human disease alleles.

Publication date: 25 September 2014
Source:Cell, Volume 159, Issue 1
Author(s): Schraga Schwartz , Douglas A. Bernstein , Maxwell R. Mumbach , Marko Jovanovic , Rebecca H. Herbst , Brian X. León-Ricardo , Jesse M. Engreitz , Mitchell Guttman , Rahul Satija , Eric S. Lander , Gerald Fink , Aviv Regev
Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function.

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Teaser

Transcriptome-wide pseudouridine mapping reveals extensive, dynamic pseudouridylation of mRNA and noncoding RNA in yeast and human.

Publication date: 14 August 2014
Source:Cell, Volume 158, Issue 4
Author(s): Bin Yang , Jennifer B. Treweek , Rajan P. Kulkarni , Benjamin E. Deverman , Chun-Kan Chen , Eric Lubeck , Sheel Shah , Long Cai , Viviana Gradinaru
Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies.

Graphical abstract

Teaser

Methodology for rendering whole organs and bodies optically transparent and macromolecule-permeable while maintaining intact connectivity allows tissue visualization, immunolabeling, and RNA FISH at single-cell resolution and should be broadly applicable to extend imaging to anatomy that was previously optically inaccessible.

Structure of an Rrp6–RNA exosome complex bound to poly(A) RNA

Nature 511, 7510 (2014). doi:10.1038/nature13406

Authors: Elizabeth V. Wasmuth, Kurt Januszyk & Christopher D. Lima

The eukaryotic RNA exosome processes and degrades RNA by directing substrates to the distributive or processive 3′ to 5′ exoribonuclease activities of Rrp6 or Rrp44, respectively. The non-catalytic nine-subunit exosome core (Exo9) features a prominent central channel. Although RNA can pass through the channel to

Employment: PhD overdrive

Nature 511, 7508 (2014). doi:10.1038/nj7508-255a

Author: Paul Smaglik

An excess of graduates means that job-seekers need to be versatile.

Nature advance online publication 09 July 2014. doi:10.1038/nature13429

Authors: Ashton Trey Belew, Arturas Meskauskas, Sharmishtha Musalgaonkar, Vivek M. Advani, Sergey O. Sulima, Wojciech K. Kasprzak, Bruce A. Shapiro & Jonathan D. Dinman

Nature Structural & Molecular Biology 21, 577 (2014). doi:10.1038/nsmb.2850

Authors: Sophie Martin & Jeff Coller

Post-transcriptional mRNA regulation is often attained by lengthening or shortening the 3′ poly(A) tail of a transcript. Eukaryotic mRNAs show a spectrum of deadenylation rates, thus allowing intricate control of gene expression, but the mechanisms that determine such rates are unclear. Three new studies highlight the structural and biochemical features of a key enzyme in removing poly(A) tails, the PAN2–PAN3 complex, providing clues to how different mRNA deadenylation rates can be achieved.