Hyeshik Chang

Nature Biotechnology 32, 569 (2014). doi:10.1038/nbt.2908

Authors: Shengdar Q Tsai, Nicolas Wyvekens, Cyd Khayter, Jennifer A Foden, Vishal Thapar, Deepak Reyon, Mathew J Goodwin, Martin J Aryee & J Keith Joung

Nature Biotechnology 32, 577 (2014). doi:10.1038/nbt.2909

Authors: John P Guilinger, David B Thompson & David R Liu

Nature Biotechnology 32, 543 (2014). doi:10.1038/nbt.2921

Authors: Paul Ginart & Arjun Raj

Using a cell as an RNA sequencing chip enables spatial analyses of the transcriptome at subcellular resolution.

Article

Wilms tumour (WT) is the most common paediatric kidney cancer and few driver genes related to its development have been identified. Here, the authors identify DROSHA mutations that may contribute to WT tumorigenesis through their effect on primary microRNA processing.

Nature Communications doi: 10.1038/ncomms5039

Authors: Giovana T. Torrezan, Elisa N. Ferreira, Adriana M. Nakahata, Bruna D. F. Barros, Mayra T. M. Castro, Bruna R. Correa, Ana C. V. Krepischi, Eloisa H. R. Olivieri, Isabela W. Cunha, Uri Tabori, Paul E. Grundy, Cecilia M. L. Costa, Beatriz de Camargo, Pedro A. F. Galante, Dirce M. Carraro

Background: Annotating mammalian genomes for noncoding RNAs (ncRNAs) is nontrivial since far from all ncRNAs are known and the computational models are resource demanding. Currently, the human genome holds the best mammalian ncRNA annotation, a result of numerous efforts by several groups. However, a more direct strategy is desired for the increasing number of sequenced mammalian genomes of which some, such as the pig, are relevant as disease models and production animals. Results: We present a comprehensive annotation of structured RNAs in the pig genome. Combining sequence and structure similarity search as well as class specific methods, we obtained a conservative set with a total of 3,391 structured RNA loci of which 1,011 and 2,314, respectively, hold strong sequence and structure similarity to structured RNAs in existing databases. The RNA loci cover 139 cis-regulatory element loci, 58 lncRNA loci, 11 conflicts of annotation, and 3,183 ncRNA genes. The ncRNA genes comprise 359 miRNAs, 8 ribozymes, 185 rRNAs, 638 snoRNAs, 1,030 snRNAs, 810 tRNAs and 153 ncRNA genes not belonging to the here fore mentioned classes . When running the pipeline on a local shuffled version of the genome, we obtained no matches at the highest confidence level. Additional analysis of RNA-seq data from a pooled library from 10 different pig tissues added another 165 miRNA loci, yielding an overall annotation of 3,556 structured RNA loci. This annotation represents our best effort at making an automated annotation. To further enhance the reliability, 571 of the 3,556 structured RNAs were manually curated by methods depending on the RNA class while 1,581 were declared as pseudogenes. We further created a multiple alignment of pig against 20 representative vertebrates, from which RNAz predicted 83,859 de novo RNA loci with conserved RNA structures. 528 of the RNAz predictions overlapped with the homology based annotation or novel miRNAs. We further present a substantial synteny analysis which includes 1,004 lineage specific de novo RNA loci and 4 ncRNA loci in the known annotation specific for Laurasiatheria (pig, cow, dolphin, horse, cat, dog, hedgehog). Conclusions: We have obtained one of the most comprehensive annotations for structured ncRNAs of a mammalian genome, which is likely to play central roles in both health modelling and production. The core annotation is available in Ensembl 70 and the complete annotation is available at http://rth.dk/resources/rnannotator/susscr102/version1.02.

Nature advance online publication 11 June 2014. doi:10.1038/nature13428

Authors: Jin Chen, Alexey Petrov, Magnus Johansson, Albert Tsai, Seán E. O’Leary & Joseph D. Puglisi

Spontaneous changes in the reading frame of translation are rare (frequency of 10−3 to 10−4 per codon), but can be induced by specific features in the messenger RNA (mRNA). In the presence of mRNA secondary structures, a heptanucleotide ‘slippery sequence’ usually defined by the motif X XXY YYZ, and (in some prokaryotic cases) mRNA sequences that base pair with the 3′ end of the 16S ribosomal rRNA (internal Shine–Dalgarno sequences), there is an increased probability that a specific programmed change of frame occurs, wherein the ribosome shifts one nucleotide backwards into an overlapping reading frame (−1 frame) and continues by translating a new sequence of amino acids. Despite extensive biochemical and genetic studies, there is no clear mechanistic description for frameshifting. Here we apply single-molecule fluorescence to track the compositional and conformational dynamics of individual ribosomes at each codon during translation of a frameshift-inducing mRNA from the dnaX gene in Escherichia coli. Ribosomes that frameshift into the −1 frame are characterized by a tenfold longer pause in elongation compared to non-frameshifted ribosomes, which translate through unperturbed. During the pause, interactions of the ribosome with the mRNA stimulatory elements uncouple EF-G catalysed translocation from normal ribosomal subunit reverse-rotation, leaving the ribosome in a non-canonical intersubunit rotated state with an exposed codon in the aminoacyl-tRNA site (A site). tRNALys sampling and accommodation to the empty A site and EF-G action either leads to the slippage of the tRNAs into the −1 frame or maintains the ribosome into the 0 frame. Our results provide a general mechanistic and conformational framework for −1 frameshifting, highlighting multiple kinetic branchpoints during elongation.

Nature advance online publication 11 June 2014. doi:10.1038/nature13440

Authors: Masahiro Naganuma, Shun-ichi Sekine, Yeeting Esther Chong, Min Guo, Xiang-Lei Yang, Howard Gamper, Ya-Ming Hou, Paul Schimmel & Shigeyuki Yokoyama

An in vivo transcriptional pause consensus sequence determined in Escherichia coli is functional across prokaryotes. [Also see Perspective by Roberts] Authors: Irina O. Vvedenskaya, Hanif Vahedian-Movahed, Jeremy G. Bird, Jared G. Knoblauch, Seth R. Goldman, Yu Zhang, Richard H. Ebright, Bryce E. Nickels

In PNAS, Hussain and Asgari claim they identified a “microRNA-like” 23-nucleotide RNA expressed by Dengue virus 2 (DENV2), called viral small RNA (vsRNA)-5, in infected mosquito and mammalian cells (1). vsRNA-5, which could also be detected by Northern blot or RT-PCR, was reported to repress DENV2 replication in insect Aag2...

Publication date: July 2010
Source:Trends in Cell Biology, Volume 20, Issue 7
Author(s): Timothy T. Weil , Richard M. Parton , Ilan Davis
Localized mRNA provides spatial and temporal protein expression essential to cell development and physiology. To explore the mechanisms involved, considerable effort has been spent in establishing new and improved methods for visualizing mRNA. Here, we discuss how these techniques have extended our understanding of intracellular mRNA localization in a variety of organisms. In addition to increased ease and specificity of detection in fixed tissue, in situ hybridization methods now enable examination of mRNA distribution at the ultrastructural level with electron microscopy. Most significantly, methods for following the movement of mRNA in living cells are now in widespread use. These include the introduction of labeled transcripts by microinjection, hybridization based methods using labeled antisense probes and complementary transgenic methods for tagging endogenous mRNAs using bacteriophage components. These technical innovations are now being coupled with super-resolution light microscopy methods and promise to revolutionize our understanding of the dynamics and complexity of the molecular mechanism of mRNA localization.

Publication date: July 2014
Source:Trends in Genetics, Volume 30, Issue 7
Author(s): Molly F. Thomas , Noelle D. L’Etoile , K. Mark Ansel
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.

Related Articles

Highly ordered architecture of microRNA cluster.

Biomed Res Int. 2013;2013:463168

Authors: Shi B, Zhu M, Liu S, Zhang M

Abstract
Although it is known that the placement of genes in a cluster may be critical for proper expression patterns, it remains largely unclear whether the orders of members in an miRNA cluster have biological insights. By investigating the relationship between expression and orders for miRNAs from the oncogenic miR-17-92 cluster, we observed a highly ordered architecture in this cluster. A significant correlation between miRNA expression level and its placement was revealed. More importantly, the placement of these miRNAs is associated with their dysregulation in cancer. Here, we presented the opinion that miRNA clusters are not arranged randomly but show highly ordered architectures, which may have critical roles in physiology and pathology.

PMID: 24195073 [PubMed - indexed for MEDLINE]

Related Articles

Revisiting the coding potential of the E. coli genome through Hfq co-immunoprecipitation.

RNA Biol. 2014 Jun 12;11(5)

Authors: Bilusic I, Popitsch N, Rescheneder P, Schroeder R, Lybecker M

Abstract
Hfq is a global regulator of gene expression in bacteria undergoing adaptation to changing environmental conditions. Its major function is to promote RNA-RNA interactions between regulatory small RNAs (sRNAs) and their target mRNAs. Previously, we demonstrated that Hfq binds many antisense RNAs (asRNAs) in vitro and hypothesized that Hfq may play a role in regulating gene expression via asRNAs. To investigate the E. coli Hfq-binding transcriptome in more detail, we co-immunoprecipitated and deep-sequenced RNAs bound to Hfq in vivo. We detected many new Hfq-binding sRNAs and observed that almost 300 mRNAs bind to Hfq. Among these, several are known to be sRNA targets. We identified 25 novel RNAs, which are transcribed from within protein coding regions and named them intragenic RNAs (intraRNAs). Furthermore, 67 asRNAs were co-immunoprecipitated with Hfq, demonstrating that Hfq binds antisense transcripts in vivo. Northern blot analyses confirmed the deep-sequencing results and demonstrated that many of the novel Hfq-binding RNAs identified are regulated by Hfq.

PMID: 24922322 [PubMed - as supplied by publisher]

Related Articles

RNA regulatory networks in animals and plants: a long noncoding RNA perspective.

Brief Funct Genomics. 2014 Jun 9;

Authors: Bai Y, Dai X, Harrison AP, Chen M

Abstract
A recent highlight of genomics research has been the discovery of many families of transcripts which have function but do not code for proteins. An important group is long noncoding RNAs (lncRNAs), which are typically longer than 200 nt, and whose members originate from thousands of loci across genomes. We review progress in understanding the biogenesis and regulatory mechanisms of lncRNAs. We describe diverse computational and high throughput technologies for identifying and studying lncRNAs. We discuss the current knowledge of functional elements embedded in lncRNAs as well as insights into the lncRNA-based regulatory network in animals. We also describe genome-wide studies of large amount of lncRNAs in plants, as well as knowledge of selected plant lncRNAs with a focus on biotic/abiotic stress-responsive lncRNAs.

PMID: 24914100 [PubMed - as supplied by publisher]

Regulation of pri-miRNA Processing by a Long Noncoding RNA Transcribed from an Ultraconserved Region.

Mol Cell. 2014 Jun 4;

Authors: Liz J, Portela A, Soler M, Gómez A, Ling H, Michlewski G, Calin GA, Guil S, Esteller M

Abstract
Noncoding RNAs (ncRNAs) control cellular programs by affecting protein-coding genes, but evidence increasingly points to their involvement in a network of ncRNA-ncRNA interactions. Here, we show that a long ncRNA, Uc.283+A, controls pri-miRNA processing. Regulation requires complementarity between the lower stem region of the pri-miR-195 transcript and an ultraconserved sequence in Uc.283+A, which prevents pri-miRNA cleavage by Drosha. Mutation of the site in either RNA molecule uncouples regulation in vivo and in vitro. We propose a model in which lower-stem strand invasion by Uc.283+A impairs microprocessor recognition and efficient pri-miRNA cropping. In addition to identifying a case of RNA-directed regulation of miRNA biogenesis, our study reveals regulatory networks involving different ncRNA classes of importance in cancer.

PMID: 24910097 [PubMed - as supplied by publisher]

The DGCR8 RNA-Binding Heme Domain Recognizes Primary MicroRNAs by Clamping the Hairpin.

Cell Rep. 2014 Jun 5;

Authors: Quick-Cleveland J, Jacob JP, Weitz SH, Shoffner G, Senturia R, Guo F

Abstract
Canonical primary microRNA transcripts (pri-miRNAs) are characterized by a ∼30 bp hairpin flanked by single-stranded regions. These pri-miRNAs are recognized and cleaved by the Microprocessor complex consisting of the Drosha nuclease and its obligate RNA-binding partner DGCR8. It is not well understood how the Microprocessor specifically recognizes pri-miRNA substrates. Here, we show that in addition to the well-known double-stranded RNA-binding domains, DGCR8 uses a dimeric heme-binding domain to directly contact pri-miRNAs. This RNA-binding heme domain (Rhed) directs two DGCR8 dimers to bind each pri-miRNA hairpin. The two Rhed-binding sites are located at both ends of the hairpin. The Rhed and its RNA-binding surface are important for pri-miRNA processing activity. Additionally, the heme cofactor is required for formation of processing-competent DGCR8-pri-miRNA complexes. Our study reveals a unique protein-RNA interaction central to pri-miRNA recognition. We propose a unifying model in which two DGCR8 dimers clamp a pri-miRNA hairpin using their Rheds.

PMID: 24910438 [PubMed - as supplied by publisher]

Publication date: 26 June 2014
Source:Cell Reports, Volume 7, Issue 6
Author(s): Jenna E. Smith , Juan R. Alvarez-Dominguez , Nicholas Kline , Nathan J. Huynh , Sarah Geisler , Wenqian Hu , Jeff Coller , Kristian E. Baker
High-throughput gene expression analysis has revealed a plethora of previously undetected transcripts in eukaryotic cells. In this study, we investigate >1,100 unannotated transcripts in yeast predicted to lack protein-coding capacity. We show that a majority of these RNAs are enriched on polyribosomes akin to mRNAs. Ribosome profiling demonstrates that many bind translocating ribosomes within predicted open reading frames 10–96 codons in size. We validate expression of peptides encoded within a subset of these RNAs and provide evidence for conservation among yeast species. Consistent with their translation, many of these transcripts are targeted for degradation by the translation-dependent nonsense-mediated RNA decay (NMD) pathway. We identify lncRNAs that are also sensitive to NMD, indicating that translation of noncoding transcripts also occurs in mammals. These data demonstrate transcripts considered to lack coding potential are bona fide protein coding and expand the proteome of yeast and possibly other eukaryotes.

Graphical abstract

Teaser

Genome-wide gene expression analyses have revealed that eukaryotes express a wide range of previously unidentified RNA transcripts, many of which are considered to lack protein-coding capacity. In this study, Smith et al. identify hundreds of unannotated RNA transcripts in yeast, many of which are shown to be engaged by the translational machinery and harbor short open reading frames. These findings provide evidence for translation of predicted noncoding RNA and unveil additional coding capacity within the yeast genome.

Publication date: 12 June 2014
Source:Cell Reports, Volume 7, Issue 5
Author(s): Natalia Gromak , Martin Dienstbier , Sara Macias , Mireya Plass , Eduardo Eyras , Javier F. Cáceres , Nicholas J. Proudfoot

Publication date: 19 June 2014
Source:Cell, Volume 157, Issue 7
Author(s): Rebecca M. Voorhees , Israel S. Fernández , Sjors H.W. Scheres , Ramanujan S. Hegde
Cotranslational protein translocation is a universally conserved process for secretory and membrane protein biosynthesis. Nascent polypeptides emerging from a translating ribosome are either transported across or inserted into the membrane via the ribosome-bound Sec61 channel. Here, we report structures of a mammalian ribosome-Sec61 complex in both idle and translating states, determined to 3.4 and 3.9 Å resolution. The data sets permit building of a near-complete atomic model of the mammalian ribosome, visualization of A/P and P/E hybrid-state tRNAs, and analysis of a nascent polypeptide in the exit tunnel. Unprecedented chemical detail is observed for both the ribosome-Sec61 interaction and the conformational state of Sec61 upon ribosome binding. Comparison of the maps from idle and translating complexes suggests how conformational changes to the Sec61 channel could facilitate translocation of a secreted polypeptide. The high-resolution structure of the mammalian ribosome-Sec61 complex provides a valuable reference for future functional and structural studies.

Graphical abstract

Teaser

High-resolution structures of the cotranslational translocation complex in two states containing either an idle or actively translating ribosome provide an unprecedented view of the mammalian ribosome, the translocation channel, and molecular details of how they are coupled.

Related Articles

Loss of the multifunctional RNA-binding protein RBM47 as a source of selectable metastatic traits in breast cancer.

Elife. 2014 Jun 4;:e02734

Authors: Vanharanta S, Marney CB, Shu W, Valiente M, Zou Y, Mele A, Darnell RB, Massagué J

Abstract
The mechanisms through which cancer cells lock in altered transcriptional programs in support of metastasis remain largely unknown. Through integrative analysis of clinical breast cancer gene expression datasets, cell line models of breast cancer progression, and mutation data from cancer genome resequencing studies, we identified RNA binding motif protein 47 (RBM47) as a suppressor of breast cancer progression and metastasis. RBM47 inhibited breast cancer re-initiation and growth in experimental models. Transcriptome-wide HITS-CLIP analysis revealed widespread RBM47 binding to mRNAs, most prominently in introns and 3'UTRs. RBM47 altered splicing and abundance of a subset of its target mRNAs. Some of the mRNAs stabilized by RBM47, as exemplified by dickkopf WNT signaling pathway inhibitor 1, inhibit tumor progression downstream of RBM47. Our work identifies RBM47 as an RNA-binding protein that can suppress breast cancer progression and demonstrates how the inactivation of a broadly targeted RNA chaperone enables selection of a pro-metastatic state.

PMID: 24898756 [PubMed - as supplied by publisher]

Publication date: 26 June 2014
Source:Cell Reports, Volume 7, Issue 6
Author(s): Jen Quick-Cleveland , Jose P. Jacob , Sara H. Weitz , Grant Shoffner , Rachel Senturia , Feng Guo
Canonical primary microRNA transcripts (pri-miRNAs) are characterized by a ∼30 bp hairpin flanked by single-stranded regions. These pri-miRNAs are recognized and cleaved by the Microprocessor complex consisting of the Drosha nuclease and its obligate RNA-binding partner DGCR8. It is not well understood how the Microprocessor specifically recognizes pri-miRNA substrates. Here, we show that in addition to the well-known double-stranded RNA-binding domains, DGCR8 uses a dimeric heme-binding domain to directly contact pri-miRNAs. This RNA-binding heme domain (Rhed) directs two DGCR8 dimers to bind each pri-miRNA hairpin. The two Rhed-binding sites are located at both ends of the hairpin. The Rhed and its RNA-binding surface are important for pri-miRNA processing activity. Additionally, the heme cofactor is required for formation of processing-competent DGCR8-pri-miRNA complexes. Our study reveals a unique protein-RNA interaction central to pri-miRNA recognition. We propose a unifying model in which two DGCR8 dimers clamp a pri-miRNA hairpin using their Rheds.

Graphical abstract

Teaser

Recognition of microRNA primary transcripts (pri-miRNAs) by the processing machinery is essential for production and biological functions of canonical microRNAs, for evolution of new microRNA genes, and for RNAi applications based on artificial pri-miRNAs. Quick-Cleveland et al. report that a previously unsuspected heme-binding domain specifically targets key structural features of a pri-miRNA located at both ends of the hairpin helix. Their study demonstrates how extensive pri-miRNA features may be recognized by processing proteins along with a universal biological cofactor.

Publication date: 3 July 2014
Source:Molecular Cell, Volume 55, Issue 1
Author(s): Julia Liz , Anna Portela , Marta Soler , Antonio Gómez , Hui Ling , Gracjan Michlewski , George A. Calin , Sònia Guil , Manel Esteller
Noncoding RNAs (ncRNAs) control cellular programs by affecting protein-coding genes, but evidence increasingly points to their involvement in a network of ncRNA-ncRNA interactions. Here, we show that a long ncRNA, Uc.283+A, controls pri-miRNA processing. Regulation requires complementarity between the lower stem region of the pri-miR-195 transcript and an ultraconserved sequence in Uc.283+A, which prevents pri-miRNA cleavage by Drosha. Mutation of the site in either RNA molecule uncouples regulation in vivo and in vitro. We propose a model in which lower-stem strand invasion by Uc.283+A impairs microprocessor recognition and efficient pri-miRNA cropping. In addition to identifying a case of RNA-directed regulation of miRNA biogenesis, our study reveals regulatory networks involving different ncRNA classes of importance in cancer.

Graphical abstract

Teaser

miRNA biogenesis is a multistep process that affects the final amount of mature, functional miRNA. Liz et al. find that a noncoding RNA transcribed from an ultraconserved region base pairs with a pri-miRNA to inhibit pri-mRNA cleavage by the microprocessor.

Publication date: 19 June 2014
Source:Molecular Cell, Volume 54, Issue 6
Author(s): Adam D. Norris , Shangbang Gao , Megan L. Norris , Debashish Ray , Arun K. Ramani , Andrew G. Fraser , Quaid Morris , Timothy R. Hughes , Mei Zhen , John A. Calarco
Alternative splicing is important for the development and function of the nervous system, but little is known about the differences in alternative splicing between distinct types of neurons. Furthermore, the factors that control cell-type-specific splicing and the physiological roles of these alternative isoforms are unclear. By monitoring alternative splicing at single-cell resolution in Caenorhabditis elegans, we demonstrate that splicing patterns in different neurons are often distinct and highly regulated. We identify two conserved RNA-binding proteins, UNC-75/CELF and EXC-7/Hu/ELAV, which regulate overlapping networks of splicing events in GABAergic and cholinergic neurons. We use the UNC-75 exon network to discover regulators of synaptic transmission and to identify unique roles for isoforms of UNC-64/Syntaxin, a protein required for synaptic vesicle fusion. Our results indicate that combinatorial regulation of alternative splicing in distinct neurons provides a mechanism to specialize metazoan nervous systems.

Graphical abstract

Teaser

By monitoring alternative pre-mRNA splicing in vivo and at single-cell resolution in C. elegans, Norris et al. demonstrate that splicing is frequently differentially regulated in individual neurons. Combinations of RNA-binding proteins are required to create regulatory specificity, and these factors control splicing networks critical for fine-tuning neuronal physiology.

Competitive Endogenous RNAs Cannot Alter MicroRNA Function In Vivo.

Mol Cell. 2014 Jun 5;54(5):711-713

Authors: Broderick JA, Zamore PD

Abstract
In this issue of Molecular Cell, Denzler et al. (2014) report a quantitative study of microRNA function in adult mouse liver, suggesting that the natural abundance of miRNAs and their binding sites generally excludes the previously proposed regulation of miRNAs by competitive endogenous RNAs (ceRNAs).

PMID: 24905003 [PubMed - as supplied by publisher]

MicroRNA Machinery Genes as Novel Biomarkers for Cancer.

Front Oncol. 2014;4:113

Authors: Huang JT, Wang J, Srivastava V, Sen S, Liu SM

Abstract
MicroRNAs (miRNAs) directly and indirectly affect tumorigenesis. To be able to perform their myriad roles, miRNA machinery genes, such as Drosha, DGCR8, Dicer1, XPO5, TRBP, and AGO2, must generate precise miRNAs. These genes have specific expression patterns, protein-binding partners, and biochemical capabilities in different cancers. Our preliminary analysis of data from The Cancer Genome Atlas consortium on multiple types of cancer revealed significant alterations in these miRNA machinery genes. Here, we review their biological structures and functions with an eye toward understanding how they could serve as cancer biomarkers.

PMID: 24904827 [PubMed - as supplied by publisher]

Related Articles

Digital expression profiling of the compartmentalized translatome of Purkinje neurons.

Genome Res. 2014 Jun 5;

Authors: Kratz A, Beguin P, Kaneko M, Chimura T, Suzuki AM, Matsunaga A, Kato S, Bertin N, Lassmann T, Vigot R, Carninci P, Plessy C, Launey T

Abstract
Underlying the complexity of the mammalian brain is its network of neuronal connections, but also the molecular networks of signaling pathways, protein interactions and regulated gene expression within each individual neuron. The diversity and complexity of the spatially intermingled neurons poses a serious challenge to the identification and quantification of single neuron components. To address this challenge, we present a novel approach for the study of the ribosome-associated transcriptome - the translatome - from selected sub-cellular domains of specific neurons, and apply it to the Purkinje cells (PC) in the rat cerebellum. We combined microdissection, Translating Ribosome Affinity Purification (TRAP) in non-transgenic animals and quantitative nanoCAGE sequencing to obtain a snapshot of RNAs bound to cytoplasmic or rough endoplasmic reticulum (rER)-associated ribosomes, in the PC and its dendrites. This allowed us to discover novel markers of PCs, to determine structural aspects of genes, to find hitherto uncharacterized transcripts, and to quantify biophysically relevant genes of membrane proteins controlling ion homeostasis and neuronal electrical activities.

PMID: 24904046 [PubMed - as supplied by publisher]

Related Articles

Cell cycle-dependent regulation of Aurora kinase B mRNA by the Microprocessor complex.

Biochem Biophys Res Commun. 2014 Mar 28;446(1):241-7

Authors: Jung E, Seong Y, Seo JH, Kwon YS, Song H

Abstract
Aurora kinase B regulates the segregation of chromosomes and the spindle checkpoint during mitosis. In this study, we showed that the Microprocessor complex, which is responsible for the processing of the primary transcripts during the generation of microRNAs, destabilizes the mRNA of Aurora kinase B in human cells. The Microprocessor-mediated cleavage kept Aurora kinase B at a low level and prevented premature entrance into mitosis. The cleavage was reduced during mitosis leading to the accumulation of Aurora kinase B mRNA and protein. In addition to Aurora kinase B mRNA, the processing of other primary transcripts of miRNAs were also decreased during mitosis. We found that the cleavage was dependent on an RNA helicase, DDX5, and the association of DDX5 and DDX17 with the Microprocessor was reduced during mitosis. Thus, we propose a novel mechanism by which the Microprocessor complex regulates stability of Aurora kinase B mRNA and cell cycle progression.

PMID: 24589731 [PubMed - indexed for MEDLINE]

Related Articles

Cell Cycle-Regulated Transcription: Effectively Using a Genomics Toolbox.

Methods Mol Biol. 2014;1170:3-27

Authors: Bristow SL, Leman AR, Haase SB

Abstract
The cell cycle comprises a series of temporally ordered events that occur sequentially, including DNA replication, centrosome duplication, mitosis, and cytokinesis. What are the regulatory mechanisms that ensure proper timing and coordination of events during the cell cycle? Biochemical and genetic screens have identified a number of cell-cycle regulators, and it was recognized early on that many of the genes encoding cell-cycle regulators, including cyclins, were transcribed only in distinct phases of the cell cycle. Thus, "just in time" expression is likely an important part of the mechanism that maintains the proper temporal order of cell cycle events. New high-throughput technologies for measuring transcript levels have revealed that a large percentage of the Saccharomyces cerevisiae transcriptome (~20 %) is cell cycle regulated. Similarly, a substantial fraction of the mammalian transcriptome is cell cycle-regulated. Over the past 25 years, many studies have been undertaken to determine how gene expression is regulated during the cell cycle. In this review, we discuss contemporary models for the control of cell cycle-regulated transcription, and how this transcription program is coordinated with other cell cycle events in S. cerevisiae. In addition, we address the genomic approaches and analytical methods that enabled contemporary models of cell cycle transcription. Finally, we address current and future technologies that will aid in further understanding the role of periodic transcription during cell cycle progression.

PMID: 24906306 [PubMed - as supplied by publisher]

Background: Gene expression in vertebrate cells may be controlled post-transcriptionally through regulatory elements in mRNAs. These are usually located in the untranslated regions (UTRs) of mRNA sequences, particularly the 3[prime]UTRs. Results: Scan for Motifs (SFM) simplifies the process of identifying a wide range of regulatory elements on alignments of vertebrate 3[prime]UTRs. SFM includes identification of both RNA Binding Protein (RBP) sites and targets of miRNAs. In addition to searching pre-computed alignments, the tool provides users the flexibility to search their own sequences or alignments. The regulatory elements may be filtered by expected value cutoffs and are cross-referenced back to their respective sources and literature. The output is an interactive graphical representation, highlighting potential regulatory elements and overlaps between them. The output also provides simple statistics and links to related resources for complementary analyses. The overall process is intuitive and fast. As SFM is a free web-application, the user does not need to install any software or databases. Conclusions: Visualisation of the binding sites of different classes of effectors that bind to 3[prime]UTRs will facilitate the study of regulatory elements in 3[prime] UTRs.