Hyeshik Chang

Unusual base pairing during the decoding of a stop codon by the ribosome

Nature 500, 7460 (2013). doi:10.1038/nature12302

Authors: Israel S. Fernández, Chyan Leong Ng, Ann C. Kelley, Guowei Wu, Yi-Tao Yu & V. Ramakrishnan

During normal translation, the binding of a release factor to one of the three stop codons (UGA, UAA or UAG) results in the termination of protein synthesis. However, modification of the initial uridine to a pseudouridine (Ψ) allows efficient recognition and read-through of these stop codons by a transfer RNA (tRNA), although it requires the formation of two normally forbidden purine–purine base pairs. Here we determined the crystal structure at 3.1 Å resolution of the 30S ribosomal subunit in complex with the anticodon stem loop of tRNASer bound to the ΨAG stop codon in the A site. The ΨA base pair at the first position is accompanied by the formation of purine–purine base pairs at the second and third positions of the codon, which show an unusual Watson–Crick/Hoogsteen geometry. The structure shows a previously unsuspected ability of the ribosomal decoding centre to accommodate non-canonical base pairs.

Nature Methods 10, 768 (2013). doi:10.1038/nmeth.2529

Authors: Nicholas P Gauthier, Boumediene Soufi, William E Walkowicz, Virginia A Pedicord, Konstantinos J Mavrakis, Boris Macek, David Y Gin, Chris Sander & Martin L Miller

Attention enhances synaptic efficacy and the signal-to-noise ratio in neural circuits

Nature 499, 7459 (2013). doi:10.1038/nature12276

Authors: Farran Briggs, George R. Mangun & W. Martin Usrey

Attention is a critical component of perception. However, the mechanisms by which attention modulates neuronal communication to guide behaviour are poorly understood. To elucidate the synaptic mechanisms of attention, we developed a sensitive assay of attentional modulation of neuronal communication. In alert monkeys performing a visual spatial attention task, we probed thalamocortical communication by electrically stimulating neurons in the lateral geniculate nucleus of the thalamus while simultaneously recording shock-evoked responses from monosynaptically connected neurons in primary visual cortex. We found that attention enhances neuronal communication by increasing the efficacy of presynaptic input in driving postsynaptic responses, by increasing synchronous responses among ensembles of postsynaptic neurons receiving independent input, and by decreasing redundant signals between postsynaptic neurons receiving common input. The results demonstrate that attention finely tunes neuronal communication at the synaptic level by selectively altering synaptic weights, enabling enhanced detection of salient events in the noisy sensory environment.

Alexander Leitner, Ruedi Aebersold.

Quantification of microRNA Expression with Next-Generation Sequencing.

Curr Protoc Mol Biol. 2013 Jul;Chapter 4:Unit4.17

Authors: Eminaga S, Christodoulou DC, Vigneault F, Church GM, Seidman JG

Abstract
Rapid advancement of next-generation sequencing technologies has made it possible to study expression profiles of microRNAs (miRNAs) comprehensively and efficiently. Multiplexing miRNA libraries by barcoding can significantly reduce sequencing cost per sample without compromising library quality. This unit provides a step-by-step protocol for isolating miRNAs and constructing multiplexed miRNA libraries. Also described is a custom computational pipeline for analyzing the multiplexed miRNA library sequencing reads generated by Illumina-based technology. Curr. Protoc. Mol. Biol. 103:4.17.1-4.17.14. © 2013 by John Wiley & Sons, Inc.

PMID: 23821442 [PubMed - as supplied by publisher]

Remodeling of Ago2-mRNA interactions upon cellular stress reflects miRNA complementarity and correlates with altered translation rates.

Genes Dev. 2013 Jul 3;

Authors: Karginov FV, Hannon GJ

Abstract
When adapting to environmental stress, cells attenuate and reprogram their translational output. In part, these altered translation profiles are established through changes in the interactions between RNA-binding proteins and mRNAs. The Argonaute 2 (Ago2)/microRNA (miRNA) machinery has been shown to participate in stress-induced translational up-regulation of a particular mRNA, CAT-1; however, a detailed, transcriptome-wide understanding of the involvement of Ago2 in the process has been lacking. Here, we profiled the overall changes in Ago2-mRNA interactions upon arsenite stress by cross-linking immunoprecipitation (CLIP) followed by high-throughput sequencing (CLIP-seq). Ago2 displayed a significant remodeling of its transcript occupancy, with the majority of 3' untranslated region (UTR) and coding sequence (CDS) sites exhibiting stronger interaction. Interestingly, target sites that were destined for release from Ago2 upon stress were depleted in miRNA complementarity signatures, suggesting an alternative mode of interaction. To compare the changes in Ago2-binding patterns across transcripts with changes in their translational states, we measured mRNA profiles on ribosome/polysome gradients by RNA sequencing (RNA-seq). Increased Ago2 occupancy correlated with stronger repression of translation for those mRNAs, as evidenced by a shift toward lighter gradient fractions upon stress, while release of Ago2 was associated with the limited number of transcripts that remained translated. Taken together, these data point to a role for Ago2 and the mammalian miRNAs in mediating the translational component of the stress response.

PMID: 23824327 [PubMed - as supplied by publisher]

Bunyaviruses are an emerging group of medically important viruses, many of which are transmitted from insects to mammals. To identify host factors that impact infection, we performed a genome-wide RNAi screen in Drosophila and identified 131 genes that impacted infection of the mosquito-transmitted bunyavirus Rift Valley fever virus (RVFV). Dcp2, the catalytic component of the mRNA decapping machinery, and two decapping activators, DDX6 and LSM7, were antiviral against disparate bunyaviruses in both insect cells and adult flies. Bunyaviruses 5' cap their mRNAs by "cap-snatching" the 5' ends of poorly defined host mRNAs. We found that RVFV cap-snatches the 5' ends of Dcp2 targeted mRNAs, including cell cycle-related genes. Loss of Dcp2 allows increased viral transcription without impacting viral mRNA stability, while ectopic expression of Dcp2 impedes viral transcription. Furthermore, arresting cells in late S/early G2 led to increased Dcp2 mRNA targets and increased RVFV replication. Therefore, RVFV competes for the Dcp2-accessible mRNA pool, which is dynamically regulated and can present a bottleneck for viral replication.

For a number of human genes that encode transcripts containing inverted repeat Alu elements (IRAlus) within their 3' untranslated region (UTR), product mRNA is efficiently exported to the cytoplasm when the IRAlus, which mediate nuclear retention, are removed by alternative polyadenylation. Here we report a new mechanism that promotes gene expression by targeting mRNAs that maintain their 3' UTR IRAlus: Binding of the dsRNA-binding protein Staufen1 (STAU1) to 3' UTR IRAlus inhibits nuclear retention so as to augment the nuclear export of 3' UTR IRAlus-containing mRNAs (IRAlus mRNAs). Moreover, we found that 3' UTR IRAlus-bound STAU1 enhances 3' UTR IRAlus mRNA translation by precluding protein kinase R (PKR) binding, which obviates PKR activation, eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, and repression of global cell translation. Thus, STAU1 binding to 3' UTR IRAlus functions along with 3' UTR IRAlus-mediated nuclear retention to suppress the shutdown of cellular translation triggered by PKR binding to endogenous cytoplasmic dsRNAs. We also show that a changing STAU1/PKR ratio contributes to myogenesis via effects on the 3' UTR IRAlus of mRNA encoding the microRNA-binding protein LIN28.

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MicroRNA biogenesis: regulating the regulators.

Crit Rev Biochem Mol Biol. 2013 Jan-Feb;48(1):51-68

Authors: Finnegan EF, Pasquinelli AE

Abstract
MicroRNAs (miRNAs) function as 21-24 nucleotide guide RNAs that use partial base-pairing to recognize target messenger RNAs and repress their expression. As a large fraction of protein-coding genes are under miRNA control, production of the appropriate level of specific miRNAs at the right time and in the right place is integral to most gene regulatory pathways. MiRNA biogenesis initiates with transcription, followed by multiple processing steps to produce the mature miRNA. Every step of miRNA production is subject to regulation and disruption of these control mechanisms has been linked to numerous human diseases, where the balance between the expression of miRNAs and their targets becomes distorted. Here we review the basic steps of miRNA biogenesis and describe the various factors that control miRNA transcription, processing, and stability in animal cells. The tremendous effort put into producing the appropriate type and level of specific miRNAs underscores the critical role of these small RNAs in gene regulation.

PMID: 23163351 [PubMed - indexed for MEDLINE]

Victor Ambros: the broad scope of microRNAs. Interview by Caitlin Sedwick.

J Cell Biol. 2013 May 13;201(4):492-3

Authors: Ambros V

PMID: 23671307 [PubMed - in process]

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Eukaryote-Specific Insertion Elements Control Human ARGONAUTE Slicer Activity.

Cell Rep. 2013 Jun 27;3(6):1893-900

Authors: Nakanishi K, Ascano M, Gogakos T, Ishibe-Murakami S, Serganov AA, Briskin D, Morozov P, Tuschl T, Patel DJ

Abstract
We have solved the crystal structure of human ARGONAUTE1 (hAGO1) bound to endogenous 5'-phosphorylated guide RNAs. To identify changes that evolutionarily rendered hAGO1 inactive, we compared our structure with guide-RNA-containing and cleavage-active hAGO2. Aside from mutation of a catalytic tetrad residue, proline residues at positions 670 and 675 in hAGO1 introduce a kink in the cS7 loop, forming a convex surface within the hAGO1 nucleic-acid-binding channel near the inactive catalytic site. We predicted that even upon restoration of the catalytic tetrad, hAGO1-cS7 sterically hinders the placement of a fully paired guide-target RNA duplex into the endonuclease active site. Consistent with this hypothesis, reconstitution of the catalytic tetrad with R805H led to low-level hAGO1 cleavage activity, whereas combining R805H with cS7 substitutions P670S and P675Q substantially augmented hAGO1 activity. Evolutionary amino acid changes to hAGO1 were readily reversible, suggesting that loading of guide RNA and pairing of seed-based miRNA and target RNA constrain its sequence drift.

PMID: 23809764 [PubMed - in process]

Computational design of RNAs with complex energy landscapes.

Biopolymers. 2013 Jul 2;

Authors: Zu Siederdissen CH, Hammer S, Abfalter I, Hofacker IL, Flamm C, Stadler PF

Abstract
RNA has become an integral building material in synthetic biology. Dominated by their secondary structures, which can be computed efficiently, RNA molecules are amenable not only to in vitro and in vivo selection, but also to rational, computation-based design. While the inverse folding problem of constructing an RNA sequence with a prescribed ground-state structure has received considerable attention for nearly two decades, there have been few efforts to design RNAs that can switch between distinct prescribed conformations. We introduce a sure-friendly tool for designing RNA sequences that fold into multiple target structures. The underlying algorithm makes use of a combination of graph coloring and heuristic local optimization to find sequences whose energy landscapes are dominated by the prescribed conformations. A flexible interface allows the specification of a wide range of design goals. We demonstrate that bi- and tri-stable "switches" can be designed easily with moderate computational effort for the vast majority of compatible combinations of desired target structures. RNAdesign is freely available under the GPL-v3 license.

PMID: 23818234 [PubMed - as supplied by publisher]

Creating an miR30-Based shRNA Vector.

Cold Spring Harb Protoc. 2013;2013(7)

Authors: Chang K, Marran K, Valentine A, Hannon GJ

Abstract
Generating expression constructs for artificial microRNAs (miRNAs) is relatively straightforward. This protocol describes the creation of miR-30-based short hairpin RNA (shRNA) cassettes that are compatible with a number of standard vector systems. The principles outlined here can also be easily applied to other miRNA scaffolds or to simple snapback shRNAs. It is important to note that one must understand the processing of the artificial scaffold and be able to predict precisely the small RNAs that will be generated. Otherwise, no design principles can be effectively applied and the probability that any individual shRNA clone will work effectively will be greatly reduced.

PMID: 23818675 [PubMed - in process]

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Ribosome traffic on mRNAs maps to gene ontology: genome-wide quantification of translation initiation rates and polysome size regulation.

PLoS Comput Biol. 2013;9(1):e1002866

Authors: Ciandrini L, Stansfield I, Romano MC

Abstract
To understand the complex relationship governing transcript abundance and the level of the encoded protein, we integrate genome-wide experimental data of ribosomal density on mRNAs with a novel stochastic model describing ribosome traffic dynamics during translation elongation. This analysis reveals that codon arrangement, rather than simply codon bias, has a key role in determining translational efficiency. It also reveals that translation output is governed both by initiation efficiency and elongation dynamics. By integrating genome-wide experimental data sets with simulation of ribosome traffic on all Saccharomyces cerevisiae ORFs, mRNA-specific translation initiation rates are for the first time estimated across the entire transcriptome. Our analysis identifies different classes of mRNAs characterised by their initiation rates, their ribosome traffic dynamics, and by their response to ribosome availability. Strikingly, this classification based on translational dynamics maps onto key gene ontological classifications, revealing evolutionary optimisation of translation responses to be strongly influenced by gene function.

PMID: 23382661 [PubMed - indexed for MEDLINE]

Nature Methods 10, 597 (2013). doi:10.1038/nmeth.2517

Authors: Zhiao Shi, Jing Wang & Bing Zhang

Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of ¹⁸O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging ¹⁸O atom into the phosphate group during the oxidation step of the synthetic cycle by using ¹⁸O water as the oxygen donor. The ¹⁸O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The ¹⁸O/¹⁶O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of ¹⁸O-labeled RNA, and this technique was used to determine the blood concentration of ¹⁸O-labeled RNA after administration to mice. ¹⁸O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies.

The transcriptional landscape in embryonic stem cells (ESCs) and during ESC differentiation has received considerable attention, albeit mostly confined to the polyadenylated fraction of RNA, whereas the non-polyadenylated (NPA) fraction remained largely unexplored. Notwithstanding, the NPA RNA super-family has every potential to participate in the regulation of pluripotency and stem cell fate. We conducted a comprehensive analysis of NPA RNA in ESCs using a combination of whole-genome tiling arrays and deep sequencing technologies. In addition to identifying previously characterized and new non-coding RNA members, we describe a group of novel conserved RNAs (snacRNAs: small NPA conserved), some of which are differentially expressed between ESC and neuronal progenitor cells, providing the first evidence of a novel group of potentially functional NPA RNA involved in the regulation of pluripotency and stem cell fate. We further show that minor spliceosomal small nuclear RNAs, which are NPA, are almost completely absent in ESCs and are upregulated in differentiation. Finally, we show differential processing of the minor intron of the polycomb group gene Eed. Our data suggest that NPA RNA, both known and novel, play important roles in ESCs.

Diversifying microRNA sequence and function.

Nat Rev Mol Cell Biol. 2013 Jun 26;

Authors: Ameres SL, Zamore PD

Abstract
MicroRNAs (miRNAs) regulate the expression of most genes in animals, but we are only now beginning to understand how they are generated, assembled into functional complexes and destroyed. Various mechanisms have now been identified that regulate miRNA stability and that diversify miRNA sequences to create distinct isoforms. The production of different isoforms of individual miRNAs in specific cells and tissues may have broader implications for miRNA-mediated gene expression control. Rigorously testing the many discrepant models for how miRNAs function using quantitative biochemical measurements made in vivo and in vitro remains a major challenge for the future.

PMID: 23800994 [PubMed - as supplied by publisher]

Synthetic shuffling and in vitro selection reveal the rugged adaptive fitness landscape of a kinase ribozyme.

RNA. 2013 Jun 24;

Authors: Curtis EA, Bartel DP

Abstract
The relationship between genotype and phenotype is often described as an adaptive fitness landscape. In this study, we used a combination of recombination, in vitro selection, and comparative sequence analysis to characterize the fitness landscape of a previously isolated kinase ribozyme. Point mutations present in improved variants of this ribozyme were recombined in vitro in more than 10(14) different arrangements using synthetic shuffling, and active variants were isolated by in vitro selection. Mutual information analysis of 65 recombinant ribozymes isolated in the selection revealed a rugged fitness landscape in which approximately one-third of the 91 pairs of positions analyzed showed evidence of correlation. Pairs of correlated positions overlapped to form densely connected networks, and groups of maximally connected nucleotides occurred significantly more often in these networks than they did in randomized control networks with the same number of links. The activity of the most efficient recombinant ribozyme isolated from the synthetically shuffled pool was 30-fold greater than that of any of the ribozymes used to build it, which indicates that synthetic shuffling can be a rich source of ribozyme variants with improved properties.

PMID: 23798664 [PubMed - as supplied by publisher]

The ability to purify, analyze, and manipulate RNA is now essential for many laboratories working in the life sciences; however, the skills and practices required to work with RNA are not present in every laboratory, and initiating RNA research can be intimidating. In this article, we provide an overview of RNA purification procedures and discuss strategies to prevent RNA degradation, so that any competent researcher can confidently purify RNA and use it to perform meaningful experiments from the most basic to the highly sophisticated.

Nature Reviews Genetics. doi:10.1038/nrg3471

Authors: Kailiang Sun & Eric C. Lai

Nature advance online publication 30 June 2013. doi:10.1038/nature12302

Authors: Israel S. Fernández, Chyan Leong Ng, Ann C. Kelley, Guowei Wu, Yi-Tao Yu & V. Ramakrishnan

During normal translation, the binding of a release factor to one of the three stop codons (UGA, UAA or UAG) results in the termination of protein synthesis. However, modification of the initial uridine to a pseudouridine (Ψ) allows efficient recognition and read-through of these stop codons by a transfer RNA (tRNA), although it requires the formation of two normally forbidden purine–purine base pairs. Here we determined the crystal structure at 3.1 Å resolution of the 30S ribosomal subunit in complex with the anticodon stem loop of tRNASer bound to the ΨAG stop codon in the A site. The ΨA base pair at the first position is accompanied by the formation of purine–purine base pairs at the second and third positions of the codon, which show an unusual Watson–Crick/Hoogsteen geometry. The structure shows a previously unsuspected ability of the ribosomal decoding centre to accommodate non-canonical base pairs.

Molecular Systems Biology 9, (2013). doi:10.1038/msb.2013.31

Authors: Michael C Jewett, Brian R Fritz, Laura E Timmerman & George M Church

This Methods paper describes a novel approach for the isolation of ribonucleoprotein (RNP) complexes mediated by microRNAs (miRNAs). The authors developed a cell-free translation system to study interactions between hepatitis C virus (HCV) RNA and miRNA-122, which binds to the 5'-UTR region of HCV and stimulates its replication. This method could potentially be applicable to the characterization of miRNA–mRNA interactions.

Motivation: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally. MiRNAs were shown to play an important role in development and disease, and accurately determining the networks regulated by these miRNAs in a specific condition is of great interest. Early work on miRNA target prediction has focused on using static sequence information. More recently, researchers have combined sequence and expression data to identify such targets in various conditions.

Results: We developed the Protein Interaction-based MicroRNA Modules (PIMiM), a regression-based probabilistic method that integrates sequence, expression and interaction data to identify modules of mRNAs controlled by small sets of miRNAs. We formulate an optimization problem and develop a learning framework to determine the module regulation and membership. Applying PIMiM to cancer data, we show that by adding protein interaction data and modeling cooperative regulation of mRNAs by a small number of miRNAs, PIMiM can accurately identify both miRNA and their targets improving on previous methods. We next used PIMiM to jointly analyze a number of different types of cancers and identified both common and cancer-type-specific miRNA regulators.

Contact: zivbj@cs.cmu.edu

Supplementary information: Supplementary data are available at Bioinformatics online.

by Daria V. Zhernakova, Eleonora de Klerk, Harm-Jan Westra, Anastasios Mastrokolias, Shoaib Amini, Yavuz Ariyurek, Rick Jansen, Brenda W. Penninx, Jouke J. Hottenga, Gonneke Willemsen, Eco J. de Geus, Dorret I. Boomsma, Jan H. Veldink, Leonard H. van den Berg, Cisca Wijmenga, Johan T. den Dunnen, Gert-Jan B. van Ommen, Peter A. C. 't Hoen, Lude Franke

Many disease-associated variants affect gene expression levels (expression quantitative trait loci, eQTLs) and expression profiling using next generation sequencing (NGS) technology is a powerful way to detect these eQTLs. We analyzed 94 total blood samples from healthy volunteers with DeepSAGE to gain specific insight into how genetic variants affect the expression of genes and lengths of 3′-untranslated regions (3′-UTRs). We detected previously unknown cis-eQTL effects for GWAS hits in disease- and physiology-associated traits. Apart from cis-eQTLs that are typically easily identifiable using microarrays or RNA-sequencing, DeepSAGE also revealed many cis-eQTLs for antisense and other non-coding transcripts, often in genomic regions containing retrotransposon-derived elements. We also identified and confirmed SNPs that affect the usage of alternative polyadenylation sites, thereby potentially influencing the stability of messenger RNAs (mRNA). We then combined the power of RNA-sequencing with DeepSAGE by performing a meta-analysis of three datasets, leading to the identification of many more cis-eQTLs. Our results indicate that DeepSAGE data is useful for eQTL mapping of known and unknown transcripts, and for identifying SNPs that affect alternative polyadenylation. Because of the inherent differences between DeepSAGE and RNA-sequencing, our complementary, integrative approach leads to greater insight into the molecular consequences of many disease-associated variants.

Nature advance online publication 19 June 2013. doi:10.1038/nature12234

Authors: Xiao Tian, Jorge Azpurua, Christopher Hine, Amita Vaidya, Max Myakishev-Rempel, Julia Ablaeva, Zhiyong Mao, Eviatar Nevo, Vera Gorbunova & Andrei Seluanov

The naked mole rat (Heterocephalus glaber) displays exceptional longevity, with a maximum lifespan exceeding 30 years. This is the longest reported lifespan for a rodent species and is especially striking considering the small body mass of the naked mole rat. In comparison, a similarly sized house mouse has a maximum lifespan of 4 years. In addition to their longevity, naked mole rats show an unusual resistance to cancer. Multi-year observations of large naked mole-rat colonies did not detect a single incidence of cancer. Here we identify a mechanism responsible for the naked mole rat’s cancer resistance. We found that naked mole-rat fibroblasts secrete extremely high-molecular-mass hyaluronan (HA), which is over five times larger than human or mouse HA. This high-molecular-mass HA accumulates abundantly in naked mole-rat tissues owing to the decreased activity of HA-degrading enzymes and a unique sequence of hyaluronan synthase 2 (HAS2). Furthermore, the naked mole-rat cells are more sensitive to HA signalling, as they have a higher affinity to HA compared with mouse or human cells. Perturbation of the signalling pathways sufficient for malignant transformation of mouse fibroblasts fails to transform naked mole-rat cells. However, once high-molecular-mass HA is removed by either knocking down HAS2 or overexpressing the HA-degrading enzyme, HYAL2, naked mole-rat cells become susceptible to malignant transformation and readily form tumours in mice. We speculate that naked mole rats have evolved a higher concentration of HA in the skin to provide skin elasticity needed for life in underground tunnels. This trait may have then been co-opted to provide cancer resistance and longevity to this species.

Related Articles

Toward a genome-wide landscape of translational control.

Cold Spring Harb Perspect Biol. 2013 Jan;5(1):a012302

Authors: Larsson O, Tian B, Sonenberg N

Abstract
Genome-wide analysis of translational control has taken strides in recent years owing to the advent of high-throughput technologies, including DNA microarrays and deep sequencing. Global studies have unraveled a principal role, among posttranscriptional mechanisms, for mRNA translation in determining protein levels in the cell. The impact of translational control in dynamic regulation of the proteome under different conditions is increasingly appreciated. Here we review genome-wide studies that use high-throughput techniques and bioinformatics to assess the role of mRNA translation in the regulation of protein levels; we also discuss how genome-wide data on mRNA translation can be obtained, analyzed, and used to identify mechanisms of translational control.

PMID: 23209130 [PubMed - indexed for MEDLINE]

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The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing.

EMBO J. 2013 Apr 17;32(8):1115-27

Authors: Stalder L, Heusermann W, Sokol L, Trojer D, Wirz J, Hean J, Fritzsche A, Aeschimann F, Pfanzagl V, Basselet P, Weiler J, Hintersteiner M, Morrissey DV, Meisner-Kober NC

Abstract
Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations co-sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon-induced protein kinase (PACT). Fractionation and membrane co-immune precipitations further confirm that siRNA-loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC-associated double-stranded siRNA, diagnostic of RISC loading, and RISC-mediated mRNA cleavage products exclusively co-sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA-independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA-mediated RNA silencing.

PMID: 23511973 [PubMed - indexed for MEDLINE]

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RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators.

Proc Natl Acad Sci U S A. 2013 Apr 16;110(16):6536-41

Authors: Redfern AD, Colley SM, Beveridge DJ, Ikeda N, Epis MR, Li X, Foulds CE, Stuart LM, Barker A, Russell VJ, Ramsay K, Kobelke SJ, Li X, Hatchell EC, Payne C, Giles KM, Messineo A, Gatignol A, Lanz RB, O'Malley BW, Leedman PJ

Abstract
The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing.

PMID: 23550157 [PubMed - indexed for MEDLINE]