Table of Contents

December 2012; 18 (12)

REVIEWS

  • Alternative polyadenylation: New insights from global analyses

    • Yongsheng Shi
    RNA December 2012 18: 2105-2117; Published in Advance October 24, 2012, doi:10.1261/rna.035899.112

    Recent global studies revealed that mRNA alternative polyadenylation (APA) is widespread in eukaryotic transcriptomes and is an important mechanism for gene regulation. APA deregulation has been found in a variety of diseases. This article reviews recent progress in the field and provides a global perspective on the prevalence, mechanisms, and functional impact of APA.

  • A structure-based model of RIG-I activation

    • Daniel Kolakofsky,
    • Eva Kowalinski,
    • and Stephen Cusack
    RNA December 2012 18: 2118-2127; Published in Advance November 1, 2012, doi:10.1261/rna.035949.112
    OPEN ACCESS ARTICLE

    A series of high-resolution crystal structures of RIG-I and RIG-I:dsRNA cocrystals has recently been reported. Comparison of these structures provides considerable insight into how this innate immune pattern recognition receptor is activated upon detecting and binding a certain class of viral RNAs.

LETTER TO THE EDITOR

  • Crystal structure of the primary piRNA biogenesis factor Zucchini reveals similarity to the bacterial PLD endonuclease Nuc

    • Franka Voigt,
    • Michael Reuter,
    • Anisa Kasaruho,
    • Eike C. Schulz,
    • Ramesh S. Pillai,
    • and Orsolya Barabas
    RNA December 2012 18: 2128-2134; Published in Advance October 19, 2012, doi:10.1261/rna.034967.112
    OPEN ACCESS ARTICLE

    Piwi-interacting RNAs (piRNAs) are a gonad-specific class of small RNAs that associate with the Piwi clade of Argonaute proteins and play a key role in transposon silencing in animals. Since biogenesis of piRNAs is independent of the double-stranded RNA-processing enzyme Dicer, an alternative nuclease that can process single-stranded RNA transcripts has been long sought. A Phospholipase D-like protein, Zucchini, that is essential for piRNA processing has been proposed to be a nuclease acting in piRNA biogenesis. Here the authors describe the crystal structure of Zucchini from Drosophila melanogaster and show that it is very similar to the bacterial endonuclease, Nuc. The structure also reveals that homodimerization induces major conformational changes assembling the active site. The active site is situated on the dimer interface at the bottom of a narrow groove that can likely accommodate single-stranded nucleic acid substrates. Furthermore, biophysical analysis identifies protein segments essential for dimerization and provides insights into regulation of Zucchini's activity.

BIOINFORMATICS

  • Abstract folding space analysis based on helices

    • Jiabin Huang,
    • Rolf Backofen,
    • and Björn Voß
    RNA December 2012 18: 2135-2147; Published in Advance October 25, 2012, doi:10.1261/rna.033548.112

    The authors introduce a position-specific abstraction based on helices which they term helix index shapes, or hishapes for short. Utilizing a dynamic programming framework, they have implemented this abstraction in the program RNAHeliCes. Furthermore, they developed two hishape-based methods, one for energy barrier estimation, called HiPath, and one for abstract structure comparison, termed HiTed. They demonstrate the superior performance of HiPath compared to other existing methods and the competitive accuracy of HiTed. RNAHeliCes, together with HiPath and HiTed, are available for download at http://www.cyanolab.de/software/RNAHeliCes.htm.

REPORTS

  • An RNAi screen identifies additional members of the Drosophila Integrator complex and a requirement for cyclin C/Cdk8 in snRNA 3′-end formation

    • Jiandong Chen,
    • Nader Ezzeddine,
    • Bernhard Waltenspiel,
    • Todd R. Albrecht,
    • William D. Warren,
    • William F. Marzluff,
    • and Eric J. Wagner
    RNA December 2012 18: 2148-2156; Published in Advance October 24, 2012, doi:10.1261/rna.035725.112

    This paper describes the results of a genome-wide RNAi screen designed to identify novel factors required for Drosophila snRNA 3′-end formation. The RNAi screen identified two additional core members of the fly Integrator complex as well as a previously unknown function for cyclin C/cdk8 in the processing of Drosophila small nuclear RNA.

  • Activation of PKR by RNA misfolding: HDV ribozyme dimers activate PKR

    • Laurie A. Heinicke and
    • Philip C. Bevilacqua
    RNA December 2012 18: 2157-2165; Published in Advance October 25, 2012, doi:10.1261/rna.034744.112

    Protein Kinase R (PKR), the double-stranded RNA (dsRNA)-activated protein kinase, plays important roles in innate immunity. Previous studies have shown that PKR is activated by long stretches of dsRNA, RNA pseudoknots, and certain single-stranded RNAs; however, regulation of PKR by RNAs with globular tertiary structure has not been reported. In this study, the HDV ribozyme is used as a model of a mostly globular RNA. In addition to a catalytic core, the ribozyme contains a peripheral 13-bp pairing region (P4), which, upon shortening, affects neither the catalytic activity of the ribozyme nor its ability to crystallize. The authors report that the HDV ribozyme sequence alone can activate PKR. Through native gel mobility and enzymatic structure mapping experiments the authors implicate misfolded HDV ribozyme dimers as the PKR-activating species, and show that the shortened P4 leads to enhanced occupancy of the RNA dimer. These observations have implications for how RNA misfolding relates to innate immune response and human disease.

ARTICLES

  • RNase III-independent microRNA biogenesis in mammalian cells

    • Thomas Maurin,
    • Demián Cazalla,
    • Jr-Shiuan Yang,
    • Diane Bortolamiol-Becet,
    • and Eric C. Lai
    RNA December 2012 18: 2166-2173; Published in Advance October 24, 2012, doi:10.1261/rna.036194.112

    RNase III enzymes are fundamental to the biogenesis of microRNAs (miRNAs) and small interfering RNAs (siRNAs) in all species studied. Although alternative miRNA pathways independent of Drosha or Dicer exist, each still requires one RNase III-type enzyme. Here, the authors describe two strategies that marry either RNase Z or the Integrator complex with the slicing activity of Argonaute2 to generate highly functional mature miRNAs. They provide stringent validation of their RNase III independence by demonstrating efficient miRNA biogenesis and activity in Drosha and Dicer knockout cells. These data provide proof-of-principle evidence for additional mechanistic possibilities for efficient generation of small regulatory RNAs, and represent novel silencing triggers that may be exploited for technical purposes.

  • Cotranscriptional splicing efficiency differs dramatically between Drosophila and mouse

    • Yevgenia L. Khodor,
    • Jerome S. Menet,
    • Michael Tolan,
    • and Michael Rosbash
    RNA December 2012 18: 2174-2186; Published in Advance October 24, 2012, doi:10.1261/rna.034090.112

    Spliceosome assembly and/or splicing of a nascent transcript may be crucial for proper isoform expression and gene regulation in higher eukaryotes. It has been shown that cotranscriptional splicing occurs efficiently in Drosophila, but there are not comparable genome-wide nascent splicing data from mammals. To provide this comparison, the authors analyzed a recently generated, high-throughput sequencing data set of mouse liver nascent RNA. Cotranscriptional splicing is approximately twofold less efficient in mouse liver than in Drosophila. Absolute gene length correlates positively with cotranscriptional splicing efficiency independently of intron location and position, in flies as well as in mice. The gene length and distance effects indicate that more “nascent time” gives rise to greater cotranscriptional splicing efficiency in both systems.

  • Kinetic analysis of pre-ribosome structure in vivo

    • Agata Swiatkowska,
    • Wiebke Wlotzka,
    • Alex Tuck,
    • J. David Barrass,
    • Jean D. Beggs,
    • and David Tollervey
    RNA December 2012 18: 2187-2200; Published in Advance October 23, 2012, doi:10.1261/rna.034751.112
    OPEN ACCESS ARTICLE

    The authors combine in vivo DMS modification with metabolic labeling with 4-thiouracil in order to follow kinetically the structural changes occurring during the maturation of RNA–protein complexes. They apply the method successfully to the pre-ribosomal particle assembly.

  • Transcriptomic profiling of the oyster pathogen Vibrio splendidus opens a window on the evolutionary dynamics of the small RNA repertoire in the Vibrio genus

    • Claire Toffano-Nioche,
    • An N. Nguyen,
    • Claire Kuchly,
    • Alban Ott,
    • Daniel Gautheret,
    • Philippe Bouloc,
    • and Annick Jacq
    RNA December 2012 18: 2201-2219; Published in Advance October 24, 2012, doi:10.1261/rna.033324.112

    The extent to which regulatory RNAs are conserved and how they evolved is not yet well understood. To be able to answer questions about regulatory RNA evolution, more information about the expression and functions of regulatory RNAs in a wider array of related organisms is needed. In this study, the authors carried out transcriptome analysis of the oyster pathogen Vibrio splendidus and compared the complement of regulatory RNAs in V. splendidus with the complement characterized in other Vibrio species. The authors report that while there is a core of 28 Vibrio small RNAs, the majority of the regulatory RNA elements are species-specific.

  • Post-developmental microRNA expression is required for normal physiology, and regulates aging in parallel to insulin/IGF-1 signaling in C. elegans

    • Nicolas J. Lehrbach,
    • Cecilia Castro,
    • Kenneth J. Murfitt,
    • Cei Abreu-Goodger,
    • Julian L. Griffin,
    • and Eric A. Miska
    RNA December 2012 18: 2220-2235; Published in Advance October 24, 2012, doi:10.1261/rna.035402.112
    OPEN ACCESS ARTICLE

    This article reports the isolation and phenotypic characterization of a temperature-sensitive allele of the Microprocessor component PASH-1 (known as DGCR8 in mammals) in the nematode Caenorhabditis elegans. This allows rapid and reversible inactivation of miRNA production in vivo. Interestingly, a shift of pash-1 mutant adults to restrictive temperature results in reduced lifespan, which can be rescued by tissue-specific expression of pash-1 in neurons, hypodermis, and muscle. The effect of miRNAs on lifespan is not acting through the insuling signaling pathway. Their results strongly suggest a post-developmental role for miRNAs in C. elegans affecting lifespan.

  • Inhibition of polyadenylation reduces inflammatory gene induction

    • Alexander Kondrashov,
    • Hedda A. Meijer,
    • Adeline Barthet-Barateig,
    • Hannah N. Parker,
    • Asma Khurshid,
    • Sarah Tessier,
    • Marie Sicard,
    • Alan J. Knox,
    • Linhua Pang,
    • and Cornelia H. de Moor
    RNA December 2012 18: 2236-2250; Published in Advance November 1, 2012, doi:10.1261/rna.032391.112
    OPEN ACCESS ARTICLE

    The adenosine analog cordycepin is thought to be the active ingredient of the caterpillar fungi, which are highly prized in Chinese traditional medicine. This study shows that cordycepin reduces the expression of inflammatory genes after the recruitment of RNA polymerase and that other methods of inhibiting polyadenylation also specifically affect inflammatory gene expression. These findings therefore indicate that the inhibition of polyadenylation has gene-specific effects and that polyadenylation may be a novel target for anti-inflammatory drugs.

  • E. coli 6S RNA release from RNA polymerase requires σ70 ejection by scrunching and is orchestrated by a conserved RNA hairpin

    • Shanker Shyam S. Panchapakesan and
    • Peter J. Unrau
    RNA December 2012 18: 2251-2259; Published in Advance November 1, 2012, doi:10.1261/rna.034785.112

    The 6S RNA plays a key role in the global regulation of eubacterial transcription. This RNA in Escherichia coli suppresses housekeeping transcription by binding to RNA polymerase holoenzyme (core polymerase + σ70) under low nutrient conditions and rescues σ70-dependent transcription in high nutrient conditions by the synthesis of a short product RNA (pRNA) using itself as a template. Previous studies have observed core polymerase bound to 6S RNA and lacking σ70, but the implications of this state have remained unclear. Here the authors show that this state is a kinetic intermediate that arises during the process of 6S RNA release. This state is related to the formation of a top-strand “release” hairpin that is conserved across the γ-proteobacteria.

  • Human tRNASec associates with HeLa membranes, cell lipid liposomes, and synthetic lipid bilayers

    • Teresa Janas,
    • Tadeusz Janas,
    • and Michael Yarus
    RNA December 2012 18: 2260-2268; Published in Advance October 24, 2012, doi:10.1261/rna.035352.112
    OPEN ACCESS ARTICLE

    The authors have shown previously that simple RNA structures bind pure phospholipid liposomes. However, binding of bona fide cellular RNA under physiological ionic conditions is shown here for the first time. Human tRNASec contains a hydrophobic anticodon-loop modification: N6-isopentenyladenosine (i6A) adjacent to its anticodon. Using a highly specific double-probe hybridization assay, the authors show mature human tRNASec specifically retained in HeLa intermediate-density membranes. Further, isolated human tRNASec rebinds to liposomes from isolated HeLa membrane lipids, to a much greater extent than an unmodified tRNASec transcript.

  • Trmt61B is a methyltransferase responsible for 1-methyladenosine at position 58 of human mitochondrial tRNAs

    • Takeshi Chujo and
    • Tsutomu Suzuki
    RNA December 2012 18: 2269-2276; Published in Advance October 24, 2012, doi:10.1261/rna.035600.112

    In human mitochondria, 1-methyladenosine (m1A) occurs at position 58 of tRNALeu(UUR). In addition, partial m1A58 modifications have been found in human mitochondrial tRNALys and tRNASer(UCN). The authors identified human Trmt61B, which encodes a mitochondria-specific tRNA methyltransferase responsible for m1A58 in these three tRNAs. Trmt61B is dominantly localized to the mitochondria. m1A58 formation in human mitochondrial tRNALeu(UUR) could be reconstituted in vitro using recombinant Trmt61B in the presence of Ado-Met as a methyl donor. Unlike the cytoplasmic tRNA m1A58 methyltransferase that consists of an α2β2 heterotetramer formed by Trmt61A and Trmt6, Trmt61B formed a homo-oligomer (presumably a homotetramer) that resembled the bacterial homotetrameric m1A58 methyltransferase.

  • Structure and mechanism of the polynucleotide kinase component of the bacterial Pnkp-Hen1 RNA repair system

    • Li Kai Wang,
    • Ushati Das,
    • Paul Smith,
    • and Stewart Shuman
    RNA December 2012 18: 2277-2286; Published in Advance November 1, 2012, doi:10.1261/rna.036061.112

    Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 5′-kinase, a central 2′,3′ phosphatase, and a C-terminal ligase. Here the authors report the crystal structure of the kinase domain of Clostridium thermocellum Pnkp bound to ATP•Mg2+ (substrate complex) and ADP•Mg2+ (product complex). The protein consists of a core P-loop phosphotransferase fold embellished by a distinctive homodimerization module composed of secondary structure elements derived from the N and C termini of the kinase domain. The studies suggest a catalytic mechanism whereby Asp38 (as general base) activates the polynucleotide 5′-OH for its nucleophilic attack on the γ phosphorus and Lys21 and Mg2+ stabilize the transition state.

  • Twist-joints and double twist-joints in RNA structure

    • Yury I. Boutorine and
    • Sergey V. Steinberg
    RNA December 2012 18: 2287-2298; Published in Advance October 11, 2012, doi:10.1261/rna.030940.111

    Analysis of available RNA crystal structures has allowed the authors to identify a new family of RNA arrangements that they call double twist-joints, or DTJs. Each DTJ arrangement is composed of a double helix that contains two bulges incorporated into different strands and separated from each other by 2 or 3 bp. At each bulge, the double helix is over-twisted, while the unpaired nucleotides of both bulges form a complex network of stacking and hydrogen-bonding with nucleotides of helical regions. In total, the authors identified 14 DTJ cases, which can be combined in three groups based on common structural characteristics. Two DTJs form parts of functional centers in the ribosome and in the RNase P.

  • Alternative transcription start site selection leads to large differences in translation activity in yeast

    • Maria F. Rojas-Duran and
    • Wendy V. Gilbert
    RNA December 2012 18: 2299-2305; Published in Advance October 25, 2012, doi:10.1261/rna.035865.112

    mRNA levels are known to be an incomplete predictor of protein levels in eukaryotic cells. Here, it was demonstrated that there is a significant contribution of heterogeneity in the 5′ UTRs of various yeast genes to protein expression as evaluated using quantitative in vitro and in vivo translation assays. These data argue that alternative transcriptional start site (TSS) selection is an under-appreciated mechanism for the regulation of gene expression.

  • Novel insights into the architecture and protein interaction network of yeast eIF3

    • Sohail Khoshnevis,
    • Florian Hauer,
    • Pohl Milón,
    • Holger Stark,
    • and Ralf Ficner
    RNA December 2012 18: 2306-2319; Published in Advance October 26, 2012, doi:10.1261/rna.032532.112

    eIF3 is a large multimeric protein that forms the scaffold for other initiation factors during the complex process of eukaryotic translation initiation. Here the authors present an in vitro reconstitution of Saccharomyces cerevisiae eIF3 from its five recombinantly expressed and purified subunits. Detailed biochemical characterization confirms its suitability for future biochemical and structural analysis.

  • Regulation of release factor expression using a translational negative feedback loop: A systems analysis

    • Russell Betney,
    • Eric de Silva,
    • Christina Mertens,
    • Yvonne Knox,
    • J. Krishnan,
    • and Ian Stansfield
    RNA December 2012 18: 2320-2334; Published in Advance October 25, 2012, doi:10.1261/rna.035113.112

    Eukaryotic eRF1 is an essential protein (encoded by the yeast gene SUP45) that recognizes stop codons during translation termination and catalyzes peptide release. Interestingly, nonsense alleles of SUP45 are known to be viable because of feedback-regulated readthrough of the premature termination codon when overall concentrations of eRF1 are low. Here, a deterministic mathematical model was developed for this feedback regulation, and several predictions from the model were experimentally tested.

METHOD

  • RNA-ID, a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay

    • Kimberly M. Dean and
    • Elizabeth J. Grayhack
    RNA December 2012 18: 2335-2344; Published in Advance October 24, 2012, doi:10.1261/rna.035907.112

    The authors have developed a robust and sensitive method, called RNA-ID, to screen for cis-regulatory sequences in RNA using fluorescence-activated cell sorting (FACS) of yeast cells bearing a reporter in which expression of both superfolder green fluorescent protein (GFP) and yeast codon-optimized mCherry red fluorescent protein (RFP) is driven by the bidirectional GAL1,10 promoter. This method recapitulates previously reported progressive inhibition of translation mediated by increasing numbers of CGA codon pairs and restoration of expression by introduction of a tRNA with an anticodon that base pairs exactly with the CGA codon. This method also reproduces effects of paromomycin and context on stop codon read-through. This method is widely applicable to the study of both RNA-mediated and codon-mediated effects on expression.

ERRATUM

Reviewer Index

Author Index

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This Issue

December 2012; 18 (12)
Cover
  1. REVIEWS
  2. LETTER TO THE EDITOR
  3. BIOINFORMATICS
  4. REPORTS
  5. ARTICLES
  6. METHOD
  7. ERRATUM
  8. Reviewer Index
  9. Author Index

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