Table of Contents

March 2012; 18 (3)

HYPOTHESIS

  • Rationalization and prediction of selective decoding of pseudouridine-modified nonsense and sense codons

    • Marc Parisien,
    • Chengqi Yi,
    • and Tao Pan
    RNA March 2012 18: 355-367; Published in Advance January 26, 2012, doi:10.1261/rna.031351.111

    A stop or nonsense codon is an in-frame triplet within a messenger RNA that signals the termination of translation. One common feature shared among all three nonsense codons (UAA, UAG, and UGA) is a uridine present at the first codon position. It has been recently shown that the conversion of this uridine into pseudouridine (Ψ) suppresses translation termination, both in vitro and in vivo. Furthermore, decoding of the pseudouridylated nonsense codons is accompanied by the incorporation of two specific amino acids in a nonsense codon-dependent fashion. How Ψ suppresses termination and, more importantly, enables selective decoding is poorly understood. Here, the authors provide molecular rationales for how pseudouridylated stop codons are selectively decoded. Their analysis enables prediction of potential decoding properties for Ψ-modified sense codons.

BIOINFORMATICS

  • Known and novel post-transcriptional regulatory sequences are conserved across plant families

    • Justin N. Vaughn,
    • Sally R. Ellingson,
    • Flavio Mignone,
    • and Albrecht von Arnim
    RNA March 2012 18: 368-384; Published in Advance January 11, 2012, doi:10.1261/rna.031179.111

    Messenger RNAs contain a myriad of cis-regulatory RNA sequences that interact with RNA binding proteins and small RNAs. These elements and their binding partners participate in the regulation of RNA localization, RNA stability, and translation, among other processes. In this paper, the authors use bioinformatics to identify nearly 300 conserved regulatory sequence elements, many of which are novel, in the 5′ and 3′ UTRs of plant mRNAs. This work provides a rich catalog of conserved sequence elements that will be the focus of future functional studies.

  • Improved prediction of RNA tertiary structure with insights into native state dynamics

    • John Paul Bida and
    • L. James Maher III
    RNA March 2012 18: 385-393; Published in Advance January 25, 2012, doi:10.1261/rna.027201.111
    OPEN ACCESS ARTICLE

    The importance of RNA tertiary structure is evident from the growing number of published high resolution NMR and X-ray crystallographic structures of RNA molecules. These structures provide insights into function and create a knowledge base that is leveraged by programs such as Assemble, ModeRNA, RNABuilder, NAST, FARNA, Mc-Sym, RNA2D3D, and iFoldRNA for tertiary structure prediction and design. While these methods sample native-like RNA structures during simulations, all struggle to capture the native RNA conformation after scoring. The authors propose RSIM, an improved RNA fragment assembly method that preserves RNA global secondary structure while sampling conformations. This approach enhances the quality of predicted RNA tertiary structure, provides insights into the native state dynamics, and generates a powerful visualization of the RNA conformational space.

  • Widespread RNA 3′-end oligouridylation in mammals

    • Yun S. Choi,
    • Weronika Patena,
    • Andrew D. Leavitt,
    • and Michael T. McManus
    RNA March 2012 18: 394-401; Published in Advance January 30, 2012, doi:10.1261/rna.029306.111

    Nontemplated 3′-end oligouridylation is an RNA modification that occurs in many species, including humans. Unlike the familiar phenomenon of polyadenylation, nontemplated addition of uridines to RNA is poorly characterized in higher eukaryotes. To determine the prevalence of oligouridylation in higher eukaryotes, the authors used next-generation sequencing technology to deeply examine the population of small RNAs in human cells. The data reveal widespread nontemplated nucleotide addition to the 3′ ends of many classes of RNA, with short stretches of uridine or adenine being the most frequently added nucleotides.

REPORTS

  • Discovery of Pyrobaculum small RNA families with atypical pseudouridine guide RNA features

    • David L. Bernick,
    • Patrick P. Dennis,
    • Matthias Höchsmann,
    • and Todd M. Lowe
    RNA March 2012 18: 402-411; Published in Advance January 26, 2012, doi:10.1261/rna.031385.111

    Eukaryotic H/ACA box small nucleolar RNAs (snoRNAs) and their homologs in Archaea have been shown to guide pseudouridine modification in ribosomal RNAs and other essential noncoding RNAs. In this study, the authors use comparative genomics and deep RNA sequencing of hyperthermophilic Archaea to reveal a novel class of abbreviated H/ACA box small RNA that breaks previously believed invariant properties of this RNA family. The results shed new light on the minimal form of these guide RNAs and provide a comprehensive view of H/ACA-like small RNA from a crenarchaeal genus that is also noted for other atypical noncoding RNAs.

  • Identification of the enzyme responsible for N1-methylation of pseudouridine 54 in archaeal tRNAs

    • Jan Philip Wurm,
    • Marco Griese,
    • Ute Bahr,
    • Martin Held,
    • Alexander Heckel,
    • Michael Karas,
    • Jörg Soppa,
    • and Jens Wöhnert
    RNA March 2012 18: 412-420; Published in Advance January 24, 2012, doi:10.1261/rna.028498.111

    This is one of two papers in this issue to identify and characterize the enzyme responsible for N1-methylation of pseudouridine 54 in archaeal tRNAs. The recombinant methyltransferase is shown to have specificity for the sequence context of the single pseudouridine that is modified, expanding the structure/function analysis of this important family of RNA modification enzymes.

ARTICLES

  • The archaeal COG1901/DUF358 SPOUT-methyltransferase members, together with pseudouridine synthase Pus10, catalyze the formation of 1-methylpseudouridine at position 54 of tRNA

    • Kunal Chatterjee,
    • Ian K. Blaby,
    • Patrick C. Thiaville,
    • Mrinmoyee Majumder,
    • Henri Grosjean,
    • Y. Adam Yuan,
    • Ramesh Gupta,
    • and Valérie de Crécy-Lagard
    RNA March 2012 18: 421-433; Published in Advance January 24, 2012, doi:10.1261/rna.030841.111

    This is one of two papers in this issue to identify and characterize the enzyme responsible for N1-methylation of pseudouridine 54 in archaeal tRNAs. The recombinant methyltransferase is shown to have specificity for the sequence context of the single pseudouridine that is modified, expanding the structure/function analysis of this important family of RNA modification enzymes.

  • Folding of the hammerhead ribozyme: Pyrrolo-cytosine fluorescence separates core folding from global folding and reveals a pH-dependent conformational change

    • Iwona A. Buskiewicz and
    • John M. Burke
    RNA March 2012 18: 434-448; Published in Advance January 24, 2012, doi:10.1261/rna.030999.111

    The catalytic activity of the hammerhead ribozyme is limited by its ability to fold into the native tertiary structure. Analysis of folding has been hampered by a lack of assays that can independently monitor the environment of nucleobases throughout the ribozyme–substrate complex in real time. Here, the authors report the development and application of a new folding assay in which they use pyrrolo-cytosine (pyC) fluorescence to (1) probe active-site formation, (2) examine the ability of peripheral ribozyme domains to support native folding, (3) identify a pH-dependent conformational change within the ribozyme, and (4) explore its influence on the equilibrium between the folded and unfolded core of the hammerhead ribozyme. The data indicate that the natural ribozyme folds in two distinct noncooperative steps and the pH-dependent correlation between core folding and activity is linked to formation of the G8-C3 base pair.

  • La-motif–dependent mRNA association with Slf1 promotes copper detoxification in yeast

    • Luca Schenk,
    • Dominik M. Meinel,
    • Katja Strässer,
    • and André P. Gerber
    RNA March 2012 18: 449-461; Published in Advance January 23, 2012, doi:10.1261/rna.028506.111

    Post-transcriptional gene regulation by RNA-binding proteins (RBPs) is increasingly recognized to have strong implications for cell physiology. In this paper, the authors took a global approach to elucidate the previously suggested role of the yeast La-related protein (LARP) Slf1p in copper homeostasis. Use of RNA-binding protein immunopurification-microarray (RIP-Chip) allowed the identification of RNA targets for Slf1p, including a set of copper-related transcripts. Interaction of Slf1p with those RNA targets is dependent on the La-motif (LAM), which is the RNA-binding domain for the LARP proteins. Thus, Slf1 stabilizes messages for proteins involved in copper homeostasis and confers increased resistance of cells to copper toxicity. These results highlight the importance of post-transcriptional regulation of factors/pathways implicated in copper homeostasis by a cytoplasmic RBP.

  • Tudor-SN and ADAR1 are components of cytoplasmic stress granules

    • Rebekka Weissbach and
    • A.D.J. Scadden
    RNA March 2012 18: 462-471; Published in Advance January 12, 2012, doi:10.1261/rna.027656.111
    OPEN ACCESS ARTICLE

    The role for inosine-containing hyperedited dsRNA (IU-dsRNA) in cells is poorly understood. Previous studies have shown that IU-dsRNA binds specifically to various components of cytoplasmic stress granules, as well as to other proteins such as Tudor Staphylococcal Nuclease (Tudor-SN). Here the authors show that Tudor-SN localizes to cytoplasmic stress granules in HeLa cells undergoing arsenite-induced oxidative stress, or following transfection with long dsRNA (poly[IC]), which initiates an interferon cascade. They also demonstrate a novel interaction between Tudor-SN and ADAR1 and show that ADAR1 is also localized to stress granules in HeLa cells following various stresses.

  • Deep-sequencing of endothelial cells exposed to hypoxia reveals the complexity of known and novel microRNAs

    • Christine Voellenkle,
    • Jeroen van Rooij,
    • Alessandro Guffanti,
    • Elena Brini,
    • Pasquale Fasanaro,
    • Eleonora Isaia,
    • Larry Croft,
    • Matei David,
    • Maurizio C. Capogrossi,
    • Anna Moles,
    • Armando Felsani,
    • and Fabio Martelli
    RNA March 2012 18: 472-484; Published in Advance January 26, 2012, doi:10.1261/rna.027615.111

    Deep-sequencing of endothelial cells exposed to hypoxia reveals the complexity of known and novel microRNAs. MicroRNAs are regulators of gene expression with an impact on many different cellular processes. In this article, the authors use deep-sequencing techniques to profile microRNAs and miRNA variants expressed by endothelial cells exposed to hypoxia. The authors present a novel customized bioinformatic analysis pipeline for miRNA annotation. Results presented here revealed the high degree of variability of the miRNAs expressed in endothelial cells, giving an insight into the role of miRNAs in vascular physiopathology.

  • Role of helix 44 of 16S rRNA in the fidelity of translation initiation

    • Daoming Qin,
    • Qi Liu,
    • Aishwarya Devaraj,
    • and Kurt Fredrick
    RNA March 2012 18: 485-495; Published in Advance January 25, 2012, doi:10.1261/rna.031203.111

    The molecular mechanisms that govern translation initiation to ensure accuracy remain unclear. Here, the authors provide evidence that the subunit-joining step of initiation is controlled in part by a conformational change in the 1408 region of helix h44. Chemical probing of 30S initiation complexes formed with either a cognate (AUG) or near-cognate (AUC) start codon shows that an IF1-dependent enhancement at A1408 is reduced in the presence of AUG. This change in reactivity is due to a conformational change rather than loss of IF1, because other portions of the IF1 footprint are unchanged and high concentrations of IF1 fail to diminish the reactivity difference seen at A1408. These data support a model in which IF1 alters the A1408 region of h44 in a way that makes 50S docking unfavorable, and canonical codon–anticodon pairing in the P site restores h44 to a docking-favorable conformation.

  • An energetically beneficial leader–linker interaction abolishes ligand-binding cooperativity in glycine riboswitches

    • Eileen M. Sherman,
    • Jackie Esquiaqui,
    • Galal Elsayed,
    • and Jing-Dong Ye
    RNA March 2012 18: 496-507; Published in Advance January 25, 2012, doi:10.1261/rna.031286.111

    Comprised of two aptamers connected by a short nucleotide linker, the glycine riboswitch was reported to bind two glycine molecules cooperatively. Here, the authors describe the discovery and characterization of the 5′ extended glycine riboswitch containing a highly conserved leader–linker duplex involving leader nucleotides upstream of the previously reported consensus glycine riboswitch sequences. The leader–linker interaction improved the ligand binding affinities by 4.5- to 86-fold in three glycine riboswitches. In-line probing and native gel assays with two aptamers in trans, together with mutational analysis, support synergistic action between glycine-binding and interaptamer interaction during global folding of the glycine riboswitch, while the two glycine sites do not bind cooperatively.

  • A 5′-terminal phosphate is required for stable ternary complex formation and translation of leaderless mRNA in Escherichia coli

    • Jacqueline Giliberti,
    • Sean O'Donnell,
    • William J. Van Etten,
    • and Gary R. Janssen
    RNA March 2012 18: 508-518; Published in Advance January 30, 2012, doi:10.1261/rna.027698.111

    The bacteriophage (λ)'s cI mRNA was used to examine the importance of the 5′-terminal phosphate on expression of leadered and leaderless mRNA in Escherichia coli. A hammerhead ribozyme was used to produce leadered and leaderless mRNAs, in vivo and in vitro, that contain a 5′-hydroxyl. Although these mRNAs may not occur naturally in the bacterial cell, they allow for the study of the importance of the 5′-phosphorylation state in ribosome binding and translation of leadered and leaderless mRNAs. Analyses with mRNAs containing either a 5′-phosphate or a 5′-hydroxyl indicate that leaderless cI mRNA requires a 5′-phosphate for stable ribosome binding in vitro as well as expression in vivo.

  • The 5′ UTR of HIV-1 full-length mRNA and the Tat viral protein modulate the programmed −1 ribosomal frameshift that generates HIV-1 enzymes

    • Johanie Charbonneau,
    • Karine Gendron,
    • Gerardo Ferbeyre,
    • and Léa Brakier-Gingras
    RNA March 2012 18: 519-529; Published in Advance January 27, 2012, doi:10.1261/rna.030346.111

    Translation of the full-length messenger RNA (mRNA) of the human immunodeficiency virus type 1 (HIV-1) generates the precursor of the viral enzymes via a programmed −1 ribosomal frameshift. Here the authors investigated whether the highly structured 5′ untranslated region (UTR) of this mRNA, which interferes with translation initiation, can modulate HIV-1 frameshift efficiency and showed that, when the 5′ UTR of HIV-1 mRNA occupies the 5′ end of the reporter mRNA, HIV-1 frameshift efficiency is increased about fourfold, compared with a control dual-luciferase reporter with a short unstructured 5′ UTR. This increase was related to an interference with cap-dependent translation initiation by the TAR-Poly(A) region at the 5′ end of the messenger. A model is presented that accounts for the variations in frameshift efficiency depending on the 5′ UTR and the presence of Tat, and it is proposed that a range of frameshift efficiencies is compatible with the virus replication.

  • Identification of 88 regulatory small RNAs in the TIGR4 strain of the human pathogen Streptococcus pneumoniae

    • Paloma Acebo,
    • Antonio J. Martin-Galiano,
    • Sara Navarro,
    • Ángel Zaballos,
    • and Mónica Amblar
    RNA March 2012 18: 530-546; Published in Advance January 24, 2012, doi:10.1261/rna.027359.111

    Genome sequencing of several pneumococcal strains has revealed valuable information about the potential proteins and genetic diversity of this prevalent human pathogen. However, little is known about its transcriptional regulation and its small regulatory noncoding RNAs. In this study, the authors performed deep sequencing of the Streptococcus pneumoniae TIGR4 strain RNome to identify small regulatory RNA candidates expressed in this pathogen. They discovered 1047 potential small RNAs including intragenic, 5′- and/or 3′-overlapping RNAs and 88 small RNAs encoded in intergenic regions. In addition to previously identified intergenic small RNAs, 68 novel candidates were identified, most of which are conserved in both sequence and genomic context in other S. pneumoniae strains.

  • Alternative ferritin mRNA translation via internal initiation

    • Alina Daba,
    • Antonis E. Koromilas,
    • and Kostas Pantopoulos
    RNA March 2012 18: 547-556; Published in Advance January 23, 2012, doi:10.1261/rna.029322.111

    Ferritin stores and detoxifies an excess of intracellular iron and thereby plays an important role in the metabolism of this metal. As unshielded iron promotes oxidative stress, ferritin is crucial in maintaining cellular redox balance and may also modulate cell growth, survival, and apoptosis. The expression of ferritin is controlled by transcriptional and post-transcriptional mechanisms. Here, the authors examined how ferritin mRNA translation responds to stress conditions. Under global inhibition of protein synthesis or specific inhibition of ferritin mRNA translation in cells overexpressing PKR or IRP1, respectively, they demonstrate association of ferritin mRNA with heavy polysomes. Further experiments revealed that the 5′ untranslated region (5′ UTR) of ferritin mRNA contains a bona fide internal ribosomal entry site (IRES).

  • Comprehensive evaluation of canonical versus Dicer-substrate siRNA in vitro and in vivo

    • Donald J. Foster,
    • Scott Barros,
    • Rick Duncan,
    • Sarfraz Shaikh,
    • William Cantley,
    • Amy Dell,
    • Elena Bulgakova,
    • Jonathan O'Shea,
    • Nate Taneja,
    • Satya Kuchimanchi,
    • Christopher B. Sherrill,
    • Akin Akinc,
    • Gregory Hinkle,
    • Amy C. Seila White,
    • Bo Pang,
    • Klaus Charisse,
    • Rachel Meyers,
    • Muthiah Manoharan,
    • and Sayda M. Elbashir
    RNA March 2012 18: 557-568; Published in Advance January 31, 2012, doi:10.1261/rna.031120.111

    Small interfering RNAs (siRNAs) have been used as a major tool to silence gene expression, offering important therapeutic prospects. Here, the authors present a thorough in vitro and in vivo comparison of the silencing activities of canonical 21/21-mer siRNAs and 25/27-mer Dicer-substrate siRNAs (dsiRNAs). This article conclusively shows that both types of compounds are very effective, displaying similar effects; however, dsiRNAs are more immunogenic.

METHODS

  • Kinetics of tRNA folding monitored by aminoacylation

    • Hari Bhaskaran,
    • Annia Rodriguez-Hernandez,
    • and John J. Perona
    RNA March 2012 18: 569-580; Published in Advance January 27, 2012, doi:10.1261/rna.030080.111

    The authors describe a strategy for tracking Mg2+-initiated folding of 32P-labeled tRNA molecules to their native structures based on the capacity for aminoacylation by the cognate aminoacyl-tRNA synthetase enzyme. The approach directly links folding to function, paralleling a common strategy used to study the folding of catalytic RNAs. The application of the approach to study the influence of post-transcriptional modifications in folding of Escherichia coli tRNA1Gln reveals that the modified bases increase the folding rate but do not affect either the equilibrium between properly folded and misfolded states or the folding pathway. This assay allows the use of 32P-labeled tRNA in integrated studies combining folding, post-transcriptional processing, and aminoacylation reactions.

  • An in vivo selection method to optimize trans-splicing ribozymes

    • Karen E. Olson and
    • Ulrich F. Müller
    RNA March 2012 18: 581-589; Published in Advance January 24, 2012, doi:10.1261/rna.028472.111

    Trans-splicing group I intron ribozymes are able to replace the 3′ portion of an mRNA with their own 3′-exon, which may be useful for medical applications. Because the trans-splicing efficiency is currently too low for such applications, the authors show here the construction of an in vivo selection system and demonstrate its function with the optimization of the ribozyme 5′-terminal sequence. This in vivo selection system can now be used to optimize any sequence on trans-splicing ribozymes.

  • Computational prediction of efficient splice sites for trans-splicing ribozymes

    • Dario Meluzzi,
    • Karen E. Olson,
    • Gregory F. Dolan,
    • Gaurav Arya,
    • and Ulrich F. Müller
    RNA March 2012 18: 590-602; Published in Advance January 24, 2012, doi:10.1261/rna.029884.111

    The efficiency of trans-splicing ribozymes, which is currently too low for therapeutic applications, can be improved by careful choice of the targeted splice sites on the mRNA substrate. Here, the authors show that binding free energies derived from predictions of RNA secondary structure correlate well with the trans-splicing efficiencies experimentally determined on chloramphenicol acetyl transferase mRNA. Thus, the proposed computation of binding free energies provides a rapid and inexpensive method to identify efficient splice sites for trans-splicing ribozymes.

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This Issue

March 2012; 18 (3)
Cover
  1. HYPOTHESIS
  2. BIOINFORMATICS
  3. REPORTS
  4. ARTICLES
  5. METHODS

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