Table of Contents

May 2014; 20 (5)

BIOINFORMATICS

  • Highly conserved RNA pseudoknots at the gag-pol junction of HIV-1 suggest a novel mechanism of −1 ribosomal frameshifting

    • Xiaolan Huang,
    • Yang Yang,
    • Guan Wang,
    • Qiang Cheng,
    • and Zhihua Du
    RNA May 2014 20: 587-593; Published in Advance March 26, 2014, doi:10.1261/rna.042457.113

    −1 programmed ribosomal frameshifting (PRF) is utilized by a large number of viruses to synthesize the viral enzymatic (Pol) and structural (Gag) proteins at a defined ratio. For efficient −1 PRF, two cis-acting elements are often required: a heptanucleotide frameshift site and a downstream stimulator, often in the form of a pseudoknot. The sequences at the gag-pol junctions from 4254 different HIV-1 strains were analyzed. Approximately ninety-five percent of the sequences have the potential to form four pseudoknots PK1–PK4 (∼97% contain PK1, PK3, and PK4). A novel mechanism is suggested for the possible roles of these pseudoknots that incorporates several exquisite new features while being consistent with the current paradigm of pseudoknot-dependent −1 PRF.

ARTICLES

  • Translation of the human erythropoietin transcript is regulated by an upstream open reading frame in response to hypoxia

    • Cristina Barbosa and
    • Luísa Romão
    RNA May 2014 20: 594-608; Published in Advance March 19, 2014, doi:10.1261/rna.040915.113

    This study examines translational regulation of erythropoietin (EPO). New findings show that EPO translation is controlled by an upstream small open reading frame (ORF). In unstressed cells, the ORF represses translation. However, under hypoxic stress, this repression is largely relieved by a mechanism of enhanced ribosome bypass of the small ORF.

  • RF3:GTP promotes rapid dissociation of the class 1 termination factor

    • Kristin S. Koutmou,
    • Megan E. McDonald,
    • Julie L. Brunelle,
    • and Rachel Green
    RNA May 2014 20: 609-620; Published in Advance March 25, 2014, doi:10.1261/rna.042523.113
    OPEN ACCESS ARTICLE

    The precise role of the bacterial class 2 release factor, RF3, in translation termination has been unclear. This study shows that RF3 complexed with GTP but not GDP enhances RF1 dissociation from ribosomes by 500-fold. Importantly, only catalytically active RF1, but not catalytically inactive RF1, is dissociated. This result suggests that a rotated state of the ribosome is required for rapid dissociation.

  • miRNA sensitivity to Drosha levels correlates with pre-miRNA secondary structure

    • Henrik Sperber,
    • Alan Beem,
    • Sandra Shannon,
    • Ross Jones,
    • Pratyusha Banik,
    • Yu Chen,
    • Sherman Ku,
    • Gabriele Varani,
    • Shuyuan Yao,
    • and Hannele Ruohola-Baker
    RNA May 2014 20: 621-631; Published in Advance March 27, 2014, doi:10.1261/rna.043943.113

    In this paper, the authors analyze how the secondary structure of primary microRNAs (pri-miRNAs) affects their processing by the Microprocessor. They show that pri-miRNAs with central mismatches in the upper stem are more sensitive to changes in Drosha level. They conclude that the secondary structure of pri-miRNA affects Drosha processing efficiency when Drosha is limiting. This raises the possibility that differentiating cells may use this as a means of miRNA regulation.

  • A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids

    • Ka-Weng Ieong,
    • Michael Y. Pavlov,
    • Marek Kwiatkowski,
    • Måns Ehrenberg,
    • and Anthony C. Forster
    RNA May 2014 20: 632-643; Published in Advance March 26, 2014, doi:10.1261/rna.042234.113
    OPEN ACCESS ARTICLE

    Earlier studies showed that tRNA isoacceptor bodies have evolved to reduce differences between natural aminoacyl-tRNAs in their affinity to EF-Tu. Simply put, “weaker” amino acids tend to be paired with “stronger” tRNAs, and vice versa. This study explores the coupling of different tRNA bodies with unnatural amino acids to determine their biochemical behavior during tRNA selection on the ribosome. The results generally argue that the coupling of a strong tRNAAla body with several unnatural amino acids (allyl-glycine, methyl-serine, and biotinyl-lysine) can increase the overall extent of incorporation by increasing the affinity of these aminoacyl-tRNAs for EF-Tu. The same strong tRNAAla body failed to increase the efficiency of N-methyl-phenylalanine incorporation, arguing that its deficits are not related to EF-Tu affinity. These results have broad implications for unnatural amino acid incorporation during translation elongation.

  • RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template

    • Dave A. Pai,
    • Craig D. Kaplan,
    • Hye Kyong Kweon,
    • Kenji Murakami,
    • Philip C. Andrews,
    • and David R. Engelke
    RNA May 2014 20: 644-655; Published in Advance March 10, 2014, doi:10.1261/rna.040444.113

    This study reports the findings that RNA Pol II can be inhibited by nonstructured RNAs. These RNAs function as inhibitors by binding to the template binding cleft of the polymerase. Only polymerase that is not engaged with a DNA template is subject to this inhibition.

  • The crystal structure of S. cerevisiae Sad1, a catalytically inactive deubiquitinase that is broadly required for pre-mRNA splicing

    • Haralambos Hadjivassiliou,
    • Oren S. Rosenberg,
    • and Christine Guthrie
    RNA May 2014 20: 656-669; Published in Advance March 28, 2014, doi:10.1261/rna.042838.113

    Ubiquitin-related domains are present in many spliceosomal proteins, but the roles they play remain largely unknown. This study provides intriguing insights into the function of Sad1, a spliceosomal protein that contains both a Zn finger ubiquitin binding domain and a noncatalytic ubiquitin-specific protease domain. This work demonstrates that Sad1 is required for pre-mRNA splicing in vivo and for spliceosome assembly. Unexpectedly, Sad1 is unable to bind ubiquitin in vitro. By determining the crystal structure of Sad1, the authors show how the Sad1 domains indeed deviate from canonical ubiquitin binding domains.

  • In vitro characterization of 6S RNA release-defective mutants uncovers features of pRNA-dependent release from RNA polymerase in E. coli

    • Mariana Oviedo Ovando,
    • Lindsay Shephard,
    • and Peter J. Unrau
    RNA May 2014 20: 670-680; Published in Advance March 28, 2014, doi:10.1261/rna.036343.112

    6S is a noncoding RNA that sequesters RNA polymerase in low-nutrient conditions. This study analyzes the effects of mutations in 6S RNA that are defective in release of RNA polymerase. Expression of these RNAs in E. coli has deleterious phenotypes including delayed entry into exponential phase and decreased cell survival. These results show that 6S release rate plays important in vivo roles.

  • A conserved role for the zinc finger polyadenosine RNA binding protein, ZC3H14, in control of poly(A) tail length

    • Seth M. Kelly,
    • Sara W. Leung,
    • ChangHui Pak,
    • Ayan Banerjee,
    • Kenneth H. Moberg,
    • and Anita H. Corbett
    RNA May 2014 20: 681-688; Published in Advance March 26, 2014, doi:10.1261/rna.043984.113

    This paper examines the function of the mammalian protein ZC3H14, which is a zinc finger polyadenosine binding protein that may be orthologous to the yeast protein Nab2. Studies in a neuronal cell line demonstrate that the protein controls poly(A) tail length. Furthermore, complementation analyses in Drosophila indicate that ZC3H14 plays an evolutionarily conserved role in controlling poly(A) tail length in neurons.

  • The β-actin mRNA zipcode regulates epithelial adherens junction assembly but not maintenance

    • Natasha Gutierrez,
    • Itua Eromobor,
    • Ryan J. Petrie,
    • Pavan Vedula,
    • Lissette Cruz,
    • and Alexis J. Rodriguez
    RNA May 2014 20: 689-701; Published in Advance March 28, 2014, doi:10.1261/rna.043208.113

    This paper examines requirements for cytoskeletal remodeling during epithelial cell-cell contact. Antisense oligonucleotides that inhibit β-actin mRNA zipcode/ZBP1 interactions cause defects in adherens junction assembly.

  • Prevalent RNA recognition motif duplication in the human genome

    • Yihsuan S. Tsai,
    • Shawn M. Gomez,
    • and Zefeng Wang
    RNA May 2014 20: 702-712; Published in Advance March 25, 2014, doi:10.1261/rna.044081.113

    This paper provides an analysis of the origin of multiple RNA recognition motifs (RRMs) within single RNA-binding proteins. One main finding is that such RRMs (sibling RRMs) have significantly higher similarity than expected by chance. These results suggest that sibling RRMs arose through domain duplication.

METHODS

  • Mod-seq: high-throughput sequencing for chemical probing of RNA structure

    • Jason Talkish,
    • Gemma May,
    • Yizhu Lin,
    • John L. Woolford, Jr.,
    • and C. Joel McManus
    RNA May 2014 20: 713-720; Published in Advance March 24, 2014, doi:10.1261/rna.042218.113

    A new method and data analysis pipeline, called Mod-seq, for assaying the structure of RNAs by high-throughput sequencing is described. It can be utilized both in vivo and in vitro, with any small molecule that modifies RNA and consequently impedes reverse transcriptase. As proof-of-principle, we used dimethyl sulfate (DMS) to probe the in vivo structure of total cellular RNAs in Saccharomyces cerevisiae. While this method is applicable to RNAs of any length, its high-throughput nature makes Mod-seq ideal for studying long RNAs and complex RNA mixtures.

  • A versatile assay for RNA-binding proteins in living cells

    • Claudia Strein,
    • Anne-Marie Alleaume,
    • Ulrich Rothbauer,
    • Matthias W. Hentze,
    • and Alfredo Castello
    RNA May 2014 20: 721-731; Published in Advance March 24, 2014, doi:10.1261/rna.043562.113
    OPEN ACCESS ARTICLE

    This method paper describes a quantitative dual fluorescence approach to analyze protein–mRNA interactions in vivo.

  • Rapid identification of mRNA processing defects with a novel single-cell yeast reporter

    • Matthew R. Sorenson and
    • Scott W. Stevens
    RNA May 2014 20: 732-745; Published in Advance March 26, 2014, doi:10.1261/rna.042663.113

    This manuscript describes the design of a versatile reporter that exhibits, via multicolor flow cytometry, distinctive signatures for defects in a wide variety of processes involved in gene expression. The reporter is capable of performing high-throughput, rapid, and large-scale screens in budding yeast.

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May 2014; 20 (5)
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