Table of Contents

June 2012; 18 (6)

BIOINFORMATICS

  • RNA secondary structure mediates alternative 3′ss selection in Saccharomyces cerevisiae

    • Mireya Plass,
    • Carles Codony-Servat,
    • Pedro Gabriel Ferreira,
    • Josep Vilardell,
    • and Eduardo Eyras
    RNA June 2012 18: 1103-1115; Published in Advance April 26, 2012, doi:10.1261/rna.030767.111

    Alternative splicing is generally mediated by splicing factors, which can bind the pre-mRNA and affect the recognition of its splicing signals. Saccharomyces species lack many of the regulatory factors present in metazoans. Using a computational classifier based on sequence and structure features of the pre-mRNA, the authors are able to predict the usage of alternative 3′ss, some which they can confirm experimentally. This work provides evidence for the regulation of 3′ss selection in yeast by the pre-mRNA secondary structure, which can be responsive to external cues, like temperature, and which is possibly related to the control of gene expression.

REPORT

  • In vivo structure–function analysis of human Dicer reveals directional processing of precursor miRNAs

    • Allan M. Gurtan,
    • Victoria Lu,
    • Arjun Bhutkar,
    • and Phillip A. Sharp
    RNA June 2012 18: 1116-1122; Published in Advance April 30, 2012, doi:10.1261/rna.032680.112

    Dicer is an RNase III family endoribonuclease and haploinsufficient tumor suppressor that processes mature miRNAs from the 5′ (5p) or 3′ (3p) arm of hairpin precursors. In murine Dicer knockout fibroblasts, the authors expressed human Dicer with point mutations in the RNase III, helicase, and PAZ domains and characterized miRNA expression by Northern blot and massively parallel sequencing of small RNAs. They show that inactivation of the RNase IIIA domain results in complete loss of 3p-derived mature miRNAs, but only partial reduction in 5p-derived mature miRNAs. Conversely, inactivation of the RNase IIIB domain by mutation of D1709, a residue mutated in a subset of nonepithelial ovarian cancers, results in complete loss of 5p-derived mature miRNAs, including the tumor-suppressive let-7 family, but only partial reduction in 3p-derived mature miRNAs. These results suggest that mutation of the clinically relevant residue D1709 within the RNase IIIB results in a uniquely miRNA-haploinsufficient state in which the let-7 family of tumor suppressor miRNAs is lost, while a complement of 3p-derived miRNAs remains expressed.

ARTICLES

  • Metal-ion rescue revisited: Biochemical detection of site-bound metal ions important for RNA folding

    • John K. Frederiksen,
    • Nan-Sheng Li,
    • Rhiju Das,
    • Daniel Herschlag,
    • and Joseph A. Piccirilli
    RNA June 2012 18: 1123-1141; Published in Advance April 26, 2012, doi:10.1261/rna.028738.111

    Metal-ion rescue experiments have provided a powerful approach to identify and distinguish catalytic metal ions within RNA active sites, but the ability of such experiments to identify metal ions that contribute to tertiary structure acquisition and structural stability is less developed and has been challenged. Here, the authors used the well-defined P4–P6 RNA domain of the Tetrahymena group I intron to reevaluate prior evidence against the discriminatory power of metal-ion rescue experiments and to advance thermodynamic descriptions necessary for interpreting these experiments. The approach successfully identifies ligands within the RNA that occupy the inner coordination sphere of divalent metal ions and distinguishes them from ligands that occupy the outer coordination sphere. These results establish metal-ion rescue as a rigorous tool for identifying and dissecting energetically important metal-ion interactions in RNAs that are noncatalytic but critical for RNA tertiary structure.

  • Meiosis-induced alterations in transcript architecture and noncoding RNA expression in S. cerevisiae

    • Karen S. Kim Guisbert,
    • Yong Zhang,
    • Jared Flatow,
    • Sara Hurtado,
    • Jonathan P. Staley,
    • Simon Lin,
    • and Erik J. Sontheimer
    RNA June 2012 18: 1142-1153; Published in Advance April 26, 2012, doi:10.1261/rna.030510.111

    Changes in transcript architecture can have profound effects on RNA fate and protein expression. The authors used high-density, genome-wide tiling microarrays to define the transcriptome at distinct stages of meiosis and sporulation in Saccharomyces cerevisiae. Hundreds of RNAs exhibit changes in 5′-terminal, 3′-terminal, or intron boundaries, indicating that transcript architecture is unexpectedly dynamic in budding yeast. These results point toward a multitude of novel transcriptional and post-transcriptional control mechanisms that are triggered during meiosis and sporulation.

  • Crystal structure of the Rna14–Rna15 complex

    • Ashley R. Paulson and
    • Liang Tong
    RNA June 2012 18: 1154-1162; Published in Advance April 18, 2012, doi:10.1261/rna.032524.112

    This article reports the crystal structure of the complex between the K. lactis 3′-end processing factors Rna14 and Rna15, the orthologs of mammalian CstF-77 and CstF-64. Rna15 is bound to the C-terminal region of Rna14 and connected to the Rna14 HAT domain by a disordered linker. The results highlight the structural and functional independence of the Rna14 HAT domain dimer core and the Rna14–Rna15 complex.

  • INT6 interacts with MIF4GD/SLIP1 and is necessary for efficient histone mRNA translation

    • Julia Neusiedler,
    • Vincent Mocquet,
    • Taran Limousin,
    • Theophile Ohlmann,
    • Christelle Morris,
    • and Pierre Jalinot
    RNA June 2012 18: 1163-1177; Published in Advance April 24, 2012, doi:10.1261/rna.032631.112

    The INT6 protein, formerly characterized as the e subunit of the eIF3 translation initiation factor, has been implicated in mouse and human breast carcinogenesis and plays a role in genome stability. This article describes an interaction of INT6 with the MIF4GD/SLIP1 protein that was previously shown to associate with SLBP. This latter protein binds the stem–loop located at the C-terminal end of the histone mRNAs and plays a key role in processing, nuclear export, and translation of cell cycle–regulated histone mRNAs that lack a poly(A) tail. In line with its interaction with MIF4GD, INT6 contributes to S-phase histone mRNA translation.

  • Properties of small rRNA methyltransferase RsmD: Mutational and kinetic study

    • Olga V. Sergeeva,
    • Irina V. Prokhorova,
    • Yerdos Ordabaev,
    • Philipp O. Tsvetkov,
    • Petr V. Sergiev,
    • Alexey A. Bogdanov,
    • Alexander A. Makarov,
    • and Olga A. Dontsova
    RNA June 2012 18: 1178-1185; Published in Advance April 25, 2012, doi:10.1261/rna.032763.112

    Ribosomal RNA modification is performed by various enzymes during all stages of ribosome assembly. Among those, the Escherichia coli RsmD targets a single nucleotide G966 of the 16S rRNA in a completely assembled 30S subunit. The authors show that the enzyme displays remarkable binding properties toward unmodified 30S subunit but is unable to bind 30S subunit modified at G966. The efficiency of RsmD may be useful to complete the modification before the involvement of the 30S subunit in translation.

  • Identification of the human PMR1 mRNA endonuclease as an alternatively processed product of the gene for peroxidasin-like protein

    • Shan-Qing Gu,
    • Baskar Bakthavachalu,
    • Joonhee Han,
    • Deepak P. Patil,
    • Yuichi Otsuka,
    • Chittibabu Guda,
    • and Daniel R. Schoenberg
    RNA June 2012 18: 1186-1196; Published in Advance April 27, 2012, doi:10.1261/rna.031369.111

    The PMR1 endonuclease was discovered in Xenopus liver and identified as a member of the large and diverse peroxidase gene family. The sequences of functionally important regions were used to inform a bioinformatics search that identified two related genes as potential candidates for human PMR1: peroxidasin homolog (PXDN) and peroxidasin homolog-like (PXDNL) protein. Although each of these genes is predicted to encode a large, multidomain membrane-bound peroxidase, alternative splicing of PXDNL pre-mRNA yields a transcript whose predicted product is a 57-kDa protein with 42% sequence identity to Xenopus PMR1. Results presented here confirm the existence of the predicted 57-kDa protein, show this is the only form of PXDNL detected in any of the human cell lines examined, and confirm its identity as human PMR1.

  • A short PPR protein required for the splicing of specific group II introns in angiosperm chloroplasts

    • Anastassia Khrouchtchova,
    • Rita-Ann Monde,
    • and Alice Barkan
    RNA June 2012 18: 1197-1209; Published in Advance April 10, 2012, doi:10.1261/rna.032623.112

    In maize, thylakoid assembly 8 (tha8) causes defects in the biogenesis of chloroplast thylakoid membranes. THA8 contains more than 10 pentatricopeptide repeats (PPR). THA8 is the first member of this subfamily with a defined molecular function. THA8 localizes to chloroplasts where it associates specifically with the ycf3-2 and trnA group II introns.

  • Identification of eRF1 residues that play critical and complementary roles in stop codon recognition

    • Sara E. Conard,
    • Jessica Buckley,
    • Mai Dang,
    • Gregory J. Bedwell,
    • Richard L. Carter,
    • Mohamed Khass,
    • and David M. Bedwell
    RNA June 2012 18: 1210-1221; Published in Advance April 27, 2012, doi:10.1261/rna.031997.111

    Translation termination in eukaryotes is carried out by the class 1 release factor eRF1. A cavity model for stop codon recognition argues that three distinct binding pockets on the surface of eRF1 are important in recognition of the three specific nucleotides of stop codons. The binding pocket sequence motifs are typically divergent in organisms that depend on an altered code. Here, evidence supporting the cavity model is provided through a mutational analysis performed on reporter proteins in Saccharomyces cerevisiae. Moreover, the identified key residues extend beyond the previously identified TASNIKS and YCF motifs.

  • Exploring purine N7 interactions via atomic mutagenesis: The group I ribozyme as a case study

    • Marcello Forconi,
    • Tara Benz-Moy,
    • Kristin Rule Gleitsman,
    • Eliza Ruben,
    • Clyde Metz,
    • and Daniel Herschlag
    RNA June 2012 18: 1222-1229; Published in Advance April 27, 2012, doi:10.1261/rna.031567.111

    Atomic mutagenesis has emerged as a powerful tool to unravel specific interactions in complex RNA molecules. The authors found that the 7-deaza substitution lowers binding by ∼20-fold, relative to the cognate exogenous guanosine nucleophile, whereas binding and reaction with 7-methyl and 8-aza-7-deaza substitutions have no effect. These and additional results suggest that there is no functionally important contact between the N7 atom of the exogenous guanosine and the ribozyme. Rather, they are consistent with indirect effects introduced by the N7 substitution on stacking interactions and/or solvation that are important for binding.

  • Basis for ligand discrimination between ON and OFF state riboswitch conformations: The case of the SAM-I riboswitch

    • Vamsi Krishna Boyapati,
    • Wei Huang,
    • Jessica Spedale,
    • and Fareed Aboul-ela
    RNA June 2012 18: 1230-1243; Published in Advance April 27, 2012, doi:10.1261/rna.032177.111

    Riboswitches are RNA elements that bind to effector ligands and control gene expression. S-Adenosyl Methionine (SAM) binds the aptamer domain of the SAM-I riboswitch and induces conformational changes in the expression domain to form an intrinsic terminator (transcription OFF state). Without SAM the riboswitch forms the transcription ON state, allowing read-through transcription. The mechanistic link between the SAM/aptamer recognition event and subsequent secondary structure rearrangement by the riboswitch is unclear. The authors present binding measurements and in-line probing that are consistent with the hypothesis that when SAM is present, stacking interactions with the AT helix stabilize a partially formed P1 helix in the hybrids. Molecular modeling indicates that continuous stacking between the P1 and the AT helices is plausible with SAM bound. These findings raise the possibility that conformational intermediates may play a role in ligand-induced aptamer folding.

  • Prp2-mediated protein rearrangements at the catalytic core of the spliceosome as revealed by dcFCCS

    • Thomas Ohrt,
    • Mira Prior,
    • Julia Dannenberg,
    • Peter Odenwälder,
    • Olexandr Dybkov,
    • Nicolas Rasche,
    • Jana Schmitzová,
    • Ingo Gregor,
    • Patrizia Fabrizio,
    • Jörg Enderlein,
    • and Reinhard Lührmann
    RNA June 2012 18: 1244-1256; Published in Advance April 25, 2012, doi:10.1261/rna.033316.112

    The compositional and conformational changes during catalytic activation of the spliceosome promoted by the DEAH box ATPase Prp2 are only poorly understood. Here, the authors show by dual-color fluorescence cross-correlation spectroscopy (dcFCCS) that the binding affinity of several proteins is significantly changed during the Prp2-mediated transition of precatalytic Bact spliceosomes to catalytically activated B* spliceosomes from Saccharomyces cerevisiae. During this step, several proteins, including the zinc-finger protein Cwc24, are quantitatively displaced from the B* complex. Conversely, high-affinity binding sites are created for Yju2 and Cwc25 during catalytic activation, consistent with their requirement for step 1 catalysis.

  • Structure and folding of a rare, natural kink turn in RNA with an A•A pair at the 2b•2n position

    • Kersten T. Schroeder,
    • Peter Daldrop,
    • Scott A. McPhee,
    • and David M.J. Lilley
    RNA June 2012 18: 1257-1266; Published in Advance April 26, 2012, doi:10.1261/rna.032409.112

    Introduction of a rare variant into the SAM-I riboswitch results in normal binding of SAM ligand, indicating that tertiary RNA contacts lead to a k-turn folding with a standard k-turn geometry.

METHODS

  • Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry

    • Joanna L. McKenzie,
    • Johanna M. Duyvestyn,
    • Tony Smith,
    • Katerina Bendak,
    • Joel MacKay,
    • Ray Cursons,
    • Gregory M. Cook,
    • and Vickery L. Arcus
    RNA June 2012 18: 1267-1278; Published in Advance April 26, 2012, doi:10.1261/rna.031229.111

    VapC is a metal-dependent ribonuclease that is inhibited by its cognate antitoxin, VapB. The authors have developed a sensitive and robust method for determining VapC ribonuclease sequence-specificity. This method is general and can be equally applied to mRNA interferases such as VapC and MazF, or unrelated ribonuclease enzymes, to determine rapidly the sequence-specificity of ribonucleases from a range of organisms.

  • Evaluation of normalization methods in mammalian microRNA-Seq data

    • Lana Xia Garmire and
    • Shankar Subramaniam
    RNA June 2012 18: 1279-1288; Published in Advance April 24, 2012, doi:10.1261/rna.030916.111

    Whereas normalization methods for mRNA-Seq data have been properly described, there has been no unbiased evaluation of normalization methods on microRNA-Seq data. Furthermore, it was not known whether the normalization methods used for mRNA-Seq data can be adequately adapted to process microRNA-Seq data. This paper evaluates seven commonly used normalization methods for high-throughput data of microRNA-Seq data. The authors concluded that Lowess normalization and quantile normalization are recommended for normalizing microRNA-Seq data, whereas the Trimmed Mean Method, which is most appropriate for RNA-Seq data, should be used with caution when applied to miRNA-Seq data.

  • ePAT: A simple method to tag adenylated RNA to measure poly(A)-tail length and other 3′ RACE applications

    • Amrei Jänicke,
    • John Vancuylenberg,
    • Peter R. Boag,
    • Ana Traven,
    • and Traude H. Beilharz
    RNA June 2012 18: 1289-1295; Published in Advance April 27, 2012, doi:10.1261/rna.031898.111

    The length of the poly(A)-tail that terminates most mRNA can serve as a readout of its metabolism. A long poly(A)-tail is generally associated with actively translating mRNA, whereas a short poly(A)-tail often indicates a translationally repressed state. Here, the authors present a simple new method to measure transcript-specific poly(A)-tail length using standard molecular biology reagents.

  • Development of a siRNA and shRNA screening system based on a kinase fusion protein

    • Sofie Pieraets,
    • Luk Cox,
    • Olga Gielen,
    • and Jan Cools
    RNA June 2012 18: 1296-1306; Published in Advance April 26, 2012, doi:10.1261/rna.030015.111

    This manuscript reports the development of a novel screening system for the selection of functional shRNAs/siRNAs. This method is based on targeting a fusion gene that comprises an open reading frame of the gene of interest fused to a mutant PDGFR kinase, which constitutively promotes cell proliferation and survival of the leukemia cell line Ba/F3. This cell line depends on the expression of PDGFRα for survival; thus, cell proliferation is used as a read-out of the efficiency of the tested siRNA/shRNA at knocking down the fusion gene. This system could be useful to perform medium to high throughput screens to identify functional siRNAs or shRNAs.

ERRATUM

  • Alternative ferritin mRNA translation via internal initiation

    • Alina Daba,
    • Antonis E. Koromilas,
    • and Kostas Pantopoulos
    RNA June 2012 18: 1307
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This Issue

June 2012; 18 (6)
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  1. BIOINFORMATICS
  2. REPORT
  3. ARTICLES
  4. METHODS
  5. ERRATUM

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