Table of Contents

October 2012; 18 (10)

REVIEW

  • Biogenesis of telomerase ribonucleoproteins

    • Emily D. Egan and
    • Kathleen Collins
    RNA October 2012 18: 1747-1759; Published in Advance August 8, 2012, doi:10.1261/rna.034629.112

    Telomerase adds simple-sequence repeats to the ends of linear chromosomes to counteract the loss of end sequence inherent in conventional DNA replication. Catalytic activity for repeat synthesis arises from the cooperation of the telomerase reverse transcriptase protein (TERT) and the template-containing telomerase RNA (TER). TERs vary widely in sequence and structure but share a set of motifs required for TERT binding and catalytic activity. Species-specific TER motifs play essential roles in RNP biogenesis, stability, trafficking, and regulation. Remarkably, the biogenesis pathways that generate mature TER differ between and even within phylogenetic groups. This review highlights the diversity of strategies for telomerase RNP biogenesis, RNP assembly, and telomere recruitment among ciliates, yeasts, and vertebrates and suggests common themes in these pathways and their regulation.

BIOINFORMATICS

  • Analysis of the accessibility of CLIP bound sites reveals that nucleation of the miRNA:mRNA pairing occurs preferentially at the 3′-end of the seed match

    • Ray M. Marín,
    • Franziska Voellmy,
    • Thibaud von Erlach,
    • and Jiří Vaníček
    RNA October 2012 18: 1760-1770; Published in Advance August 22, 2012, doi:10.1261/rna.033282.112

    In this article, the authors analyze in detail the predicted accessibility of binding sites within the seed region (opposite microRNA positions 2–8) using a large set of miRNA binding sites identified in HITS-CLIP and PAR-CLIP experiments. Their analysis revealed that nucleotides at the 3′-end of bound seed matches are significantly more accessible than nucleotides at the 5′-end and also than nucleotides at any positions in the unbound seed matches. Of importance the authors concluded that in order to discriminate between a functional and a nonfunctional binding site, the accessibility of a single nucleotide at the 3′-end is more effective than the accessibility of several nucleotides at the 5′-end. Based on these results, the authors propose a model whereby nucleation of the binding between microRNAs and their mRNA targets begins at the 5′-end of the miRNA and continues along the length of the seed. These findings can be applied to improved miRNA target prediction algorithms.

ARTICLES

  • MicroRNAs in Amoebozoa: Deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs

    • Lotta Avesson,
    • Johan Reimegård,
    • E. Gerhart H. Wagner,
    • and Fredrik Söderbom
    RNA October 2012 18: 1771-1782; Published in Advance August 8, 2012, doi:10.1261/rna.033175.112

    Micro (mi)RNAs are important regulators of gene expression in plants and animals; however, miRNAs have previously only been identified in a handful of unicellular eukaryotes. This study describes the first high-throughput sequencing of small RNAs from the unicellular social amoeba Dictyostelium discoideum, establishing the presence of miRNAs in the Amoebozoa supergroup. Identified miRNAs were further analyzed with regard to developmental regulation and biogenesis.

  • The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy

    • Alfonso Benítez-Páez,
    • Magda Villarroya,
    • and M.-Eugenia Armengod
    RNA October 2012 18: 1783-1795; Published in Advance August 13, 2012, doi:10.1261/rna.033266.112

    While most of the enzymes responsible for tRNA modifications in Escherichia coli were known, the enzyme that introduces the m2A modification at purine 37 of some tRNAs was not. The authors of this study show that the missing enzyme is the methyltransferase RlmN responsible for introducing the m2A modification at position 2503 of the 23S rRNA, thus demonstrating that RlmN is a dual-specificity enzyme. The authors go on to use a tRNA chimera that should also be useful for the study of other tRNA modifications, to identify the specificity determinants. They also report that strains lacking rlmN have an error-prone phenotype (expected from loss of m2A2503 modification of rRNA) rather than a hyperaccurate phenotype (expected from loss of m2A37 modification of tRNA).

  • Single-stranded microRNA mimics

    • Guillaume Chorn,
    • Molly Klein-McDowell,
    • Lihong Zhao,
    • Matthew A. Saunders,
    • W. Michael Flanagan,
    • Aarron T. Willingham,
    • and Lee P. Lim
    RNA October 2012 18: 1796-1804; Published in Advance August 21, 2012, doi:10.1261/rna.031278.111

    miRNAs are ∼22-nt RNAs that bind to the Argonaute family of proteins and have important regulatory roles in plants and animals. Here, the authors show that miRNAs exhibit targeting activity in cells when delivered as single strands that are 5′-phosphorylated and that contain 2′-fluoro ribose modifications. Length preferences, chemical modification sensitivity, and genome-wide seed-based targeting all suggest that this activity is Ago-based. These results provide an initial step in the development of single-stranded miRNA mimics for therapeutic use.

  • Ribosomal proteins L7 and L8 function in concert with six A3 assembly factors to propagate assembly of domains I and II of 25S rRNA in yeast 60S ribosomal subunits

    • Jelena Jakovljevic,
    • Uli Ohmayer,
    • Michael Gamalinda,
    • Jason Talkish,
    • Lisa Alexander,
    • Jan Linnemann,
    • Philipp Milkereit,
    • and John L. Woolford, Jr.
    RNA October 2012 18: 1805-1822; Published in Advance August 14, 2012, doi:10.1261/rna.032540.112

    Ribosomal proteins L7 and L8 are required for some early steps in pre-rRNA processing during assembly of large subunits in yeast but are found in distinct domains within the ribosome. Here, following depletion of L7 and L8 in yeast, assembly of the large ribosomal subunit is broadly analyzed. In addition to previously described defects in rRNA processing, specific sets of proteins in the neighborhoods of L7 and L8, respectively, fail to assemble into the large subunit structure. These observations argue that localized RNP structures first assemble and then independently come together in the final ribosome structure.

  • Maf1-mediated repression of RNA polymerase III transcription inhibits tRNA degradation via RTD pathway

    • Tomasz W. Turowski,
    • Iwona Karkusiewicz,
    • Justyna Kowal,
    • and Magdalena Boguta
    RNA October 2012 18: 1823-1832; Published in Advance August 23, 2012, doi:10.1261/rna.033597.112

    tRNA precursors, which are transcribed by RNA polymerase III, undergo end-maturation, splicing, and base modifications. Hypomodified tRNAs, such as tRNAVal(AAC), lacking 7-methylguanosine and 5-methylcytidine modifications, are subject to degradation by a rapid tRNA decay pathway. Here the authors searched for genes which, when overexpressed, restored stability of tRNAVal(AAC) molecules in a modification-deficient trm4Δtrm8Δ mutant. They identified TEF1 and VAS1, encoding elongation factor eEF1A and valyl-tRNA synthetase respectively, which likely protect hypomodified tRNAVal(AAC) by direct interactions. They also identified MAF1 whose product is a general negative regulator of RNA polymerase III. Expression of a Maf1-7A mutant that constitutively repressed RNA polymerase III transcription resulted in increased stability of hypomodified tRNAVal(AAC). These results support a model whereby inhibition of tRNA transcription leads to stabilization of hypomodified tRNAVal(AAC) due to more efficient protection by tRNA-interacting proteins.

  • Mechanism of the AAA+ ATPases pontin and reptin in the biogenesis of H/ACA RNPs

    • Rosario Machado-Pinilla,
    • Dominique Liger,
    • Nicolas Leulliot,
    • and U. Thomas Meier
    RNA October 2012 18: 1833-1845; Published in Advance August 24, 2012, doi:10.1261/rna.034942.112

    The authors bring new insight about the roles of SHQ1, pontin, and reptin in the chaperoned cellular process of H/ACA RNP biogenesis. They show that pontin and reptin are required to pry SHQ1 from its grip on NAP57/dyskerin, subsequently allowing dyskerin to form an H/ACA RNP.

  • Functional characterization of two paralogs that are novel RNA binding proteins influencing mitochondrial transcripts of Trypanosoma brucei

    • Lucie Kafková,
    • Michelle L. Ammerman,
    • Drahomíra Faktorová,
    • John C. Fisk,
    • Sara L. Zimmer,
    • Roman Sobotka,
    • Laurie K. Read,
    • Julius Lukeš,
    • and Hassan Hashimi
    RNA October 2012 18: 1846-1861; Published in Advance August 16, 2012, doi:10.1261/rna.033852.112

    A majority of Trypanosoma brucei proteins have unknown functions, a consequence of its independent evolutionary history within the order Kinetoplastida that allowed for the emergence of several unique biological properties. Among these is RNA editing, needed for expression of mitochondrial-encoded genes. The recently discovered mitochondrial RNA binding complex 1 (MRB1) is composed of proteins with several functions in processing organellar RNA. The authors characterize two MRB1 subunits, referred to herein as MRB8170 and MRB4160, and show that they are both novel RNA binding proteins, possibly representing a new class of these proteins.

  • Mechanistic characterization of the 5′-triphosphate-dependent activation of PKR: Lack of 5′-end nucleobase specificity, evidence for a distinct triphosphate binding site, and a critical role for the dsRBD

    • Rebecca Toroney,
    • Chelsea M. Hull,
    • Joshua E. Sokoloski,
    • and Philip C. Bevilacqua
    RNA October 2012 18: 1862-1874; Published in Advance August 21, 2012, doi:10.1261/rna.034520.112

    The protein kinase PKR is activated by RNA to phosphorylate eIF-2α, inhibiting translation initiation. Long dsRNA activates PKR via interactions with the dsRNA-binding domain (dsRBD). Weakly structured RNA also activates PKR and does so in a 5′-triphosphate (ppp)-dependent fashion, however relatively little is known about this pathway. The authors used a mutant T7 RNA polymerase to incorporate all four triphosphate-containing nucleotides into the first position of a largely single-stranded RNA and found absence of selectivity, in that all four transcripts activate PKR. Recognition of 5′-triphosphate, but not the nucleobase at the 5′-most position, makes this RNA-mediated innate immune response sensitive to a broad array of viruses.

  • Lin28-mediated control of let-7 microRNA expression by alternative TUTases Zcchc11 (TUT4) and Zcchc6 (TUT7)

    • James E. Thornton,
    • Hao-Ming Chang,
    • Elena Piskounova,
    • and Richard I. Gregory
    RNA October 2012 18: 1875-1885; Published in Advance August 16, 2012, doi:10.1261/rna.034538.112

    Precursor let-7 miRNAs are degraded in undifferentiated cells and cancer cells in the presence of the small RNA-binding protein Lin28 and the Terminal Uridyl Transferase (TUTase) Zcchc11/TUTase 4. While recent efforts have focused on the role of Lin28 in the repression of let-7, little is known regarding the mechanism by which Zcchc11 mediates the uridylation of this miRNA. Here the authors perform mutational analyses in vitro to identify the domains of Zcchc11 necessary and sufficient for the uridylation of pre-let-7. The authors find that another TUTase, Zcchc6/TUTase 7, shares these critical domains and functions redundantly with Zcchc11 in vitro and in vivo to repress let-7 levels in embryonic stem cells.

  • The yeast rapid tRNA decay pathway competes with elongation factor 1A for substrate tRNAs and acts on tRNAs lacking one or more of several modifications

    • Joshua M. Dewe,
    • Joseph M. Whipple,
    • Irina Chernyakov,
    • Laura N. Jaramillo,
    • and Eric M. Phizicky
    RNA October 2012 18: 1886-1896; Published in Advance August 15, 2012, doi:10.1261/rna.033654.112

    The structural and functional integrity of tRNA is crucial for translation. In the yeast Saccharomyces cerevisiae, certain aberrant pre-tRNA species are subject to nuclear surveillance, leading to 3′ exonucleolytic degradation, and certain mature tRNA species are subject to rapid tRNA decay (RTD) if they are appropriately hypomodified or bear specific destabilizing mutations, leading to 5′-3′ exonucleolytic degradation by Rat1 and Xrn1. For example, trm8-Δ trm4-Δ strains are temperature sensitive due to lack of m7G46 and m5C and the consequent RTD of tRNAVal(AAC). It is unknown how the RTD pathway interacts with translation and other cellular processes, and how generally this pathway acts on hypomodified tRNAs. The authors provide evidence here that elongation factor 1A (EF-1A) competes with the RTD pathway for substrate tRNAs, since its overexpression suppresses the tRNA degradation and the growth defect of strains subject to RTD.

  • Mutational analysis of Trypanosoma brucei editosome proteins KREPB4 and KREPB5 reveals domains critical for function

    • Jason Carnes,
    • Achim Schnaufer,
    • Suzanne M. McDermott,
    • Gonzalo Domingo,
    • Rose Proff,
    • Alodie G. Steinberg,
    • Irina Kurtz,
    • and Kenneth Stuart
    RNA October 2012 18: 1897-1909; Published in Advance August 23, 2012, doi:10.1261/rna.035048.112

    The transcriptome of kinetoplastid mitochondria undergoes extensive RNA editing that inserts and deletes uridine residues (U's) to produce mature mRNAs. The editosome is a multiprotein complex that provides endonuclease, TUTase, exonuclease, and ligase activities required for RNA editing. The editosome's KREPB4 and KREPB5 proteins are essential for editosome integrity and parasite viability and contain semi-conserved motifs corresponding to zinc finger, RNase III, and PUF domains, but to date no functional analysis of these domains has been reported. The authors show here that various point mutations to KREPB4 and KREPB5 identify essential domains and suggest that these proteins do not themselves perform RNase III catalysis. The data presented are consistent with the hypothesis that KREPB4 and KREPB5 form intermolecular heterodimers with the catalytically active editing endonucleases, which is unprecedented among known RNase III proteins.

  • IRES-dependent translation of egr2 is induced under inflammatory conditions

    • Daniela Rübsamen,
    • Johanna S. Blees,
    • Kathrin Schulz,
    • Claudia Döring,
    • Martin-Leo Hansmann,
    • Heinrich Heide,
    • Andreas Weigert,
    • Tobias Schmid,
    • and Bernhard Brüne
    RNA October 2012 18: 1910-1920; Published in Advance August 22, 2012, doi:10.1261/rna.033019.112

    Because the effects of inflammatory conditions on translation are only poorly characterized, the authors aimed to identify translationally deregulated targets in inflammatory settings. For this purpose, they cocultured breast tumor cells with conditioned medium of activated monocyte-derived macrophages (CM). Polysome profiling and microarray analysis identified early growth response-2 (egr2) to be regulated at the level of translation. Using bicistronic reporter assays, it was found that egr2 contains an internal ribosome entry site (IRES) within its 5′ UTR, which facilitated enhanced translation upon CM treatment. In summary, the data provide evidence that egr2 expression is translationally regulated via an IRES element, which is responsive to an inflammatory environment.

  • Yeast Trm7 interacts with distinct proteins for critical modifications of the tRNAPhe anticodon loop

    • Michael P. Guy,
    • Brandon M. Podyma,
    • Melanie A. Preston,
    • Hussam H. Shaheen,
    • Kady L. Krivos,
    • Patrick A. Limbach,
    • Anita K. Hopper,
    • and Eric M. Phizicky
    RNA October 2012 18: 1921-1933; Published in Advance August 21, 2012, doi:10.1261/rna.035287.112

    Post-transcriptional modification of the tRNA anticodon loop is critical for translation. Yeast Trm7 is required for 2′-O-methylation of C32 and N34 of tRNAPhe, tRNATrp, and tRNALeu(UAA) to form Cm32 and Nm34, and trm7-Δ mutants have severe growth and translation defects, but the reasons for these defects are not known. The authors show here that overproduction of tRNAPhe suppresses the growth defect of trm7-Δ mutants, suggesting that the crucial biological role of Trm7 is the modification of tRNAPhe.

  • Air proteins control differential TRAMP substrate specificity for nuclear RNA surveillance

    • Karyn Schmidt,
    • Zhenjiang Xu,
    • David H. Mathews,
    • and J. Scott Butler
    RNA October 2012 18: 1934-1945; Published in Advance August 24, 2012, doi:10.1261/rna.033431.112

    In the nucleus of Saccharomyces cerevisiae, TRAMP complexes recognize and polyadenylate RNAs, which enhances RNA degradation by the exosome and may contribute to its specificity. TRAMPs contain either of two putative RNA-binding factors called Air proteins. Previous studies suggested that these proteins function interchangeably in targeting the poly(A)-polymerase activity of TRAMPs to RNAs. Experiments reported here show that the Air proteins govern separable functions. Phenotypic analysis and RNA deep-sequencing results from air mutants reveal specific requirements for each Air protein in the regulation of the levels of noncoding and coding RNAs.

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October 2012; 18 (10)
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