RNA Uridylytransferases of Trypanosomes

Summary

Principal Investigator: RUSLAN D APHASIZHEV
Abstract: DESCRIPTION (provided by the applicant): Trypanosomatids are causative agents of major diseases affecting developing countries around the world. Uridine insertion/deletion editing of mitochondria! mRNAs is unique to these parasites and is necessary for their viability. Our long-term goal is to elucidate the mechanisms of two key editing reactions: posttranscriptional formation of the 3'oligo[U] tail in guide RNAs and U-insertions into mRNA. These reactions are catalyzed by distinct RET1 and RET2 terminal uridylyltransferases (TUTases). We hypothesize that these enzymes share the mechanism of UTP recognition and transfer but their adaptation for a particular function is conferred by additional non-catalytic domains or interacting proteins within editing complexes. Furthermore, multiple uridylyltransferases may exist in Kinetoplastids in addition to those involved in editing. Thus, an inhibitor of trypanosomal UTP-specific RNA transferases may potentially become a multi-target therapeutic agent. The specific aims of this proposal are: 1. Define the mechanisms that regulate RET1 activity during the 3'end processing of guide RNAs. The average length of the oligo[U] tail in vivo is -15 nucleotides whereas in vitro recombinant RET1 adds hundreds of uridines to the synthetic guide RNA. We will isolate factors that modulate RET1 activity in the mitochondria and assess interaction of guide RNA 3'end processing and RNA editing complexes. Ultimately, reconstruction of the regulated uridylylation of short RNAs in vitro will be performed. 2. Characterize the mechanism of U-insertion editing. Uridines are inserted into the mRNA by RET2, an integral part of the enzymatic cascade within the core editing complex. A set of RNA-protein and nucleotideprotein cross-linking agents will be utilized to investigate the recognition of UTP and RNA substrates by RET2. Catalytic and RNA binding domain swapping between RET1 and RET2 will be used to understand the structural basis of specificity and processivity of U-addition. 3. Identify novel RNA uridylyltransferases. To expand our knowledge of TUTase-specific features, genome databases will be searched with the RET1 and RET2 sequence profiles. Highest-scoring homologues will be expressed and uridylyltransferase activity assayed. Sequences of the newly identified UTP-specific transferases will be used to establish characteristic protein motifs for this family of enzymes.
Funding Period: 2005-02-01 - 2010-01-31
more information: NIH RePORT

Top Publications

  1. ncbi iCODA: RNAi-based inducible knock-in system in Trypanosoma brucei
    Gene Errol Ringpis
    Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA, USA
    Methods Mol Biol 718:23-37. 2011
  2. pmc Mechanism of U-insertion RNA editing in trypanosome mitochondria: characterization of RET2 functional domains by mutational analysis
    Gene Errol Ringpis
    Department of Microbiology and Molecular Genetics, School of Medicine, University of California Irvine, B240 Medical Sciences I, Irvine, CA 92697, USA
    J Mol Biol 399:696-706. 2010
  3. pmc Structure of the mitochondrial editosome-like complex associated TUTase 1 reveals divergent mechanisms of UTP selection and domain organization
    Jason Stagno
    Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA
    J Mol Biol 399:464-75. 2010
  4. pmc Mechanism of U insertion RNA editing in trypanosome mitochondria: the bimodal TUTase activity of the core complex
    Gene Errol Ringpis
    Department of Microbiology and Molecular Genetics, University of California Irvine School of Medicine, B240 Medical Sciences I, Irvine, CA 92697, USA
    J Mol Biol 399:680-95. 2010
  5. pmc RET1-catalyzed uridylylation shapes the mitochondrial transcriptome in Trypanosoma brucei
    Inna Aphasizheva
    Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, California 92697, USA
    Mol Cell Biol 30:1555-67. 2010
  6. pmc Novel TUTase associates with an editosome-like complex in mitochondria of Trypanosoma brucei
    Inna Aphasizheva
    Department of Microbiology and Molecular Genetics, School of Medicine, University of California at Irvine, Irvine, California 92697, USA
    RNA 15:1322-37. 2009
  7. pmc Identification and characterization of nuclear non-canonical poly(A) polymerases from Trypanosoma brucei
    Ronald D Etheridge
    Department of Microbiology and Molecular Genetics, School of Medicine, B240 Medical Sciences I, University of California, Irvine, CA 92697, USA
    Mol Biochem Parasitol 164:66-73. 2009
  8. pmc Guide RNA-binding complex from mitochondria of trypanosomatids
    James Weng
    Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, Irvine, CA 92697, USA
    Mol Cell 32:198-209. 2008
  9. pmc Terminal RNA uridylyltransferases of trypanosomes
    Ruslan Aphasizhev
    Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA 92697 4025, USA
    Biochim Biophys Acta 1779:270-80. 2008
  10. pmc Dual role of the RNA substrate in selectivity and catalysis by terminal uridylyl transferases
    Jason Stagno
    Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA
    Proc Natl Acad Sci U S A 104:14634-9. 2007

Scientific Experts

  • RUSLAN D APHASIZHEV
  • Inna Aphasizheva
  • Gene Errol Ringpis
  • Jason Stagno
  • Richard H Lathrop
  • Ronald D Etheridge
  • James Weng
  • Hartmut Luecke
  • Lan Huang
  • Paul D Gershon
  • Xiaorong Wang
  • Michel Pelletier
  • G Wesley Hatfield
  • Jessica Bruystens
  • Daniel M Clemens
  • Arnold M Falick
  • Laurie K Read
  • Anja Rosengarth

Detail Information

Publications13

  1. ncbi iCODA: RNAi-based inducible knock-in system in Trypanosoma brucei
    Gene Errol Ringpis
    Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA, USA
    Methods Mol Biol 718:23-37. 2011
    ..These methods should be readily applicable for any gene in T. brucei...
  2. pmc Mechanism of U-insertion RNA editing in trypanosome mitochondria: characterization of RET2 functional domains by mutational analysis
    Gene Errol Ringpis
    Department of Microbiology and Molecular Genetics, School of Medicine, University of California Irvine, B240 Medical Sciences I, Irvine, CA 92697, USA
    J Mol Biol 399:696-706. 2010
    ..Recognition of a double-stranded RNA, which resembles a guide RNA/mRNA duplex, is further facilitated by multipoint contacts across the RET2-specific middle domain...
  3. pmc Structure of the mitochondrial editosome-like complex associated TUTase 1 reveals divergent mechanisms of UTP selection and domain organization
    Jason Stagno
    Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA
    J Mol Biol 399:464-75. 2010
    ..Because MEAT1 is essential for the viability of the bloodstream and insect forms of Trypanosoma brucei, the unique organization of its active site renders this protein an attractive target for trypanocide development...
  4. pmc Mechanism of U insertion RNA editing in trypanosome mitochondria: the bimodal TUTase activity of the core complex
    Gene Errol Ringpis
    Department of Microbiology and Molecular Genetics, University of California Irvine School of Medicine, B240 Medical Sciences I, Irvine, CA 92697, USA
    J Mol Biol 399:680-95. 2010
    ..Therefore, RET2 distinguishes base pairs in gapped RNA substrates which may constitute an additional checkpoint contributing to overall fidelity of the editing process...
  5. pmc RET1-catalyzed uridylylation shapes the mitochondrial transcriptome in Trypanosoma brucei
    Inna Aphasizheva
    Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, California 92697, USA
    Mol Cell Biol 30:1555-67. 2010
    ..We propose that the minicircle-encoded antisense transcripts, which are stabilized by RET1-catalyzed uridylylation, may direct a nucleolytic cleavage of multicistronic precursors...
  6. pmc Novel TUTase associates with an editosome-like complex in mitochondria of Trypanosoma brucei
    Inna Aphasizheva
    Department of Microbiology and Molecular Genetics, School of Medicine, University of California at Irvine, Irvine, California 92697, USA
    RNA 15:1322-37. 2009
    ..However, preliminary RNA analysis revealed no gross effects on RNA editing in MEAT1-depleted cells and indicated its possible role in regulating the mitochondrial RNA stability...
  7. pmc Identification and characterization of nuclear non-canonical poly(A) polymerases from Trypanosoma brucei
    Ronald D Etheridge
    Department of Microbiology and Molecular Genetics, School of Medicine, B240 Medical Sciences I, University of California, Irvine, CA 92697, USA
    Mol Biochem Parasitol 164:66-73. 2009
    ..Such mechanisms are likely to play a major role in alleviating promiscuity of the transcriptional machinery...
  8. pmc Guide RNA-binding complex from mitochondria of trypanosomatids
    James Weng
    Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, Irvine, CA 92697, USA
    Mol Cell 32:198-209. 2008
    ..Systematic analysis of RNA-mediated and RNA-independent interactions involving the GRBC and MERS1 suggests a unified model for RNA processing in the kinetoplast mitochondria...
  9. pmc Terminal RNA uridylyltransferases of trypanosomes
    Ruslan Aphasizhev
    Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA 92697 4025, USA
    Biochim Biophys Acta 1779:270-80. 2008
    ..This review focuses on biological functions and structures of trypanosomal TUTases...
  10. pmc Dual role of the RNA substrate in selectivity and catalysis by terminal uridylyl transferases
    Jason Stagno
    Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA
    Proc Natl Acad Sci U S A 104:14634-9. 2007
    ..We propose a dual role for RNA substrates in TUTase-catalyzed reactions: contribution to selective incorporation of the cognate nucleoside and shaping of the catalytic metal binding site...
  11. ncbi Isolation of RNA binding proteins involved in insertion/deletion editing
    Michel Pelletier
    Department of Microbiology and Immunology, SUNY Buffalo School of Medicine, Buffalo, New York, USA
    Methods Enzymol 424:75-105. 2007
    ..This chapter describes purification and characterization of three RNA binding proteins from kinetoplastid mitochondria that have been genetically demonstrated to affect RNA editing...
  12. ncbi RNA editing uridylyltransferases of trypanosomatids
    Ruslan Aphasizhev
    Department of Microbiology and Molecular Genetics, University of California, Irvine, California, USA
    Methods Enzymol 424:55-73. 2007
    ..This chapter describes isolation of TUTases and their complexes from trypanosomatids, methods used for analysis of interactions involving RET1 and RET2, purification of recombinant proteins, and enzyme kinetic assays...
  13. pmc UTP-bound and Apo structures of a minimal RNA uridylyltransferase
    Jason Stagno
    Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA
    J Mol Biol 366:882-99. 2007
    ..We also report a 2.4 Angstroms crystal structure of TbTUT4 with the bound 2' deoxyribonucleoside, which provides the structural basis of the enzyme's preference toward ribonucleotides...