Roles for ligases in the RNA editing complex of Trypanosoma brucei: band IV is needed for U‐deletion and RNA repair

Trypanosome RNA editing utilizes a seven polypeptide complex that includes two RNA ligases, band IV and band V. We now find that band IV protein contributes to the structural stability of the editing complex, so its lethal genetic knock‐out could reflect structural or catalytic requirements. To assess the catalytic role in editing, we generated cell lines which inducibly replaced band IV protein with an enzymatically inactive but structurally conserved version. This induction halts cell growth, showing that catalytic activity is essential. These induced cells have impaired in vivo editing, specifically of RNAs requiring uridylate (U) deletion; unligated RNAs cleaved at U‐deletion sites accumulated. Additionally, mitochondrial extracts of cells with reduced band IV activity were deficient in catalyzing U‐deletion, specifically at its ligation step, but were not deficient in U‐insertion. Thus band IV ligase is needed to seal RNAs in U‐deletion. U‐insertion does not appear to require band IV, so it might use the other ligase of the editing complex. Furthermore, band IV ligase was also found to serve an RNA repair function, both in vitro and in vivo.

[1]  S. Hajduk,et al.  RNA Ligase and Its Involvement in Guide RNA/mRNA Chimera Formation , 1995, The Journal of Biological Chemistry.

[2]  S. Shuman,et al.  RNA capping enzyme and DNA ligase: a superfamily of covalent nucleotidyl transferases , 1995, Molecular microbiology.

[3]  S. Heaphy,et al.  Effect of single amino acid changes in the region of the adenylylation site of T4 RNA ligase. , 1987, Biochemistry.

[4]  S. Gygi,et al.  Association of Two Novel Proteins, TbMP52 and TbMP48, with the Trypanosoma brucei RNA Editing Complex , 2001, Molecular and Cellular Biology.

[5]  S. Hajduk.  Defining the editing 'reaction'. , 1997, Trends in Microbiology.

[6]  K. Piller,et al.  T. brucei RNA editing: adenosine nucleotides inversely affect U-deletion and U-insertion reactions at mRNA cleavage. , 1998, Molecular cell.

[7]  Kenneth Stuart,et al.  Extensive editing of the cytochrome c oxidase III transcript in Trypanosoma brucei , 1988, Cell.

[8]  L. Rusche,et al.  Trypanosome RNA Editing: Simple Guide RNA Features Enhance U Deletion 100-Fold , 2001, Molecular and Cellular Biology.

[9]  L. Rusche,et al.  Ribonuclease activities of trypanosome RNA editing complex directed to cleave specifically at a chosen site. , 2001, Methods in enzymology.

[10]  S. Shuman,et al.  Mutational analysis of vaccinia DNA ligase defines residues essential for covalent catalysis. , 1995, Virology.

[11]  K. Stuart,et al.  Direct Visualization of Uridylate Deletion In Vitro Suggests a Mechanism for Kinetoplastid RNA Editing , 1996, Cell.

[12]  G. Cross,et al.  Regulated processive transcription of chromatin by T7 RNA polymerase in Trypanosoma brucei. , 1998, Nucleic acids research.

[13]  J. Cruz-Reyes,et al.  Trypanosome U-deletional RNA editing involves guide RNA-directed endonuclease cleavage, terminal U exonuclease, and RNA ligase activities. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[14]  R. Emeson,et al.  Functions and mechanisms of RNA editing. , 2000, Annual review of genetics.

[15]  G. Cross,et al.  Trypanosoma brucei , 1998 .

[16]  H. U. Göringer,et al.  The involvement of gRNA-binding protein gBP21 in RNA editing-an in vitro and in vivo analysis. , 1999, Nucleic acids research.

[17]  Michael T. McManus,et al.  Biochemical methods for analysis of kinetoplastid RNA editing. , 1998, Methods.

[18]  R. Benne,et al.  Mechanism and evolution of RNA editing in kinetoplastida. , 1996, Biochimica et biophysica acta.

[19]  Reza Salavati,et al.  An RNA Ligase Essential for RNA Editing and Survival of the Bloodstream Form of Trypanosoma brucei , 2001, Science.

[20]  K. Stuart,et al.  Uridylate Addition and RNA Ligation Contribute to the Specificity of Kinetoplastid Insertion RNA Editing , 2000, Molecular and Cellular Biology.

[21]  K. Stuart,et al.  RNA Editing: A Mechanism for gRNA-Specified Uridylate Insertion into Precursor mRNA , 1996, Science.

[22]  L. Simpson,et al.  Partially edited mRNAs for cytochrome b and subunit III of cytochrome oxidase from leishmania tarentolae mitochondria: RNA editing intermediates , 1990, Cell.

[23]  B. Sollner-Webb,et al.  RNA editing involves indiscriminate U changes throughout precisely defined editing domains , 1990, Cell.

[24]  K. Piller,et al.  The Two RNA Ligases of the Trypanosoma brucei RNA Editing Complex: Cloning the Essential Band IV Gene and Identifying the Band V Gene , 2001, Molecular and Cellular Biology.

[25]  Michael T. McManus,et al.  Identification of candidate mitochondrial RNA editing ligases from Trypanosoma brucei. , 2001, RNA.

[26]  K. Stuart,et al.  RNA editing: transfer of genetic information from gRNA to precursor mRNA in vitro. , 1994, Science.

[27]  J. Alfonzo,et al.  The mechanism of U insertion/deletion RNA editing in kinetoplastid mitochondria. , 1997, Nucleic acids research.

[28]  Larry Simpson,et al.  A model for RNA editing in kinetoplastid mitochondria: RNA molecules transcribed from maxicircle DNA provide the edited information , 1990, Cell.

[29]  A. Tomkinson,et al.  Location of the active site for enzyme-adenylate formation in DNA ligases. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[30]  K. Stuart,et al.  RNA editing in kinetoplastid protozoa , 1991, Current opinion in genetics & development.

[31]  L. Rusche,et al.  Trypanosoma brucei U insertion and U deletion activities co-purify with an enzymatic editing complex but are differentially optimized. , 1998, Nucleic acids research.

[32]  K. Piller,et al.  Purification of a functional enzymatic editing complex from Trypanosoma brucei mitochondria , 1997, The EMBO journal.

[33]  K. Stuart,et al.  Cycles of progressive realignment of gRNA with mRNA in RNA editing , 1991, Cell.

[34]  P. Myler,et al.  Complexes from Trypanosoma brucei that exhibit deletion editing and other editing-associated properties , 1996, Molecular and cellular biology.