Reconstitution of uridine-deletion precleaved RNA editing with two recombinant enzymes.

Uridine insertion/deletion RNA editing in trypanosomatid mitochondria is a posttranscriptional RNA modification phenomenon required for translation of mitochondrial mRNAs. This process involves guide RNA-mediated cleavage at a specific site, insertion or deletion of Us from the 3' end of the 5' mRNA fragment, and ligation of the two mRNA fragments. The Leishmania major RNA ligase-containing complex protein 2 expressed in insect cells has a 3'-5' exoribonuclease activity and was therefore renamed RNA editing exonuclease 1 (REX1). Recombinant REX1 specifically trims 3' overhanging Us and stops at a duplex region. Evidence is presented that REX1 is responsible for deletion of the 3' overhanging Us from the bridged mRNA 5' cleavage fragment and that RNA editing ligase 1 is responsible for the ligation of the two mRNA cleavage fragments in U-deletion editing. The evidence involves both in vivo down-regulation of REX1 expression in Trypanosoma brucei by RNA interference and the reconstitution of precleaved U-deletion in vitro editing with only two recombinant enzymes: recombinant REX1 and recombinant RNA editing ligase 1.

[1]  John E. Coligan,et al.  Current Protocols in Protein Science , 1996 .

[2]  Reza Salavati,et al.  TbMP57 is a 3' terminal uridylyl transferase (TUTase) of the Trypanosoma brucei editosome. , 2003, Molecular cell.

[3]  L. Simpson,et al.  Isolation of kinetoplast-mitochondrial complexes from Leishmania tarentolae. , 1974, The Journal of protozoology.

[4]  Larry Simpson,et al.  A tale of two TUTases , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[5]  L. Simpson,et al.  A 100-kD complex of two RNA-binding proteins from mitochondria of Leishmania tarentolae catalyzes RNA annealing and interacts with several RNA editing components. , 2003, RNA.

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

[7]  S. Gygi,et al.  Mass spectrometric analysis of the editosome and other multiprotein complexes in Trypanosoma brucei , 2003, Journal of the American Society for Mass Spectrometry.

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

[9]  B Sollner-Webb,et al.  Roles for ligases in the RNA editing complex of Trypanosoma brucei: band IV is needed for U‐deletion and RNA repair , 2001, The EMBO journal.

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

[11]  Larry Simpson,et al.  The polarity of editing within a multiple gRNA-mediated domain is due to formation of anchors for upstream gRNAs by downstream editing , 1992, Cell.

[12]  Reza Salavati,et al.  Comparative analysis of editosome proteins in trypanosomatids. , 2003, Nucleic acids research.

[13]  J. Cruz-Reyes,et al.  Distinct Functions of Two RNA Ligases in Active Trypanosoma brucei RNA Editing Complexes , 2002, Molecular and Cellular Biology.

[14]  L. Simpson,et al.  Is the Trypanosoma brucei REL1 RNA Ligase Specific for U-deletion RNA Editing, and Is the REL2 RNA Ligase Specific for U-insertion Editing?* , 2003, Journal of Biological Chemistry.

[15]  Larry Simpson,et al.  Isolation of a U‐insertion/deletion editing complex from Leishmania tarentolae mitochondria , 2003, The EMBO journal.

[16]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

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

[18]  L. Simpson,et al.  Native gel analysis of ribonucleoprotein complexes from a Leishmania tarentolae mitochondrial extract. , 1997, Molecular and biochemical parasitology.

[19]  Larry Simpson,et al.  Mitochondrial proteins and complexes in Leishmania and Trypanosoma involved in U-insertion/deletion RNA editing. , 2004, RNA.

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

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

[22]  Jennifer M. Campbell,et al.  Matrix‐assisted laser desorption/ionization‐tandem mass spectrometry with high resolution and sensitivity for identification and characterization of proteins , 2002, Proteomics.

[23]  L Simpson,et al.  Uridine insertion/deletion RNA editing in trypanosome mitochondria--a review. , 1999, Gene.

[24]  A. Schnaufer,et al.  Identification of novel components of Trypanosoma brucei editosomes. , 2003, RNA.

[25]  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.

[26]  J. Donelson,et al.  Double-stranded RNA interference in Trypanosoma brucei using head-to-head promoters. , 2000, Molecular and biochemical parasitology.

[27]  Reza Salavati,et al.  Separate insertion and deletion subcomplexes of the Trypanosoma brucei RNA editing complex. , 2003, Molecular cell.

[28]  B. Séraphin,et al.  The tandem affinity purification (TAP) method: a general procedure of protein complex purification. , 2001, Methods.

[29]  Larry Simpson,et al.  Trypanosome Mitochondrial 3′ Terminal Uridylyl Transferase (TUTase) The Key Enzyme in U-Insertion/Deletion RNA Editing , 2002, Cell.

[30]  S. Beverley,et al.  Development of a stable Leishmania expression vector and application to the study of parasite surface antigen genes. , 1990, Proceedings of the National Academy of Sciences of the United States of America.