Naphthalene-based RNA Editing Inhibitor Blocks RNA Editing Activities and Editosome Assembly in Trypanosoma brucei

Abstract

RNA editing, catalyzed by the multiprotein editosome complex, is an essential step for the expression of most mitochondrial genes in trypanosomatid pathogens. It has been shown previously that Trypanosoma brucei RNA editing ligase 1 (TbREL1), a core catalytic component of the editosome, is essential in the mammalian life stage of these parasitic pathogens. Because of the availability of its crystal structure and absence from human, the adenylylation domain of TbREL1 has recently become the focus of several studies for designing inhibitors that target its adenylylation pocket. Here, we have studied new and existing inhibitors of TbREL1 to better understand their mechanism of action. We found that these compounds are moderate to weak inhibitors of adenylylation of TbREL1 and in fact enhance adenylylation at higher concentrations of protein. Nevertheless, they can efficiently block deadenylylation of TbREL1 in the editosome and, consequently, result in inhibition of the ligation step of RNA editing. Further experiments directly showed that the studied compounds inhibit the interaction of the editosome with substrate RNA. This was supported by the observation that not only the ligation activity of TbREL1 but also the activities of other editosome proteins such as endoribonuclease, terminal RNA uridylyltransferase, and uridylate-specific exoribonuclease, all of which require the interaction of the editosome with the substrate RNA, are efficiently inhibited by these compounds. In addition, we found that these compounds can interfere with the integrity and/or assembly of the editosome complex, opening the exciting possibility of using them to study the mechanism of assembly of the editosome components. RNA editing, catalyzed by the multiprotein editosome complex, is an essential step for the expression of most mitochondrial genes in trypanosomatid pathogens. It has been shown previously that Trypanosoma brucei RNA editing ligase 1 (TbREL1), a core catalytic component of the editosome, is essential in the mammalian life stage of these parasitic pathogens. Because of the availability of its crystal structure and absence from human, the adenylylation domain of TbREL1 has recently become the focus of several studies for designing inhibitors that target its adenylylation pocket. Here, we have studied new and existing inhibitors of TbREL1 to better understand their mechanism of action. We found that these compounds are moderate to weak inhibitors of adenylylation of TbREL1 and in fact enhance adenylylation at higher concentrations of protein. Nevertheless, they can efficiently block deadenylylation of TbREL1 in the editosome and, consequently, result in inhibition of the ligation step of RNA editing. Further experiments directly showed that the studied compounds inhibit the interaction of the editosome with substrate RNA. This was supported by the observation that not only the ligation activity of TbREL1 but also the activities of other editosome proteins such as endoribonuclease, terminal RNA uridylyltransferase, and uridylate-specific exoribonuclease, all of which require the interaction of the editosome with the substrate RNA, are efficiently inhibited by these compounds. In addition, we found that these compounds can interfere with the integrity and/or assembly of the editosome complex, opening the exciting possibility of using them to study the mechanism of assembly of the editosome components. IntroductionTrypanosoma brucei, Trypanosoma cruzi, and Leishmania major are three major trypanosomatid pathogens that cause hundreds of thousands of deaths and infect millions of people in tropical and subtropical areas of the world (1Stuart K. Brun R. Croft S. Fairlamb A. Gürtler R.E. McKerrow J. Reed S. Tarleton R. J. Clin. Investig. 2008; 118: 1301-1310Crossref PubMed Scopus (415) Google Scholar). Current trypanocidal drugs have a number of limitations such as high rate of toxicity, low rate of efficacy, and drug resistance (2Denise H. Barrett M.P. Biochem. Pharmacol. 2001; 61: 1-5Crossref PubMed Scopus (66) Google Scholar, 3Delespaux V. de Koning H.P. Drug Resist. Updat. 2007; 10: 30-50Crossref PubMed Scopus (223) Google Scholar). Therefore, it is important to look for a drug that is effective and does not produce harmful side effects. RNA editing is a unique post-transcriptional modification of mitochondrial mRNAs that is shared in all trypanosomatid pathogens (4Simpson L. Sbicego S. Aphasizhev R. RNA. 2003; 9: 265-276Crossref PubMed Scopus (140) Google Scholar, 5Stuart K.D. Schnaufer A. Ernst N.L. Panigrahi A.K. Trends Biochem. Sci. 2005; 30: 97-105Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). Modification of specific editing sites, dictated by complementary guide RNAs (gRNAs), 5The abbreviations used are: gRNA, guide RNA; U, uridylate; Cyb, cytochrome b; TbREL1, T. brucei RNA editing ligase 1; OB, oligonucleotide binding; SIE, solvated interaction energy; TEV, tobacco etch virus; FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine; TAP, tandem affinity purification. constitutes essential steps to ensure the production of translatable mRNAs that encode essential components of the mitochondrial respiratory system. Although gRNAs specify the number of uridylates (Us) to be added or deleted by base pairing at each editing block (6Kable M.L. Heidmann S. Stuart K.D. Trends Biochem. Sci. 1997; 22: 162-166Abstract Full Text PDF PubMed Scopus (48) Google Scholar, 7Seiwert S.D. Heidmann S. Stuart K. Cell. 1996; 84: 831-841Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), a 1.6-MDa multiprotein complex, the editosome, is responsible for catalysis of different steps of RNA editing. Although the complete composition of the editosome is being elucidated, most purified functional editosomes contain over 20 proteins (8Panigrahi A.K. Gygi S.P. Ernst N.L. Igo Jr., R.P. Palazzo S.S. Schnaufer A. Weston D.S. Carmean N. Salavati R. Aebersold R. Stuart K.D. Mol. Cell. Biol. 2001; 21: 380-389Crossref PubMed Scopus (112) Google Scholar, 9Panigrahi A.K. Schnaufer A. Ernst N.L. Wang B. Carmean N. Salavati R. Stuart K. RNA. 2003; 9: 484-492Crossref PubMed Scopus (118) Google Scholar). The editosomes differ in their compositions, having at least three different complexes that sediment at ∼20 S on glycerol gradients, and have the essential RNA editing activities (10Carnes J. Trotter J.R. Peltan A. Fleck M. Stuart K. Mol. Cell. Biol. 2008; 28: 122-130Crossref PubMed Scopus (93) Google Scholar). These editosomes, however, contain a similar essential catalytic core whose proteins are functionally characterized, including two endoribonucleases (KREN1 and KREN2) (11Carnes J. Trotter J.R. Ernst N.L. Steinberg A. Stuart K. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 16614-16619Crossref PubMed Scopus (108) Google Scholar, 12Trotter J.R. Ernst N.L. Carnes J. Panicucci B. Stuart K. Mol. Cell. 2005; 20: 403-412Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), two 3′-terminal uridylyltransferases (KRET1 and KRET2) (13Aphasizhev R. Sbicego S. Peris M. Jang S.H. Aphasizheva I. Simpson A.M. Rivlin A. Simpson L. Cell. 2002; 108: 637-648Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 14Ernst N.L. Panicucci B. Igo Jr., R.P. Panigrahi A.K. Salavati R. Stuart K. Mol. Cell. 2003; 11: 1525-1536Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), two 3′-exoribonucleases (U-exoribonucleases) that are called KREX1 and KREX2 (15Schnaufer A. Ernst N.L. Palazzo S.S. O'Rear J. Salavati R. Stuart K. Mol. Cell. 2003; 12: 307-319Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 16Kang X. Rogers K. Gao G. Falick A.M. Zhou S. Simpson L. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 1017-1022Crossref PubMed Scopus (52) Google Scholar), two RNA ligases (KREL1 and KREL2) (17Cruz-Reyes J. Zhelonkina A.G. Huang C.E. Sollner-Webb B. Mol. Cell. Biol. 2002; 22: 4652-4660Crossref PubMed Scopus (63) Google Scholar, 18Schnaufer A. Panigrahi A.K. Panicucci B. Igo Jr., R.P. Wirtz E. Salavati R. Stuart K. Science. 2001; 291: 2159-2162Crossref PubMed Scopus (214) Google Scholar), and six proteins with predicted oligonucleotide binding (OB) folds (KREPA1–6) (8Panigrahi A.K. Gygi S.P. Ernst N.L. Igo Jr., R.P. Palazzo S.S. Schnaufer A. Weston D.S. Carmean N. Salavati R. Aebersold R. Stuart K.D. Mol. Cell. Biol. 2001; 21: 380-389Crossref PubMed Scopus (112) Google Scholar, 19Worthey E.A. Schnaufer A. Mian I.S. Stuart K. Salavati R. Nucleic Acids Res. 2003; 31: 6392-6408Crossref PubMed Scopus (71) Google Scholar, 20Kala S. Salavati R. RNA. 2010; 16: 1951-1967Crossref PubMed Scopus (15) Google Scholar, 21Salavati R. Ernst N.L. O'Rear J. Gilliam T. Tarun Jr., S. Stuart K. RNA. 2006; 12: 819-831Crossref PubMed Scopus (42) Google Scholar, 22Guo X. Ernst N.L. Carnes J. Stuart K.D. PLoS One. 2010; 5: e8913Crossref PubMed Scopus (14) Google Scholar, 23Tarun Jr., S.Z. Schnaufer A. Ernst N.L. Proff R. Deng J. Hol W. Stuart K. RNA. 2008; 14: 347-358Crossref PubMed Scopus (34) Google Scholar, 24Niemann M. Brecht M. Schlüter E. Weitzel K. Zacharias M. Göringer H.U. Nucleic Acids Res. 2008; 36: 4465-4473Crossref PubMed Scopus (15) Google Scholar, 25Law J.A. O'Hearn S.F. Sollner-Webb B. RNA. 2008; 14: 1187-1200Crossref PubMed Scopus (15) Google Scholar). The letter K in the beginning of each protein name refers to kinetoplastid. To specify species, we have used species-specific abbreviations (e.g. TbREL1 for REL1 in T. brucei).Although other ligases such as T7 DNA ligase have a catalytic domain and an OB fold domain, KREL1 and KREL2 contain only the catalytic domain (19Worthey E.A. Schnaufer A. Mian I.S. Stuart K. Salavati R. Nucleic Acids Res. 2003; 31: 6392-6408Crossref PubMed Scopus (71) Google Scholar, 26McManus M.T. Shimamura M. Grams J. Hajduk S.L. RNA. 2001; 7: 167-175Crossref PubMed Scopus (97) Google Scholar). The OB fold domain that is essential for interaction with the substrate RNA is provided in trans by KREPA2 and KREPA1, which interact via their zinc fingers with KREL1 and KREL2, respectively, and bring the substrate RNA and the catalytic domain into proximity (15Schnaufer A. Ernst N.L. Palazzo S.S. O'Rear J. Salavati R. Stuart K. Mol. Cell. 2003; 12: 307-319Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 27Swift R.V. Durrant J. Amaro R.E. McCammon J.A. Biochemistry. 2009; 48: 709-719Crossref PubMed Scopus (9) Google Scholar, 28Gao G. Rogers K. Li F. Guo Q. Osato D. Zhou S.X. Falick A.M. Simpson L. Protist. 2010; 161: 489-496Crossref PubMed Scopus (8) Google Scholar). Although RNA editing ligases differ in their structure compared with DNA ligases (15Schnaufer A. Ernst N.L. Palazzo S.S. O'Rear J. Salavati R. Stuart K. Mol. Cell. 2003; 12: 307-319Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), their overall mechanism is similar. Ligation requires three distinct and reversible steps: (i) the ligase adenylylation step in which the conserved catalytic lysine of the ligase attacks the α-phosphate of ATP and displaces pyrophosphate, forming an enzyme-AMP intermediate through the phosphoamide linkage; (ii) the ligase deadenylylation step in which the guide RNA/nicked mRNA duplex binds to the protein and AMP is transferred from the adenylylated ligase to the 5′-phosphate of the RNA molecule, forming an adenylylated RNA with a 5′,5′-phosphoanhydride bond; and (iii) the ligation step in which the free 3′-hydroxyl at the nick site attacks the phosphoanhydride bond of the adenylylated RNA fragment, forming a phosphodiester bond, ligating the double-stranded RNA, and releasing AMP.TbREL1 is an essential enzyme for the editing process and parasite viability (18Schnaufer A. Panigrahi A.K. Panicucci B. Igo Jr., R.P. Wirtz E. Salavati R. Stuart K. Science. 2001; 291: 2159-2162Crossref PubMed Scopus (214) Google Scholar). The crystal structure of the TbREL1 N-terminal catalytic domain has been of compounds TbREL1 R.E. Schnaufer A. H. Hol W. Stuart K.D. McCammon J.A. Proc. Natl. Acad. Sci. U.S.A. 2008; PubMed Scopus Google Scholar, L. R.V. M. Schnaufer A. Amaro R.E. PLoS 2010; PubMed Scopus Google Scholar). The unique of the TbREL1 and the absence of in the TbREL1 an target for of inhibitors that block the essential RNA ligase study has found several inhibitors TbREL1 using a of in and in adenylylation R.E. Schnaufer A. H. Hol W. Stuart K.D. McCammon J.A. Proc. Natl. Acad. Sci. U.S.A. 2008; PubMed Scopus Google Scholar). of these inhibitors have been as inhibitors of RNA editing in the of the purified editosome H. Salavati R. Nucleic Acids Res. 2010; PubMed Scopus Google Scholar). we a study L. R.V. M. Schnaufer A. Amaro R.E. PLoS 2010; PubMed Scopus Google was that a number of inhibitors of In the we have studied the of of the inhibitors provided by the number to as L. R.V. M. Schnaufer A. Amaro R.E. PLoS 2010; PubMed Scopus Google and in study as on the editosome, and we that of new all the different essential catalytic steps of RNA editing are most as a result of the interaction of the core editosome with substrate the of we have the mechanism of of two inhibitors of the RNA editing and and have shown that these compounds all editosome activities that require that they block the interaction of the editosome with its substrate RNA. This is supported by experiments that the editosome interact with substrate RNA in the of and Therefore, the mechanism of of compounds to be and as the compounds that can interfere with In addition, we the of the on integrity and showed that interfere with the integrity and/or assembly of the editosome complex, the 20 S editosome to the S This the from of core editosome proteins by R. Aphasizheva I. R.E. Gao G. Simpson A.M. X. Falick A.M. Sbicego S. Simpson L. J. 2003; 22: PubMed Scopus (118) Google Scholar), that the editosome requires its substrate RNA for assembly and/or its has been that and inhibit the adenylylation step of ligation with the compounds with ATP for binding to the adenylylation R.E. Schnaufer A. H. Hol W. Stuart K.D. McCammon J.A. Proc. Natl. Acad. Sci. U.S.A. 2008; PubMed Scopus Google Scholar, L. R.V. M. Schnaufer A. Amaro R.E. PLoS 2010; PubMed Scopus Google Scholar). in purified editosome as the of TbREL1 that is to editing is which is higher the of TbREL1 used in of editosome, we not inhibition at the adenylylation we to inhibition of adenylylation we editosome that adenylylation inhibition only at low protein the other we inhibition of editing using the of editosome that is for of in RNA editing, that adenylylation inhibition is most not the mechanism through which and inhibit the editing process at higher protein that the mechanism of of is through inhibition of a interaction that is to the process of editing can to such the OB of ligases in a can to their to interact with the RNA the deadenylylation step in and DNA it has been shown that the OB fold and at a from the that the ligase to have an to to the double-stranded DNA Nucleic Acids Res. 28: PubMed Scopus Google Scholar). is that in the of the OB fold the ATP for the adenylylation the OB fold and the which does not the interaction of protein with RNA In other to the of the OB fold on the adenylylated catalytic the OB fold and is not of its binding the site nick Although to in studies that the interaction of and with TbREL1 as as R.E. Schnaufer A. H. Hol W. Stuart K.D. McCammon J.A. Proc. Natl. Acad. Sci. U.S.A. 2008; PubMed Scopus Google and L. R.V. M. Schnaufer A. Amaro R.E. PLoS 2010; PubMed Scopus Google Scholar), it is not supported by in that the target of does not to be in with TbREL1 inhibit a that is responsible for the substrate RNA into the editosome This result from of of of proteins to was to be in the adenylylation of RNA editing which can be similar to other and we have shown that in the of is a in the 20 S editosome S. This result is similar to the that of the editosome with the editosome is R. Aphasizheva I. R.E. Gao G. Simpson A.M. X. Falick A.M. Sbicego S. Simpson L. J. 2003; 22: PubMed Scopus (118) Google Scholar), that the of integrity of the editosome with is the editosome its with the compounds does not result in of RNA binding activity of that these compounds not directly and that they not block in a but target a that we found to be most by has been previously as Göringer H.U. Stuart K. Mol. Cell. Biol. 14: PubMed Scopus Google Scholar). The most protein of has been shown to be a protein. has been to be an intermediate the of the complete editosome H.U. Stuart K. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). This observation that the of high editosome target of can be a previously essential protein that has been shown to be to to and double-stranded RNA and is important for the of mitochondrial RNA S. A.M. X. Peris M. Simpson L. Cell. 2003; PubMed Scopus Google Scholar). The of protein the observation that the interaction of its target with and double-stranded which to of RNA drug as shown by the a for RNA to editosome are that that mitochondrial DNA and mitochondrial mRNA and contain editosomes Palazzo S.S. Wang B. B. Salavati R. Stuart K.D. Cell. 2003; PubMed Scopus Google Scholar). the mechanism of compounds such as a for assembly of functional IntroductionTrypanosoma brucei, Trypanosoma cruzi, and Leishmania major are three major trypanosomatid pathogens that cause hundreds of thousands of deaths and infect millions of people in tropical and subtropical areas of the world (1Stuart K. Brun R. Croft S. Fairlamb A. Gürtler R.E. McKerrow J. Reed S. Tarleton R. J. Clin. Investig. 2008; 118: 1301-1310Crossref PubMed Scopus (415) Google Scholar). Current trypanocidal drugs have a number of limitations such as high rate of toxicity, low rate of efficacy, and drug resistance (2Denise H. Barrett M.P. Biochem. Pharmacol. 2001; 61: 1-5Crossref PubMed Scopus (66) Google Scholar, 3Delespaux V. de Koning H.P. Drug Resist. Updat. 2007; 10: 30-50Crossref PubMed Scopus (223) Google Scholar). Therefore, it is important to look for a drug that is effective and does not produce harmful side effects. RNA editing is a unique post-transcriptional modification of mitochondrial mRNAs that is shared in all trypanosomatid pathogens (4Simpson L. Sbicego S. Aphasizhev R. RNA. 2003; 9: 265-276Crossref PubMed Scopus (140) Google Scholar, 5Stuart K.D. Schnaufer A. Ernst N.L. Panigrahi A.K. Trends Biochem. Sci. 2005; 30: 97-105Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). Modification of specific editing sites, dictated by complementary guide RNAs (gRNAs), 5The abbreviations used are: gRNA, guide RNA; U, uridylate; Cyb, cytochrome b; TbREL1, T. brucei RNA editing ligase 1; OB, oligonucleotide binding; SIE, solvated interaction energy; TEV, tobacco etch virus; FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine; TAP, tandem affinity purification. constitutes essential steps to ensure the production of translatable mRNAs that encode essential components of the mitochondrial respiratory system. Although gRNAs specify the number of uridylates (Us) to be added or deleted by base pairing at each editing block (6Kable M.L. Heidmann S. Stuart K.D. Trends Biochem. Sci. 1997; 22: 162-166Abstract Full Text PDF PubMed Scopus (48) Google Scholar, 7Seiwert S.D. Heidmann S. Stuart K. Cell. 1996; 84: 831-841Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), a 1.6-MDa multiprotein complex, the editosome, is responsible for catalysis of different steps of RNA editing. Although the complete composition of the editosome is being elucidated, most purified functional editosomes contain over 20 proteins (8Panigrahi A.K. Gygi S.P. Ernst N.L. Igo Jr., R.P. Palazzo S.S. Schnaufer A. Weston D.S. Carmean N. Salavati R. Aebersold R. Stuart K.D. Mol. Cell. Biol. 2001; 21: 380-389Crossref PubMed Scopus (112) Google Scholar, 9Panigrahi A.K. Schnaufer A. Ernst N.L. Wang B. Carmean N. Salavati R. Stuart K. RNA. 2003; 9: 484-492Crossref PubMed Scopus (118) Google Scholar). The editosomes differ in their compositions, having at least three different complexes that sediment at ∼20 S on glycerol gradients, and have the essential RNA editing activities (10Carnes J. Trotter J.R. Peltan A. Fleck M. Stuart K. Mol. Cell. Biol. 2008; 28: 122-130Crossref PubMed Scopus (93) Google Scholar). These editosomes, however, contain a similar essential catalytic core whose proteins are functionally characterized, including two endoribonucleases (KREN1 and KREN2) (11Carnes J. Trotter J.R. Ernst N.L. Steinberg A. Stuart K. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 16614-16619Crossref PubMed Scopus (108) Google Scholar, 12Trotter J.R. Ernst N.L. Carnes J. Panicucci B. Stuart K. Mol. Cell. 2005; 20: 403-412Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), two 3′-terminal uridylyltransferases (KRET1 and KRET2) (13Aphasizhev R. Sbicego S. Peris M. Jang S.H. Aphasizheva I. Simpson A.M. Rivlin A. Simpson L. Cell. 2002; 108: 637-648Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 14Ernst N.L. Panicucci B. Igo Jr., R.P. Panigrahi A.K. Salavati R. Stuart K. Mol. Cell. 2003; 11: 1525-1536Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), two 3′-exoribonucleases (U-exoribonucleases) that are called KREX1 and KREX2 (15Schnaufer A. Ernst N.L. Palazzo S.S. O'Rear J. Salavati R. Stuart K. Mol. Cell. 2003; 12: 307-319Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 16Kang X. Rogers K. Gao G. Falick A.M. Zhou S. Simpson L. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 1017-1022Crossref PubMed Scopus (52) Google Scholar), two RNA ligases (KREL1 and KREL2) (17Cruz-Reyes J. Zhelonkina A.G. Huang C.E. Sollner-Webb B. Mol. Cell. Biol. 2002; 22: 4652-4660Crossref PubMed Scopus (63) Google Scholar, 18Schnaufer A. Panigrahi A.K. Panicucci B. Igo Jr., R.P. Wirtz E. Salavati R. Stuart K. Science. 2001; 291: 2159-2162Crossref PubMed Scopus (214) Google Scholar), and six proteins with predicted oligonucleotide binding (OB) folds (KREPA1–6) (8Panigrahi A.K. Gygi S.P. Ernst N.L. Igo Jr., R.P. Palazzo S.S. Schnaufer A. Weston D.S. Carmean N. Salavati R. Aebersold R. Stuart K.D. Mol. Cell. Biol. 2001; 21: 380-389Crossref PubMed Scopus (112) Google Scholar, 19Worthey E.A. Schnaufer A. Mian I.S. Stuart K. Salavati R. Nucleic Acids Res. 2003; 31: 6392-6408Crossref PubMed Scopus (71) Google Scholar, 20Kala S. Salavati R. RNA. 2010; 16: 1951-1967Crossref PubMed Scopus (15) Google Scholar, 21Salavati R. Ernst N.L. O'Rear J. Gilliam T. Tarun Jr., S. Stuart K. RNA. 2006; 12: 819-831Crossref PubMed Scopus (42) Google Scholar, 22Guo X. Ernst N.L. Carnes J. Stuart K.D. PLoS One. 2010; 5: e8913Crossref PubMed Scopus (14) Google Scholar, 23Tarun Jr., S.Z. Schnaufer A. Ernst N.L. Proff R. Deng J. Hol W. Stuart K. RNA. 2008; 14: 347-358Crossref PubMed Scopus (34) Google Scholar, 24Niemann M. Brecht M. Schlüter E. Weitzel K. Zacharias M. Göringer H.U. Nucleic Acids Res. 2008; 36: 4465-4473Crossref PubMed Scopus (15) Google Scholar, 25Law J.A. O'Hearn S.F. Sollner-Webb B. RNA. 2008; 14: 1187-1200Crossref PubMed Scopus (15) Google Scholar). The letter K in the beginning of each protein name refers to kinetoplastid. To specify species, we have used species-specific abbreviations (e.g. TbREL1 for REL1 in T. brucei).Although other ligases such as T7 DNA ligase have a catalytic domain and an OB fold domain, KREL1 and KREL2 contain only the catalytic domain (19Worthey E.A. Schnaufer A. Mian I.S. Stuart K. Salavati R. Nucleic Acids Res. 2003; 31: 6392-6408Crossref PubMed Scopus (71) Google Scholar, 26McManus M.T. Shimamura M. Grams J. Hajduk S.L. RNA. 2001; 7: 167-175Crossref PubMed Scopus (97) Google Scholar). The OB fold domain that is essential for interaction with the substrate RNA is provided in trans by KREPA2 and KREPA1, which interact via their zinc fingers with KREL1 and KREL2, respectively, and bring the substrate RNA and the catalytic domain into proximity (15Schnaufer A. Ernst N.L. Palazzo S.S. O'Rear J. Salavati R. Stuart K. Mol. Cell. 2003; 12: 307-319Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 27Swift R.V. Durrant J. Amaro R.E. McCammon J.A. Biochemistry. 2009; 48: 709-719Crossref PubMed Scopus (9) Google Scholar, 28Gao G. Rogers K. Li F. Guo Q. Osato D. Zhou S.X. Falick A.M. Simpson L. Protist. 2010; 161: 489-496Crossref PubMed Scopus (8) Google Scholar). Although RNA editing ligases differ in their structure compared with DNA ligases (15Schnaufer A. Ernst N.L. Palazzo S.S. O'Rear J. Salavati R. Stuart K. Mol. Cell. 2003; 12: 307-319Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), their overall mechanism is similar. Ligation requires three distinct and reversible steps: (i) the ligase adenylylation step in which the conserved catalytic lysine of the ligase attacks the α-phosphate of ATP and displaces pyrophosphate, forming an enzyme-AMP intermediate through the phosphoamide linkage; (ii) the ligase deadenylylation step in which the guide RNA/nicked mRNA duplex binds to the protein and AMP is transferred from the adenylylated ligase to the 5′-phosphate of the RNA molecule, forming an adenylylated RNA with a 5′,5′-phosphoanhydride bond; and (iii) the ligation step in which the free 3′-hydroxyl at the nick site attacks the phosphoanhydride bond of the adenylylated RNA fragment, forming a phosphodiester bond, ligating the double-stranded RNA, and releasing AMP.TbREL1 is an essential enzyme for the editing process and parasite viability (18Schnaufer A. Panigrahi A.K. Panicucci B. Igo Jr., R.P. Wirtz E. Salavati R. Stuart K. Science. 2001; 291: 2159-2162Crossref PubMed Scopus (214) Google Scholar). The crystal structure of the TbREL1 N-terminal catalytic domain has been of compounds TbREL1 R.E. Schnaufer A. H. Hol W. Stuart K.D. McCammon J.A. Proc. Natl. Acad. Sci. U.S.A. 2008; PubMed Scopus Google Scholar, L. R.V. M. Schnaufer A. Amaro R.E. PLoS 2010; PubMed Scopus Google Scholar). The unique of the TbREL1 and the absence of in the TbREL1 an target for of inhibitors that block the essential RNA ligase study has found several inhibitors TbREL1 using a of in and in adenylylation R.E. Schnaufer A. H. Hol W. Stuart K.D. McCammon J.A. Proc. Natl. Acad. Sci. U.S.A. 2008; PubMed Scopus Google Scholar). of these inhibitors have been as inhibitors of RNA editing in the of the purified editosome H. Salavati R. Nucleic Acids Res. 2010; PubMed Scopus Google Scholar). we a study L. R.V. M. Schnaufer A. Amaro R.E. PLoS 2010; PubMed Scopus Google was that a number of inhibitors of In the we have studied the of of the inhibitors provided by the number to as L. R.V. M. Schnaufer A. Amaro R.E. PLoS 2010; PubMed Scopus Google and in study as on the editosome, and we that of new all the different essential catalytic steps of RNA editing are most as a result of the interaction of the core editosome with substrate RNA.