UNCIONES ASOCIADAS AL RETROELEMENTO L1Tc DE TRYPANOSOMA CRUZI
Trypanosoma cruzi is the etiological agent of the Chagas’ disease, which affects around 10 million people, primarily in Central and South America [1]. Apart from its impact on human health, the T. cruzi parasite has been extensively studied because of the interesting molecular characteristics shown by the members of the Trypanosomatidae. It is known that the transcription of protein-coding genes and mRNA maturation processes involve mechanisms that are distinct from those existing in most higher eukaryotes. In fact, most trypanosome mRNAs are synthesized as polycistronic precursors. Mature mRNAs are generated by trans¬-splicing and polyadenylation. It has also been reported that at least 50% of the T. cruzi genome contain repeated sequences, such as retrotransposons and gene families of surface proteins. These repeated sequences have been correlated with the large genomic polymorphism and the high degree of plasticity this parasite shows [2,3].
Retrotransposons are DNA sequences able to mobilize their own copies within a host genome by transcription and reverse transcription of an intermediate RNA. The generation of this intermediate RNA is a crucial step for the mobilization mechanism. The retro¬transposition mechanism described for non-LTR retrotransposons, called Target-Primed Reverse Transcription (TPRT), consists of a single-step reverse transcrip¬tion of the mRNA element and the synthesis of a cDNA strand to generate a new double-stranded DNA copy able to be inserted into a new position in the genome. Thus, the intermediate RNA of the non-LTR retrotransposons is likely to bear within its sequence the informa¬tion required for its own transcription.
The L1Tc element (~5kb) is the best represented autonomous non-LTR retrotransposon from the Trypanosoma cruzi genome [4]. Some L1Tc copies have been found to contain a single ORF encoding a 1574 amino acid protein that contains all functional domains [5]. L1Tc codes for the enzymatic machinery involved in its retrotransposition process including an AP (apurinic/apyrimidinic) endonuclease, a 3’ phosphatase, a 3’ phosphodiesterase (NL1Tc) [6,7], autocatalytic 2A sequence [8], a reverse transcriptase (RTL1Tc) [9] an RNaseH activity (RHL1Tc) [10] and nucleic acid chaperone (C2L1Tc) [11,12] activities. The 5’ untranslatable region (UTR) of this element contains two functional sequences at several levels: internal promoter called Pr77 (RNA pol II-dependent) at DNA level [13] and an HDV-ribozyme, termed L1TcRz at RNA level [14,15].
On the other hand, NARTc (263 nt in length) is a non-autonomous element thought to be mobilized by the gen products of L1Tc [16]. L1Tc and NARTc share 97% and 77% identity at their first 77 and last 30 nucleotides, respectively. The conserved sequence located at their 3'-end is thought to be recognized by the reverse transcrip¬tase encoded by the L1Tc element. The sequence corresponding to the first 77 nucleotides (Pr77) of L1Tc and NARTc has promoter activity and acts as an internal promoter that generates abundant, although poorly translatable, transcripts [13].
In this work we study some functional sequences present in the L1Tc retroelement and analyze the role that they play in L1Tc mobilization process in the T. cruzi genome. We have determined regions within Pr77 mRNA sequence that are implied in HDV-ribozyme co-transcriptional cleavage activity. Moreover, we have shown that L1TcRz is active by in vivo primer extension assays.
Electrophoretic mobility shift assays using specific sequences suggest that the RTL1Tc protein, encoded by L1Tc element, is implied in ribonucleoprotein (RNP) formation. Furthermore, by primer extension experiments, we have demonstrated that the C2L1Tc protein (endowed NAC activity) is able to improve NARTc mRNA reverse transcription events carried out by RTL1Tc protein, suggesting that both activities interact between them in the L1Tc and NARTc mobilization process.
Referencias
1. http://www.who.int/topics/chagas_disease/es/
2. El-Sayed,N.M., Myler,P.J., Bartholomeu,D.C., Nilsson,D., Aggarwal,G., Tran,A.N., Ghedin,E., Worthey,E.A., Delcher,A.L. et al. (2005) The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease. Science, 309, 409-415.
3. Thomas MC, Macias F, Alonso C, López MC. “The biology and evolution of transposable elements in parasites.” Trends Parasitol 2010; 26:350-62;
4. Martín F, Marañón C, Olivares M, Alonso C, López MC. “Characterization of a non-long terminal repeat retrotransposon cDNA (L1Tc) from Trypanosoma cruzi: homology of the first ORF with the ape family of DNA repair enzymes.” J Mol Biol 1995; 247: 49-59;
5. Olivares, M., Thomas, M. C., Lopez-Barajas, A., Requena, J. M., Garcia-Perez, J. L., Angel, S., Alonso, C. and Lopez, M. C. (2000) Genomic clustering of the Trypanosoma cruzi non-long terminal L1Tc retrotransposon with defined interspersed repeated DNA elements. Electrophoresis 21, 2973-2982
6. Olivares M, Alonso C, López MC. “The open reading frame 1 of the L1Tc retrotransposon of Trypanosoma cruzi codes for a protein with apurinic-apyrimidinic nuclease activity.” J Biol Chem 1997; 272:25224-8;
7. Olivares M, Thomas MC, Alonso C, López MC. “The L1Tc, long interspersed nucleotide element from Trypanosoma cruzi, encodes a protein with3’-phosphatase and 3’-phosphodiesterase enzymatic activities .” J Biol Chem 1999; 274(34):23883-6;
8. Heras SR, Thomas MC, García-Canadas M, de Felipe P, García-Pérez JL, Ryan MD, et al. “L1Tc non-LTR retrotransposons from Trypanosoma cruzi contain a functional viral-like self-cleaving 2A sequence in frame with the active proteins they encode.” Cell Mol Life Sci 2006; 63:1449-60;
9. García-Pérez JL, González CI, Thomas MC, Olivares M, López MC. “Characterization of reverse trans¬criptase activity of the L1Tc retroelement from Trypanosoma cruzi.” Cell Mol Life Sci 2003; 60: 2692-701;
10. Olivares M, García-Pérez JL, Thomas MC, Heras SR, López MC. “The non-LTR (long terminal repeat) retrotransposon L1Tc from Trypanosoma cruzi codes for a protein with RNase H activity.” J Biol Chem 2002; 277:28025-30;
11. Heras SR, López MC, García-Pérez JL, Martin SL, Thomas MC. “The L1Tc C-terminal domain from Trypanosoma cruzi non-long terminal repeat retro¬transposon codes for a protein that bears two C2H2 zinc finger motifs and is endowed with nucleic acid chaperone activity.” Mol Cell Biol 2005; 25:9209-20;
12. Heras SR, Thomas MC, Macias F, Patarroyo ME, Alonso C, López MC. “Nucleic-acid-binding properties of the C2-L1Tc nucleic acid chaperone encoded by L1Tc retrotransposon.” Biochem J 2009; 424:479-90;
13. Heras SR, López MC, Olivares M, Thomas MC. “The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts.” Nucleic Acids Res 2007; 35: 2199-214;
14. Sánchez-Luque FJ, López MC, Macias F, Alonso C, Thomas MC. “Identification of an hepatitis delta virus¬like ribozyme at the mRNA 5'-end of the L1Tc retrotransposon from Trypanosoma cruzi.” Nucleic Acids Res 2011; 39:8065-77;
15. Sánchez-Luque FJ, López MC, Macias F, Alonso C, Thomas MC. “Pr77 and L1TcRz: a dual system within the 5’-end of L1Tc retrotransposon, internal promoter and HDV-like ribozyme¬.” Mobile Genetic Elements 2012; 2:1; 1-7;
16. Bringaud F, García-Pérez JL, Heras SR, Ghedin E, El-Sayed NM, Andersson B, et al. “Identification of non-autonomous non-LTR retrotransposons in the genome of Trypanosoma cruzi.” Mol Biochem Parasitol 2002; 124:73-8;
Retrotransposons are DNA sequences able to mobilize their own copies within a host genome by transcription and reverse transcription of an intermediate RNA. The generation of this intermediate RNA is a crucial step for the mobilization mechanism. The retro¬transposition mechanism described for non-LTR retrotransposons, called Target-Primed Reverse Transcription (TPRT), consists of a single-step reverse transcrip¬tion of the mRNA element and the synthesis of a cDNA strand to generate a new double-stranded DNA copy able to be inserted into a new position in the genome. Thus, the intermediate RNA of the non-LTR retrotransposons is likely to bear within its sequence the informa¬tion required for its own transcription.
The L1Tc element (~5kb) is the best represented autonomous non-LTR retrotransposon from the Trypanosoma cruzi genome [4]. Some L1Tc copies have been found to contain a single ORF encoding a 1574 amino acid protein that contains all functional domains [5]. L1Tc codes for the enzymatic machinery involved in its retrotransposition process including an AP (apurinic/apyrimidinic) endonuclease, a 3’ phosphatase, a 3’ phosphodiesterase (NL1Tc) [6,7], autocatalytic 2A sequence [8], a reverse transcriptase (RTL1Tc) [9] an RNaseH activity (RHL1Tc) [10] and nucleic acid chaperone (C2L1Tc) [11,12] activities. The 5’ untranslatable region (UTR) of this element contains two functional sequences at several levels: internal promoter called Pr77 (RNA pol II-dependent) at DNA level [13] and an HDV-ribozyme, termed L1TcRz at RNA level [14,15].
On the other hand, NARTc (263 nt in length) is a non-autonomous element thought to be mobilized by the gen products of L1Tc [16]. L1Tc and NARTc share 97% and 77% identity at their first 77 and last 30 nucleotides, respectively. The conserved sequence located at their 3'-end is thought to be recognized by the reverse transcrip¬tase encoded by the L1Tc element. The sequence corresponding to the first 77 nucleotides (Pr77) of L1Tc and NARTc has promoter activity and acts as an internal promoter that generates abundant, although poorly translatable, transcripts [13].
In this work we study some functional sequences present in the L1Tc retroelement and analyze the role that they play in L1Tc mobilization process in the T. cruzi genome. We have determined regions within Pr77 mRNA sequence that are implied in HDV-ribozyme co-transcriptional cleavage activity. Moreover, we have shown that L1TcRz is active by in vivo primer extension assays.
Electrophoretic mobility shift assays using specific sequences suggest that the RTL1Tc protein, encoded by L1Tc element, is implied in ribonucleoprotein (RNP) formation. Furthermore, by primer extension experiments, we have demonstrated that the C2L1Tc protein (endowed NAC activity) is able to improve NARTc mRNA reverse transcription events carried out by RTL1Tc protein, suggesting that both activities interact between them in the L1Tc and NARTc mobilization process.
Referencias
1. http://www.who.int/topics/chagas_disease/es/
2. El-Sayed,N.M., Myler,P.J., Bartholomeu,D.C., Nilsson,D., Aggarwal,G., Tran,A.N., Ghedin,E., Worthey,E.A., Delcher,A.L. et al. (2005) The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease. Science, 309, 409-415.
3. Thomas MC, Macias F, Alonso C, López MC. “The biology and evolution of transposable elements in parasites.” Trends Parasitol 2010; 26:350-62;
4. Martín F, Marañón C, Olivares M, Alonso C, López MC. “Characterization of a non-long terminal repeat retrotransposon cDNA (L1Tc) from Trypanosoma cruzi: homology of the first ORF with the ape family of DNA repair enzymes.” J Mol Biol 1995; 247: 49-59;
5. Olivares, M., Thomas, M. C., Lopez-Barajas, A., Requena, J. M., Garcia-Perez, J. L., Angel, S., Alonso, C. and Lopez, M. C. (2000) Genomic clustering of the Trypanosoma cruzi non-long terminal L1Tc retrotransposon with defined interspersed repeated DNA elements. Electrophoresis 21, 2973-2982
6. Olivares M, Alonso C, López MC. “The open reading frame 1 of the L1Tc retrotransposon of Trypanosoma cruzi codes for a protein with apurinic-apyrimidinic nuclease activity.” J Biol Chem 1997; 272:25224-8;
7. Olivares M, Thomas MC, Alonso C, López MC. “The L1Tc, long interspersed nucleotide element from Trypanosoma cruzi, encodes a protein with3’-phosphatase and 3’-phosphodiesterase enzymatic activities .” J Biol Chem 1999; 274(34):23883-6;
8. Heras SR, Thomas MC, García-Canadas M, de Felipe P, García-Pérez JL, Ryan MD, et al. “L1Tc non-LTR retrotransposons from Trypanosoma cruzi contain a functional viral-like self-cleaving 2A sequence in frame with the active proteins they encode.” Cell Mol Life Sci 2006; 63:1449-60;
9. García-Pérez JL, González CI, Thomas MC, Olivares M, López MC. “Characterization of reverse trans¬criptase activity of the L1Tc retroelement from Trypanosoma cruzi.” Cell Mol Life Sci 2003; 60: 2692-701;
10. Olivares M, García-Pérez JL, Thomas MC, Heras SR, López MC. “The non-LTR (long terminal repeat) retrotransposon L1Tc from Trypanosoma cruzi codes for a protein with RNase H activity.” J Biol Chem 2002; 277:28025-30;
11. Heras SR, López MC, García-Pérez JL, Martin SL, Thomas MC. “The L1Tc C-terminal domain from Trypanosoma cruzi non-long terminal repeat retro¬transposon codes for a protein that bears two C2H2 zinc finger motifs and is endowed with nucleic acid chaperone activity.” Mol Cell Biol 2005; 25:9209-20;
12. Heras SR, Thomas MC, Macias F, Patarroyo ME, Alonso C, López MC. “Nucleic-acid-binding properties of the C2-L1Tc nucleic acid chaperone encoded by L1Tc retrotransposon.” Biochem J 2009; 424:479-90;
13. Heras SR, López MC, Olivares M, Thomas MC. “The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts.” Nucleic Acids Res 2007; 35: 2199-214;
14. Sánchez-Luque FJ, López MC, Macias F, Alonso C, Thomas MC. “Identification of an hepatitis delta virus¬like ribozyme at the mRNA 5'-end of the L1Tc retrotransposon from Trypanosoma cruzi.” Nucleic Acids Res 2011; 39:8065-77;
15. Sánchez-Luque FJ, López MC, Macias F, Alonso C, Thomas MC. “Pr77 and L1TcRz: a dual system within the 5’-end of L1Tc retrotransposon, internal promoter and HDV-like ribozyme¬.” Mobile Genetic Elements 2012; 2:1; 1-7;
16. Bringaud F, García-Pérez JL, Heras SR, Ghedin E, El-Sayed NM, Andersson B, et al. “Identification of non-autonomous non-LTR retrotransposons in the genome of Trypanosoma cruzi.” Mol Biochem Parasitol 2002; 124:73-8;