RESUMEN
RJLs represent a recently described family of the Ras-related GTP-binding proteins. The Trypanosoma cruzi orthologue, TeRjl, was isolated and its locus was characterized in a region of almost 5 kb. Its 660 bp orf, predicting a protein of 24.13 kDa, is present as a single copy gene in T. cruzi I lineage, and from 1-2 copies in T. cruzi II lineage. TcRjl shares 73% aa sequence similarity with its closest identified orthologue, T. brucei TbRjl. RT-PCR experiments revealed that TcRjl is transcribed in mRNA in the 3 main life forms of the parasite, while Northern hybridization demonstrated that TcRjl is transcribed in T. cruzi epimastigotes as at least 2 transcripts, one of around 950 nt and the other of 1500 nt. Splice-leader addition was mapped to a single site at -69 bp upstream of TcRjl orf indicating that the two mRNA types may derive in differences at the 3' of TcRjl mRNA. TcRjl locus presents considerable synteny with Rjl loci from Trypanosoma brucei and Leishmania major as available from their respective genome projects.
Asunto(s)
Proteínas de Unión al GTP/genética , Proteínas Protozoarias/genética , Trypanosoma cruzi/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Mapeo Cromosómico , Proteínas de Unión al GTP/química , Genoma de Protozoos , Datos de Secuencia Molecular , Proteínas Protozoarias/química , ARN Protozoario/química , ARN Protozoario/genética , Alineación de SecuenciaRESUMEN
Rho GTPases are members of the Ras superfamily and are involved in signal transduction pathways, including maintenance of cell morphology and motility, cell cycle progression, and transcription activation. We report the molecular identification in trypanosomatids (Trypanosoma cruzi) of the first member of the Rho family. The cloned Rho protein, TcRho1, shares approximately 40% homology with other members of the Rho family. Southern blot analysis revealed that TcRHO1 is a single copy gene per haploid genome, and Northern blot assays showed a transcript of 1200 nucleotides in length. Mapping the 5'-untranslated region of TcRHO1 transcripts revealed at least five different transcripts derived from differential trans-splicing. Three of the five transcripts contain the trans-splicing site within the coding region of the TcRHO1 gene. TcRho1 also contains the C-terminal sequence CQLF (CAAX motif), which is predicted to direct post-translation prenylation of the cysteine residue. A synthetic peptide containing this C-terminal motif, when tested against Q-Sepharose chromatography fractions from T. cruzi cytosol, was shown to be efficiently farnesylated, but not geranylgeranylated, despite the fact that the CAAX motif with X = Phe specifies geranylgeranylation by mammalian protein geranylgeranyltransferase I. Furthermore, immunoblot analyses of epimastigote protein with anti-S-farnesylcysteine methyl ester and anti-TcRho1 antisera strongly suggested that TcRho1 is farnesylated in vivo. The farnesylation of proteins such as Rho GTPases could be the basis for the selective cytotoxic action of protein farnesyltransferase inhibitors on trypanosomatids versus mammalian cells.
