A multifunctional DNA binding-protein, LaRBP38 associates in vivo with the telomeric DNA of L. amazonensis promastigotes
Siqueira Neto, J.L. 1; Lira, C.B.B.1,3; Gui, K. 1,3; Peroni L.A. 2; dos Reis, J.R.R. 2; Ramos, C.H.I. 3 and Cano, M.I.N. 1
1 Laboratório de Telômeros, Departamento de Genética, IBB, UNESP- Botucatu, SP-Brasil 2 Laboratório de de Imunologia Aplicada, Departamento de Microbiologia e Imunologia, IB, UNICAMP- Campinas, SP-Brasil 3 Laboratório Nacional de Luz Síncrotron, Campinas, SP, Brazil
Telomeres
are specialized structures located at the end of linear chromosomes.
They are composed of proteins complexed to DNA and are essential to
maintain genome stability and cell viability. A few reports have dealt with the composition of the telomeric chromatin in Leishmania spp., the causative agent of leishmaniasis. Leishmania
spp. telomeres are composed by the conserved TTAGGG sequence, which is
maintained by telomerase and present proteins that can associate with
both the single and the double-stranded form of the telomeric DNA. Rbp38 is one of the proteins that interact with the G-rich strand. It was previously isolated from affinity purified nuclear extracts of L. amazonensis
promastigotes as the protein component of LaTG2. Using blastp to search
for Rbp38 homologues in the public genome database, we verified that
Rbp38 is classified as a RNA-binding protein from trypanosomatid mitochondria that modulates RNA stability. It is
exclusively expressed in kinetoplastid protozoa parasites and shares
approximately 70% identity with Tc38, a poly(dT-dG) DNA-binding protein
of Trypanosoma cruzi that has nuclear and mitochondrial localization. Recombinant LaRBP38 expressed in E. coli bound efficiently the telomeric DNA in vitro using electrophoretic mobility shift assay. We are currently constructing truncated versions of the protein, since the in silico available tools (i.e. rps blast) were not able to define any known DNA binding domain in LaRBP38. The analysis of LaRbp38 expression by Western blotting showed
that a polyclonal antibody raised against the recombinant protein was
able to specifically recognize the native LaRbp38 protein (39.8 kDa)
present in nuclear and total extracts of L. amazonensis promastigotes. By indirect immunofluorescence it was possible to show that LaRbp38 is expressed either in the nucleus, in the kinetoplast or in both. It also co-precipitated with the telomeric sequence in a in vivo
chromatin immunoprecipitation assay. All together these results
reaffirm our previous hypothesis of LaRbp38 being a protein which may
have nuclear and mitochondrial functions. The importance of LaRBP38 at Leishmania telomeres is being investigated. Financial Support: FAPESP, UNICEF/ UNDP/World Bank/WHO (TDR)
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