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Abstract Background Telomeres are specialized structures at the end of chromosomes essential for maintaining genome stability and cell viability. The importance of telomeric proteins for telomere maintenance has increased our interest in the identification of homologues within the genus Leishmania. The mammalian TRF1 and TRF2 proteins, for example, bind double-stranded telomeres via a Myb-like DNA-binding domain and are involved with telomere length regulation and chromosome end protection. In addition, TRF2 can modulate the activity of several enzymes and influence the conformation of telomeric DNA. In this work, we identified and characterized a Leishmania protein (LaTRF) homologous to both mammalian TRF1 and TRF2. Results LaTRF was cloned using a PCR-based strategy. ClustalW and bl2seq sequence analysis showed that LaTRF shared sequence identity with the Trypanosoma brucei TRF (TbTRF) protein and had the same degree of sequence similarities with the dimerization (TRFH) and the canonical DNA-binding Myb-like domains of both mammalian TRFs. LaTRF was predicted to be an 82.5 kDa protein, indicating that it is double the size of the trypanosome TRF homologues. Western blot and indirect immunofluorescence combined with fluorescence in situ hybridization showed that LaTRF, similarly to hTRF2, is a nuclear protein that also associates with parasite telomeres. Native and full length LaTRF and a mutant bearing the putative Myb-like domain expressed in bacteria bound double-stranded telomeric DNA in vitro. Chromatin immunoprecipitation showed that LaTRF interacted specifically with telomeres in vivo. Conclusion The nuclear localization of LaTRF, its association and co-localization with parasite telomeres and its high identity with TbTRF protein, support the hypothesis that LaTRF is a Leishmania telomeric protein.

Rbp38 is a protein exclusively found in trypanosomatid parasites, including Leishmania amazonensis , the etiologic agent of tegumentar leishmaniasis in the Americas. The protein was first described as a Leishmania tarentolae mitochondrial RNA binding protein. Later, it was shown that the trypanosomes Rbp38 orthologues were exclusively found in the mitochondria and involved in the stabilization and replication of kinetoplast DNA (kDNA). In contrast, L. amazonensis Rbp38 (LaRbp38), co-purifies with telomerase activity and interacts not only with kDNA but also with telomeric DNA, although shares with its counterparts high sequence identity and a putative N-terminal mitochondrial targeting signal (MTS). To understand how LaRbp38 interacts both with nuclear and kDNA, we have first investigated its subcellular localization. Using hydroxy-urea synchronized L. amazonensis promastigotes we could show that LaRbp38 shuttles from mitochondria to the nucleus at late S and G2 phases. Further, we identified a non-classical nuclear localization signal (NLS) at LaRbp38 C-terminal that binds with importin alpha, a protein involved in the nuclear transport of several proteins. Also, we obtained LaRbp38 truncated forms among which, some of them also showed an affinity for both telomeric DNA and kDNA. Analysis of these truncated forms showed that LaRbp38 DNA-binding region is located between amino acid residues 95–235. Together, our findings strongly suggest that LaRbp38 is multifunctional with dual subcellular localization. • Rbp38 prior described as a mitochondrial protein, in Leishmania sp. is also found in the nucleus. • L. amazonensis Rbp38 (LaRbp38) dual subcellular localization is cell cycle-dependent. • LaRbp38 has a monopartite non-classical NLS that binds to importin-alpha. • LaRbp38 binds telomeric G-rich DNA and kDNA. • LaRbp38 DNA-binding region is located between amino acid residues 95–235. [ABSTRACT FROM AUTHOR]

Background: Telomeres are specialized structures at the end of chromosomes essential for maintaining genome stability and cell viability. The importance of telomeric proteins for telomere maintenance has increased our interest in the identification of homologues within the genus Leishmania. The mammalian TRF1 and TRF2 proteins, for example, bind double-stranded telomeres via a Myb-like DNA-binding domain and are involved with telomere length regulation and chromosome end protection. In addition, TRF2 can modulate the activity of several enzymes and influence the conformation of telomeric DNA. In this work, we identified and characterized a Leishmania protein (LaTRF) homologous to both mammalian TRF1 and TRF2. Results: LaTRF was cloned using a PCR-based strategy. ClustalW and bl2seq sequence analysis showed that LaTRF shared sequence identity with the Trypanosoma brucei TRF (TbTRF) protein and had the same degree of sequence similarities with the dimerization (TRFH) and the canonical DNA-binding Myb-like domains of both mammalian TRFs. LaTRF was predicted to be an 82.5 kDa protein, indicating that it is double the size of the trypanosome TRF homologues. Western blot and indirect immunofluorescence combined with fluorescence in situ hybridization showed that LaTRF, similarly to hTRF2, is a nuclear protein that also associates with parasite telomeres. Native and full length LaTRF and a mutant bearing the putative Myb-like domain expressed in bacteria bound double-stranded telomeric DNA in vitro. Chromatin immunoprecipitation showed that LaTRF interacted specifically with telomeres in vivo. Conclusion: The nuclear localization of LaTRF, its association and co-localization with parasite telomeres and its high identity with TbTRF protein, support the hypothesis that LaTRF is a Leishmania telomeric protein. [ABSTRACT FROM AUTHOR]