UtilizaÃÃo de abordagens moleculares in vivo no estudo de homÃlogos do fator de iniciaÃÃo da traduÃÃo 4G (eIF4G) de tripanosomatÃdeos

AUTOR(ES)

Danielle Maria Nascimento Moura

DATA DE PUBLICAÇÃO

2007

RESUMO

In higher eukaryotes, protein synthesis starts with the binding of the translation initiation complex eIF4F to the mRNA. This complex (formed by the eIF4A, eIF4E and eIF4G subunits) allows the recognition of the mRNA by the 40S ribosomal subunit and the initiation of translation. The eIF4G subunit is a large scaffolding protein that is responsible for the correct assembly of the eIF4F complex. So far little is known about translation initiation in trypanosomatids. To investigate this process, we have identified multiple homologues for each eIF4F subunit within the Leishmania major genome. Five homologues for the eIF4G factor were identified and their sequences showed different degrees of similarity to vertebrate factors. Nevertheless they were all conserved in Trypanosoma brucei and T. cruzi. So far, these homologues, named LmEIF4G1-5, have been studied by several approaches, including expression analyses during the L. major life cycle and pull down assays. In order to supplement in vitro data, we used the T. brucei orthologues TbEIF4G3-5 to perform in vivo analysis. The genes were amplified and cloned into different plasmids vectors, allowing protein expression in Escherichia coli, antibody production, RNA interference and subcellular localization through the detection of fluorescent fusion proteins. The RNA interference of the three homologues showed that they are essential for cellular viability but each one presented different growth curves after RNAi induction. Cells expressing TbEIF4G4-dsRNAs also exhibited reduction of mobility and change in morphology before parasite death. The TbEIF4G3-5 antibodies were used in western blots assays to verify protein depletion after RNAi. The cellular localization of the three proteins confirmed their presence in cytoplasm of T. brucei

ASSUNTO(S)

iniciaÃÃo da traduÃÃo eif4g translation initiation eif4g trypanosomatids tripanosomatÃdeos genetica

Documentos Relacionados

• CaracterizaÃÃo funcional de homÃlogos ao fator de iniciaÃÃo da traduÃÃo eIF4E de TripanosomatÃdeos
• CaracterizaÃÃo de possÃveis homÃlogos aos fatores de iniciaÃÃo da traduÃÃo eIF4G e eIF4A de Leishmania major
• IdentificaÃÃo e mapeamento de domÃnios de ligaÃÃo de homÃlogos do fator eIF4G de iniciaÃÃo da traduÃÃo de Leishmania major
• AnÃlise computacional de candidatos a homÃlogos a fatores de iniciaÃÃo da traduÃÃo em tripanossomatÃdeos
• AnÃlise da expressÃo e localizaÃÃo subcelular dos homÃlogos TbEIF4G1 e TbEIF4G2 do fator de iniciaÃÃo da traduÃÃo eIF4G de Trypanosoma brucei