title: Functional characterization of three leishmania poly(a) binding protein homologues with distinct binding properties to RNA and protein partners.
abstract: Trypanosomatid protozoans are reliant on posttranscriptional processes to control gene expression. Regulation occurs at the levels of mRNA processing, stability, and translation, events that may require the participation of the poly(A) binding protein (PABP). Here, we have undertaken a functional study of the three distinct Leishmania major PABP (LmPABP) homologues: the previously described LmPABP1; LmPABP2, orthologous to the PABP described from Trypanosoma species; and LmPABP3, unique to Leishmania. Sequence identity between the three PABPs is no greater than 40%. In assays measuring binding to A-rich sequences, LmPABP1 binding was poly(A) sensitive but heparin insensitive; LmPABP2 binding was heparin sensitive and less sensitive to poly(A), compatible with unique substitutions observed in residues implicated in poly(A) binding; and LmPABP3 displayed intermediate properties. All three homologues are simultaneously expressed as abundant cytoplasmic proteins in L. major promastigotes, but only LmPABP1 is present as multiple isoforms. Upon transcription inhibition, LmPABP2 and -3 migrated to the nucleus, while LmPABP1 remained predominantly cytoplasmic. Immunoprecipitation assays showed an association between LmPABP2 and -3. Although the three proteins bound to a Leishmania homologue of the translation initiation factor eukaryotic initiation factor 4G (eIF4G) (LmEIF4G3) in vitro, LmPABP1 was the only one to copurify with native LmEIF4G3 from cytoplasmic extracts. Functionality was tested using RNA interference (RNAi) in Trypanosoma brucei, where both orthologues to LmPABP1 and -2 are required for cellular viability. Our results indicate that these homologues have evolved divergent functions, some of which may be unique to the trypanosomatids, and reinforces a role for LmPABP1 in translation through its interaction with the eIF4G homologue.
authors.affiliation: Departamento de Microbiologia, Centro de Pesquisas Aggeu Magalhães/Fiocruz, Recife, PE, Brazil.
authors.forename: Tamara D
authors.initials: TD
authors.lastname: da Costa Lima
authors.forename: Danielle M N
authors.initials: DM
authors.lastname: Moura
authors.forename: Christian R S
authors.initials: CR
authors.lastname: Reis
authors.forename: J Ronnie C
authors.initials: JR
authors.lastname: Vasconcelos
authors.forename: Louise
authors.initials: L
authors.lastname: Ellis
authors.forename: Mark
authors.initials: M
authors.lastname: Carrington
authors.forename: Regina C B Q
authors.initials: RC
authors.lastname: Figueiredo
authors.forename: Osvaldo P
authors.initials: OP
authors.lastname: de Melo Neto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002470:Cell Survival; D039603:Eukaryotic Initiation Factor-4G / Q000378:metabolism*; D018320:Leishmania major / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D008969:Molecular Sequence Data; D011061:Poly A / Q000378:metabolism; D039102:Poly(A)-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism
chemical_list: D039603:Eukaryotic Initiation Factor-4G; D039102:Poly(A)-Binding Proteins; D012333:RNA, Messenger; D011061:Poly A