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.

Two related trypanosomatid eIF4G homologues have functional differences compatible with distinct roles during translation initiation

“…45,46,63 The very short N-terminus of the trypanosomatid eIF4Gs are not likely to be sufficient for PABP binding and indeed no interaction was observed between Leishmania EIF4G3 and PABP1 through 2-hybrid assays. 49 Nevertheless, wheat PABP binds the plant eIFiso4G homolog through its MIF4G domain 64 and the results shown here, with the GST-based co-precipitation assays, confirm a previously reported interaction 47 between EIF4G3, but not EIF4G4, and PABP1. It remains to be seen to what extent this interaction contributes to the EIF4G3 function.…”

“…In vitro co-precipitation assays have been used previously to investigate the interaction of L. major EIF4G3 with EIF4AI and PABP1, 34,47 as well as between T. brucei EIF4G3 and EIF4G4 and eIF4E partners. 36 Here, co-precipitation assays were performed to better characterize these interactions and investigate how L. major EIF4G3 and EIF4G4 differ in binding to putative or known binding partners.…”

“…The interaction between GST-tagged EIF4G3 and the 3 distinct PABPs from L. major (PABPs 1 to 3) was previously evaluated and only PABP1 bound EIF4G3, 47 however an independent 2 hybrid assay failed to confirm this interaction. 49 Here, the same interactions were evaluated comparing the 2 eIF4G homologues and using both GSTtagged PABPs and 35 S-labeled EIF4G3 and EIF4G4 and the reverse assay with GST-tagged eIF4Gs with 35 S-labeled PABPs.…”

“…34,36,47 Constructs expressing the L. major EIF4G3 MIF4G domain and its N-terminus plus MIF4G were also generated previously. 34 All cloning procedures carried out here with both sets of L. major and T. brucei EIF4G3 and EIF4G4 genes and their mutagenesis are detailed in the Supplementary Methods section.…”

Nutritional stress affects an atypical cap-binding protein in Leishmania

“…The initiation factors LeishIF2-α and seven subunits of LeishIF3 (a, c, d, e, i, k and l), as well as LeishEF1-β, were detected only in complexes that were pulled down with LeishIF4E-3. LeishPABP-2, whose role in translation is yet unclear, 25 was solely identified in the LeishIF4G-4 complex. Since no interaction between the two proteins was reported by yeast two-hybrid assays ( Fig.…”