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ABSTRACT
The Poly-A Binding Protein (PABP) is a conserved eukaryotic polypeptide involved in many aspects of mRNA metabolism. During translation initiation, PABP interacts with the translation initiation complex eIF4F and enhances the translation of polyadenylated mRNAs. Schematically, most PABPs can be divided into an N-terminal RNA-binding region, a non-conserved linker segment and the C-terminal MLLE domain. In pathogenic Leishmania protozoans, three PABP homologues have been identified, with the first one (PABP1) targeted by phosphorylation and shown to co-immunoprecipitate with an eIF4F-like complex (EIF4E4/EIF4G3) implicated in translation initiation. Here, PABP1 phosphorylation was shown to be linked to logarithmic cell growth, reminiscent of EIF4E4 phosphorylation, and coincides with polysomal association. Phosphorylation targets multiple serine-proline (SP) or threonine-proline (TP) residues within the PABP1 linker region. This is an essential protein, but phosphorylation is not needed for its association with polysomes or cell viability. Mutations which do impair PABP1 polysomal association and are required for viability do not prevent phosphorylation, although further mutations lead to a presumed inactive protein largely lacking phosphorylated isoforms. Co-immunoprecipitation experiments were carried out to investigate PABP1 function further, identifying several novel protein partners and the EIF4E4/EIF4G3 complex, but no other eIF4F-like complex or subunit. A novel, direct interaction between PABP1 and EIF4E4 was also investigated and found to be mediated by the PABP1 MLLE binding to PABP Interacting Motifs (PAM2) within the EIF4E4 N-terminus. The results shown here are consistent with phosphorylation of PABP1 being part of a novel pathway controlling its function and possibly translation in Leishmania.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Carole Dumas for technical assistance and other members of Dr. Papadopoulou's and Dr. de Melo Neto's laboratories for helpful discussions. We are also thankful to the Proteomics platform of the CHU de Quebec Research Center for preliminary mass-spectrometry analysis of PABP1 bound complexes. The work in Dr. Papadopoulou's lab was supported by the Canadian Institutes of Health Research (CIHR) operating grant MOP-12182, the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant GC100741 (418444), and the Ministère du Développement Économique de l'Innovation et de l'Exportation (MDEIE) PSR-SIIRI-439 grant awarded to BP. The Leishmania work in Dr. de Melo Neto's lab was more recently funded with grants provided by the Brazilian funding agencies FACEPE (APQ-0239-2.02/12 and APQ-1662-2.02/15), CNPq (480899/2013-4, 313934/2013-4 and 401282/2014-7) and CAPES (23038.007656/2011-92). Dr. de Melo Neto was the recipient of a Visiting Scholar grant from CAPES for a medium term stay at the University of Laval (BEX 3743/10-1). To cover their stays at the same institution, TPR, TDCL and LMN received funding from CNPq, the Agence Universitaire de la Francophonie and CAPES, respectively. Studentships for the graduate students in Brazil (TPR, KCM, POR, LAA, LMN and CCX) were provided by CNPq, FACEPE or CAPES. The authors thank the Nucleo de Plataformas Tecnológicas (NPT) at the Instituto Aggeu Magalhães for the use of its automatic sequencing facility, the Instituto Carlos Chagas for the use of its proteomics facility and FIOCRUZ for the funding support.
