Identification of novel proteins and mRNAs differentially bound to the Leishmania Poly(A) Binding Proteins reveals a direct association between PABP1, the RNA-binding protein RBP23 and mRNAs encoding ribosomal proteins.

Poly(A) Binding Proteins (PABPs) are major eukaryotic RNA-binding proteins (RBPs) with multiple roles associated with mRNA stability and translation and characterized mainly from multicellular organisms and yeasts. A variable number of PABP homologues are seen in different organisms however the biological reasons for multiple PABPs are generally not well understood. In the unicellular Leishmania, dependent on post-transcriptional mechanisms for the control of its gene expression, three distinct PABPs are found, with yet undefined functional distinctions. Here, using RNA-immunoprecipitation sequencing analysis we show that the Leishmania PABP1 preferentially associates with mRNAs encoding ribosomal proteins, while PABP2 and PABP3 bind to an overlapping set of mRNAs distinct to those enriched in PABP1. Immunoprecipitation studies combined to mass-spectrometry analysis identified RBPs differentially associated with PABP1 or PABP2, including RBP23 and DRBD2, respectively, that were investigated further. Both RBP23 and DRBD2 bind directly to the three PABPs in vitro, but reciprocal experiments confirmed preferential co-immunoprecipitation of PABP1, as well as the EIF4E4/EIF4G3 based translation initiation complex, with RBP23. Other RBP23 binding partners also imply a direct role in translation. DRBD2, in contrast, co-immunoprecipitated with PABP2, PABP3 and with RBPs unrelated to translation. Over 90% of the RBP23-bound mRNAs code for ribosomal proteins, mainly absent from the transcripts co-precipitated with DRBD2. These experiments suggest a novel and specific route for translation of the ribosomal protein mRNAs, mediated by RBP23, PABP1 and the associated EIF4E4/EIF4G3 complex. They also highlight the unique roles that different PABP homologues may have in eukaryotic cells associated with mRNA translation.

Phosphorylation and interactions associated with the control of the Leishmania Poly-A Binding Protein 1 (PABP1) function during translation initiation.

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.

The unique Leishmania EIF4E4 N-terminus is a target for multiple phosphorylation events and participates in critical interactions required for translation initiation.

The eukaryotic initiation factor 4E (eIF4E) recognizes the mRNA cap structure and, together with eIF4G and eIF4A, form the eIF4F complex that regulates translation initiation in eukaryotes. In trypanosomatids, 2 eIF4E homologues (EIF4E3 and EIF4E4) have been shown to be part of eIF4F-like complexes with presumed roles in translation initiation. Both proteins possess unique N-terminal extensions, which can be targeted for phosphorylation. Here, we provide novel insights on the Leishmania infantum EIF4E4 function and regulation. We show that EIF4E4 is constitutively expressed throughout the parasite development but is preferentially phosphorylated in exponentially grown promastigote and amastigote life stages, hence correlating with high levels of translation. Phosphorylation targets multiple serine-proline or threonine-proline residues within the N-terminal extension of EIF4E4 but does not require binding to the EIF4E4's partner, EIF4G3, or to the cap structure. We also report that EIF4E4 interacts with PABP1 through 3 conserved boxes at the EIF4E4 N-terminus and that this interaction is a prerequisite for efficient EIF4E4 phosphorylation. EIF4E4 is essential for Leishmania growth and an EIF4E4 null mutant was only obtained in the presence of an ectopically provided wild type gene. Complementation for the loss of EIF4E4 with several EIF4E4 mutant proteins affecting either phosphorylation or binding to mRNA or to EIF4E4 protein partners revealed that, in contrast to other eukaryotes, only the EIF4E4-PABP1 interaction but neither the binding to EIF4G3 nor phosphorylation is essential for translation. These studies also demonstrated that the lack of both EIF4E4 phosphorylation and EIF4G3 binding leads to a non-functional protein. Altogether, these findings further highlight the unique features of the translation initiation process in trypanosomatid protozoa.