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

Abstract

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. Author summary Flagellated protozoa belonging to the Leishmania genus are the causative agents of the Leishmaniasis, neglected tropical diseases with a worldwide distribution. These parasites are well known for unique molecular mechanisms which include a lack of regulation of mRNA synthesis, generally the major stage for the control of gene expression in eukaryotic cells. Regulation of mRNA translation is therefore critical for survival and adaptation during a life cycle involving multiple life forms. Such regulation is likely dependent on RNA binding proteins (RBPs), such as the Poly-A Binding Protein (PABP) that binds to the 3’ ends of the mRNAs. Three PABPs are found in Leishmania, more than in other unicellular organisms, with presumably distinct but undefined roles. Here we identify different mRNAs targets and specific RBPs with whom they interact. We define a specific association between one of the Leishmania PABPs, a single RBP and mRNAs encoding ribosomal proteins, an important subset of cellular mRNAs. Our results shed new light on the interactions required for the translation of these mRNAs, highlighting the relevance of regulating their translation in different organisms and the convergence of regulatory mechanisms acting through their 5’ and 3’ ends. The data also expands on the known PABP roles.