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.

Roles and interactions of the specialized initiation factors EIF4E2, EIF4E5, and EIF4E6 in Trypanosoma brucei: EIF4E2 maintains the abundances of S‐phase mRNAs

“…They also both copurified some proteins that most likely associate via the bound mRNAs. DRBD2 (Wippel et al, 2019) is a cytosolic mRNA‐binding protein (Lueong et al, 2016) that was previously reported to associate with PABP2 (Assis et al, 2021; Kramer et al, 2013) but repressed when tethered to a reporter 3′‐UTR (Lueong et al, 2016). TRRM1 has been implicated in chromatin organization and transcription regulation in procyclic forms and is predominantly nuclear (Naguleswaran et al, 2015), but it is also an mRNA‐binding protein and an activator in the tethering assay (Lueong et al, 2016).…”

“…All these observations indicate that the EIF4E3/EIF4G4 and EIF4E4/EIF4G3 complexes are active in translation initiation. Recent results suggest that in both T. brucei and Leishmania , EIF4E4 and EIF4G3 associate preferentially with PABP1 and an RNA‐binding protein, RBP23 (Figure 1d), and that this complex is preferentially associated with mRNAs encoding ribosomal proteins (Assis et al, 2021; Bezerra et al, submitted; Zoltner et al, 2018).…”

Sequences and proteins that influence mRNA processing in Trypanosoma brucei: evolutionary conservation of SR-domain and PTB protein functions

“…Intriguingly, RBSR1 also specifically pulled down only one of the cap-binding translation initiation factor complexes, EIF4E4 and EIF4G3, even though two others (EIF4E3/EIF4G4 and EIF4E6/EIF4G5) are as abundant (Tinti and Ferguson 2022), and RBSR1 is detected exclusively in the nucleus. Results from Leishmania suggest that the EIF4E4/G3 complex is implicated in translation of mRNAs encoding ribosomal proteins (Assis et al 2021). This link, as well as the apparent association of RBSR1 with the ribosome, might merit further investigation.…”

Roles and interactions of the specialized initiation factors EIF4E2, EIF4E5 and EIF4E6 inTrypanosoma brucei: EIF4E2 maintains the abundances of S-phase mRNAs

“…Our studies of EIF4E6 also revealed there were also a few additional proteins, perhaps associated with bound mRNAs. For example, ZFP2 and DRBD2 (Wippel et al ., 2019) are cytosolic proteins; DRBD2 was reported to associate with PABP2 (Assis et al ., 2021, Kramer et al ., 2013), and ZFP2 is required for differentiation of bloodstream forms the procyclic form (Hendriks et al ., 2001). DRBD3, which was present with EIF4E6 but more with EIF4E3, probably influences splicing but can also stabilize bound mRNAs (Das et al ., 2015, Fernandez-Moya et al ., 2012, Stern et al ., 2009, Estévez, 2008).…”

“…Since tagged EIF4E4 was not detected in axenic Leishmania amastigotes, it was suggested that another EIF4E might substitute (Zinoviev et al ., 2011). A more recent publication has shown copurification of Leishmania EIF4E4 and EIF4G3 with both PABP1 and an RNA-binding protein, RBP23; and also that both RBP23 and PABP1 were preferentially associated with mRNAs encoding ribosomal proteins (Assis et al ., 2021). T. brucei EIF4E4 also associates preferentially with PABP1, although some association with PABP2 was also detected (Zoltner et al ., 2018).…”