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Poly(A)-binding proteins (PABPs) regulate mRNA fate by controlling stability and translation through interactions with both the poly(A) tail and eIF4F complex. Many organisms have several paralogs of PABPs and eIF4F complex components and it is likely that different eIF4F/PABP complex combinations regulate distinct sets of mRNAs. Trypanosomes have five eIF4G paralogs, six of eIF4E and two PABPs, PABP1 and PABP2. Under starvation, polysomes dissociate and the majority of mRNAs, most translation initiation factors and PABP2 reversibly localise to starvation stress granules. To understand this more broadly we identified a protein interaction cohort for both T. brucei PABPs by cryo-mill/affinity purification-mass spectrometry. PABP1 very specifically interacts with the previously identified interactors eIF4E4 and eIF4G3 and few others. In contrast PABP2 is promiscuous, with a larger set of interactors including most translation initiation factors and most prominently eIF4G1, with its two partners TbG1-IP and TbG1-IP2. Only RBP23 was specific to PABP1, whilst 14 RNA-binding proteins were exclusively immunoprecipitated with PABP2. Significantly, PABP1 and associated proteins are largely excluded from starvation stress granules, but PABP2 and most interactors translocate to granules on starvation. We suggest that PABP1 regulates a small subpopulation of mainly small-sized mRNAs, as it interacts with a small and distinct set of proteins unable to enter the dominant pathway into starvation stress granules and localises preferentially to a subfraction of small polysomes. By contrast PABP2 likely regulates bulk mRNA translation, as it interacts with a wide range of proteins, enters stress granules and distributes over the full range of polysomes., Author summary Poly(A)-binding proteins (PABPs) bind to the poly(A) tails of eukaryotic mRNAs and function in regulating mRNA fate. Many eukaryotes have several PABP paralogs and the current view is that each PABP binds a specific subset of mRNAs. Trypanosoma brucei has two PABPs, and to understand the differential functionality of these paralogs we identified interacting proteins for each. We found unique interactors for both PABPs, and significant differences between the two interaction cohorts. Our data indicate that the two PABP paralogs of trypanosomes have very distinct roles in mediating mRNA fate.

The degree of conservation and evolution of cytoplasmic mRNA metabolism pathways across the eukaryotes remains incompletely resolved. In this study, we describe a comprehensive genome and transcriptome-wide analysis of proteins involved in mRNA maturation, translation, and mRNA decay across representative organisms from the six eukaryotic super-groups. We demonstrate that eukaryotes share common pathways for mRNA metabolism that were almost certainly present in the last eukaryotic common ancestor, and show for the first time a correlation between intron density and a selective absence of some Exon Junction Complex (EJC) components in eukaryotes. In addition, we identify pathways that have diversified in individual lineages, with a specific focus on the unique gene gains and losses in members of the Excavata and SAR groups that contribute to their unique gene expression pathways compared to other organisms.

For persistent infections of the mammalian host, African trypanosomes limit their population size by quorum sensing of the parasite-excreted stumpy induction factor (SIF), which induces development to the tsetse-infective stumpy stage. We found that besides this cell density-dependent mechanism, there exists a second path to the stumpy stage that is linked to antigenic variation, the main instrument of parasite virulence. The expression of a second variant surface glycoprotein (VSG) leads to transcriptional attenuation of the VSG expression site (ES) and immediate development to tsetse fly infective stumpy parasites. This path is independent of SIF and solely controlled by the transcriptional status of the ES. In pleomorphic trypanosomes varying degrees of ES-attenuation result in phenotypic plasticity. While full ES-attenuation causes irreversible stumpy development, milder attenuation may open a time window for rescuing an unsuccessful antigenic switch, a scenario that so far has not been considered as important for parasite survival., Author summary African trypanosomes escape the mammalian host’s immune system by antigenic variation of their variant surface glycoprotein (VSG) coat. VSGs are expressed from a specialized region in the genome, the expression site (ES), that contains essential expression site associated genes (ESAGs). So far, it was assumed that only successful antigenic switches to an intact expression site are viable. Here we show that unsuccessful VSG switches are not a dead-end, but may rather contribute to the persistence of the trypanosomes at the population level. We have simulated an unsuccessful VSG switch in pleomorphic trypanosomes by expression of a second VSG from a locus without ESAGs. The parasites responded with surprising phenotypic plasticity. All parasites immediately exchanged the surface coat and reduced the abundance of ES-derived transcripts. However, depending on the degree of ES-attenuation, the transgenic trypanosomes either resumed growth, or stopped proliferation. We show that the growth-arrested populations synchronously differentiate to the stumpy life cycle stage and become infective for the tsetse fly. This occurs at low cell densities and in the absence of the quorum sensing factor SIF. Thus, unsuccessful VSG switches are not lethal and cell density-dependent quorum sensing is not the only path to the tsetse fly competence.