Members of the eIF4E mRNA cap-binding family are involved in translation and the modulation of transcript availability in other systems as part of a three-component complex including eIF4G and eIF4A. The kinetoplastids possess four described eIF4E and five eIF4G homologs. We have identified two new eIF4E family proteins in Trypanosoma brucei, and define distinct complexes associated with the fifth member, TbEIF4E5. The cytosolic TbEIF4E5 protein binds cap 0 in vitro. TbEIF4E5 was found in association with two of the five TbEIF4Gs. TbIF4EG1 bound TbEIF4E5, a 47.5-kDa protein with two RNA-binding domains, and either the regulatory protein 14-3-3 II or a 117.5-kDa protein with guanylyltransferase and methyltransferase domains in a potentially dynamic interaction. The TbEIF4G2/TbEIF4E5 complex was associated with a 17.9-kDa hypothetical protein and both 14-3-3 variants I and II. Knockdown of TbEIF4E5 resulted in the loss of productive cell movement, as evidenced by the inability of the cells to remain in suspension in liquid culture and the loss of social motility on semisolid plating medium, as well as a minor reduction of translation. Cells appeared lethargic, as opposed to compromised in flagellar function per se. The minimal use of transcriptional control in kinetoplastids requires these organisms to implement downstream mechanisms to regulate gene expression, and the TbEIF4E5/TbEIF4G1/117.5-kDa complex in particular may be a key player in that process. We suggest that a pathway involved in cell motility is affected, directly or indirectly, by one of the TbEIF4E5 complexes.

SummaryRibosome assembly occurs mainly in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding many ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Here, we report a physical and functional interaction between locally synthesized RPs and ribosomes in the axon. We show that axonal RP translation is regulated through a sequence motif, CUIC, that forms an RNA-loop structure in the region immediately upstream of the initiation codon. Using imaging and subcellular proteomics techniques, we show that RPs synthesized in axons join axonal ribosomes in a nucleolus-independent fashion. Inhibition of axonal CUIC-regulated RP translation decreases local translation activity and reduces axon branching in the developing brain, revealing the physiological relevance of axonal RP synthesis in vivo. These results suggest that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus in neurons.

Trypanosoma brucei, as well as Trypanosoma cruzi and more than 20 species of the genus Leishmania, form a group of flagellated protists that threaten human health. These organisms are transmitted by insects that, together with mammals, are their natural hosts. This implies that during their life cycles each of them faces environments with different physical, chemical, biochemical, and biological characteristics. In this work we review how amino acids are obtained from such environments, how they are metabolized, and how they and some of their intermediate metabolites are used as a survival toolbox to cope with the different conditions in which these parasites should establish the infections in the insects and mammalian hosts.

Selective transcription of individual protein coding genes does not occur in trypanosomes and the cellular copy number of each mRNA must be determined post-transcriptionally. Here, we provide evidence that codon choice directs the levels of constitutively expressed mRNAs. First, a novel codon usage metric, the gene expression codon adaptation index (geCAI), was developed that maximised the relationship between codon choice and the measured abundance for a transcriptome. Second, geCAI predictions of mRNA levels were tested using differently coded GFP transgenes and were successful over a 25-fold range, similar to the variation in endogenous mRNAs. Third, translation was necessary for the accelerated mRNA turnover resulting from codon choice. Thus, in trypanosomes, the information determining the levels of most mRNAs resides in the open reading frame and translation is required to access this information.

T he operon arrangement used by prokaryotes is an elegant solution to the question of regulated gene expression, with coordinated transcription of genes encoding enzymes within a given metabolic pathway under the control of a single promoter. In contrast, the majority of eukaryotes evolved independent promoters to control the expression of individual genes, and promoter types fall into classes that are activated or repressed in synchrony with functionally linked genes. Kinetoplastids employ an unusual blend of these two strategies, the constitutive transcription of polycistronic gene clusters that, apart from tandem gene arrays, typically show no discernible biochemical linkage within arrays (1, 2). The result is the virtual absence of genetic control at the level of gene transcription for mRNAs transcribed by RNA polymerase II (3, 4). Trypanosoma brucei has circumvented this limitation for the expression of a set of virulence factors associated with the variant surface glycoproteins. This family provides the coat on the cell surface and cycles a single member over time to allow this parasite to evade the host immune system. RNA polymerase I promoters provide temporal control to this gene set (5, 6). This unusual choice of polymerase is available to trypanosomes because of the mechanism that also provides a complex mRNA cap structure to all nuclear transcripts, namely, trans splicing of the spliced leader (SL) RNA (7).The SL RNA is a small, independently transcribed molecule that is the source of the hypermethylated cap 4 structure that defines nucleus-derived mRNA in kinetoplastids (8). The cap 4 structure consists of cap 0 followed by 2=-O-methylation of the first four transcribed nucleotides and an additional three methylations on the first and fourth bases (9). The first 39 nucleotides are transferred by trans splicing to each gene transcript in a polycistronic array, which, coupled with 3= polyadenylation (10), results in a monocistronic mRNA population looking very much like that from any other eukaryote with a few extra 5= methylations. Other eukaryotes widely separated from each other in evolutionary terms use this combination of polycistronic transcription and trans splicing of their own flavor of SL (11-13).RNA cap formation requires a minimum of three enzymatic activities, a triphosphatase to remove the gamma phosphate of the primary transcript, a guanylyltransferase to attach an inverted GTP cap via a triphosphate bridge, and a methyltransferase to complete the m 7 G modification that defines cap 0 (14). This trio of activities is found in various combinations in different systems, including three separate proteins in yeast, a pairing of the first and second activities in metazoa and plants or the second and third

2!SUMMARY Ribosomes are known to be assembled in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Using subcellular proteomics and live-imaging, we show that locally synthesized RPs incorporate into axonal ribosomes in a nucleolus-independent fashion. We revealed that axonal RP translation is regulated through a novel sequence motif, CUIC, that forms a RNA-loop structure in the region immediately upstream of the initiation codon. Inhibition of axonal CUICregulated RP translation leads to defects in local translation activity and axon branching, demonstrating the physiological relevance of the axonal ribosome remodeling. These results indicate that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus. INTRODUCTIONRNA localization and local translation play key roles in the assembly and maintenance of neuronal connections (Campbell and Holt, 2001;Holt and Schuman, 2013;Wu et al., 2005).Recent genome-wide studies on the axonal transcriptome revealed that thousands of mRNAs are localized to the axon. A consistent but unexpected finding of these studies is the robust enrichment of mRNAs that encode ribosomal proteins (RPs), protein components of ribosomes. Axons are long neuronal processes that carry out many vital specific cellular functions far from their cell bodies, including translation, and must therefore maintain their protein synthetic machinery in good order. However, because most eukaryotic ribosome assembly is well known to occur in the nucleolus (Fromont-Racine et al., 2003;Lastick and McConkey, 1976), the physiological function of RP-coding mRNAs in a neuronal subcellular compartment far distant from the nucleus was enigmatic. RP-coding mRNAs have been abundantly detected in axons of a variety of neuron types, such as retinal ganglion cells (RGCs) (Zivraj et al., 2010), sympathetic neurons (Andreassi et al., 2010) and motor ! 5!In this study, we explore intra-ribosomal roles of axonally synthesized RPs using a range of technical approaches including live imaging, in vivo gene knockdown, bioinformatics, nascent protein labeling and mass spectrometry-based proteomics. We found that axonal translation of RPs coordinately peaks at the axon branching stage in RGCs in vivo, and their translation is regulated by a branch-promoting factor, Netrin-1, through a novel loop structure-forming sequence motif, CUIC, that is shared by ~70% of RP-coding mRNAs. Using nascent protein labeling and proteomic mass spectrometry analysis on ribosomes isolated from pure axons, together with live-imaging approaches, we show that axonally synthesized RPs are physically incorporated into axonal ribosomes in a nucleolus-independent fashion. Furthermore, we demonstrate the physiological importance of the axonal ribosome remodeling by showing that inhibition of axonal RP translation leads to a significant decrease in the level of axonal mRNA translation and s...

The fate of an mRNA is determined by its interaction with proteins and small RNAs within dynamic complexes called ribonucleoprotein complexes (mRNPs). In Trypanosoma brucei and related kinetoplastids, responses to internal and external signals are mainly mediated by post-transcriptional processes. Here, we used proximity-dependent biotin identification (BioID) combined with RNA-seq to investigate the changes resulting from ectopic expression of RBP10 and RBP9, two developmentally regulated RNA-binding proteins (RBPs). Both RBPs have reduced expression in insect procyclic forms (PCFs) compared with bloodstream forms (BSFs). Upon overexpression in PCFs, both proteins were recruited to cytoplasmic foci, co-localizing with the processing body marker SCD6. Further, both RBPs altered the transcriptome from a PCF- to a BSF-like pattern. Notably, upon expression of BirA*-RBP9 and BirA*-RBP10, BioID yielded more than 200 high confidence protein interactors (more than 10-fold enriched); 45 (RBP9) and 31 (RBP10) were directly related to mRNA metabolism. This study validates the use of BioID for investigating mRNP components but also illustrates the complexity of mRNP function.

This study reports the first cloning of T. cruzi purine and pyrimidine transporters, including the first gene encoding a pyrimidine-selective protozoan transporter.