The mechanism of eukaryotic translation initiation has been studied almost exclusively in Opisthokonts, in particular animal cells and Saccharomyces cerevisiae (reviewed in [Shirokikh & Preiss, 2018]). In these organisms, the first step is usually binding of the translation initiation factor eIF4E to the mRNA cap. eIF4E is joined by eIF4G, which binds to the helicase eIF4A. Meanwhile, small ribosomal subunits, together with a primed Met-tRNA, eIF2, eIF3, eIF1, and eIF5, together form the 43S complex. The 43S complex is recruited to the cap via an interaction between eIF4G and either eIF3, eIF5, eIF1, or rRNA. The primed small subunit complex scans across the 5′-untranslated region (5′-UTR) until it encounters a start codon.Then, a large subunit joins, an exchange of translation factors

Spliced leader trans splicing is the addition of a short, capped sequence to the 5’ end of mRNAs. It is widespread in eukaryotic evolution, but factors that influence trans splicing acceptor site choice have been little investigated. In Kinetoplastids, all protein-coding mRNAs are 5’ trans spliced. A polypyrimidine tract is usually found upstream of the AG splice acceptor, but there is no branch point consensus; moreover, splicing dictates polyadenylation of the preceding mRNA. We here describe a trans splicing reporter system that can be used for studies and screens concerning the roles of sequences and proteins in processing site choice and efficiency. Splicing was poor with poly(U) tracts less than 9 nt long, and was influenced by nearby secondary structures. A screen for signals resulted in selection of sequences that were on average 45% U and 35% C. Tethering of either the splicing factor SF1, or the cleavage and polyadenylation factor CPSF3 within the intron stimulated processing in the correct positions, while tethering of two possible homologues of Opisthokont PTB inhibited processing. In contrast, tethering of SR-domain proteins RBSR1, RBSR2, or TSR1 or its interaction partner TSR1IP, promoted use of alternative signals upstream of the tethering sites. RBSR1 interacts predominantly with proteins implicated in splicing, whereas the interactome of RBSR2 is more diverse. These results suggest that the functions of PTB and SR-domain proteins in splice site definition were already present in the last eukaryotic common ancestor.

Objective The cleavage and polyadenylation endonuclease CPSF73 is thought to be the target of the anti-trypanosomal benzoxaboroles AN7973, acoziborole and AN11736. We previously showed that AN7973 inhibits mRNA processing. We here investigated whether the drug candidates acoziborole (for human sleeping sickness) and AN11736 (for nagana in cattle) have the same effect. We also affinity purified tagged CPSF73 from parasites without, or after, AN7973 treatment, and analysed differentially co-purified proteins by mass spectrometry. Results AN11736 and acoziborole both inhibited mRNA processing, as demonstrated by decreased levels of spliced mRNAs and accumulation of di- and tri-cistronic mRNAs from the alpha-beta tubulin locus. Treating the cells with AN7973 for 30 min. did not significantly affect the proteins that copurified with CPSF73.

SummaryTrypanosoma brucei has six versions of the cap-binding translation initiation factor EIF4E. We investigated the functions of EIF4E2, EIF4E3, EIF4E5 and EIF4E6 in bloodstream forms. We confirmed the protein associations previously found in procyclic forms, and detected specific co-purification of some RNA-binding proteins. Bloodstream forms lacking EIF4E5 grew normally and differentiated to replication-incompetent procyclic forms. Depletion of EIF4E6 inhibited bloodstream-form trypanosome growth and translation. EIF4E2 co-purified only the putative RNA binding protein SLBP2. Bloodstream forms lacking EIF4E2 multiplied slowly, had a low maximal cell density, and expressed the stumpy-form marker PAD1, but showed no evidence for enhanced stumpy-form signalling. EIF4E2 knock-out cells differentiated readily to replication-competent procyclic forms. EIF4E2 was strongly associated with mRNAs that are maximally abundant in S-phase, three of which are bound and stabilized by the Pumilio domain protein PUF9. The same mRNAs had decreased abundances in EIF4E2 knock-out cells. Yeast 2-hybrid results suggested that PUF9 interacts directly with SLBP2, but PUF9 was not detected in EIF4E2 pull-downs. We suggest that the EIF4E2-SLBP2 complex might interact with PUF9, and its bound RNAs, only early during G1/S, stabilizing the mRNAs in preparation for translation later in S-phase or in early G2.

Background Spliced leader trans splicing is the addition of a short, capped sequence to the 5’ end of mRNAs. It is widespread in eukaryotic evolution, but factors that influence trans splicing acceptor site choice have been little investigated. In Kinetoplastids, all protein-coding mRNAs are 5’ trans spliced. A polypyrimidine tract is usually found upstream of the AG splice acceptor, but there is no branch point consensus; moreover, splicing dictates polyadenylation of the preceding mRNA, which is a validated drug target. Methodology and principal findings We here describe a trans splicing reporter system that can be used for studies and screens concerning the roles of sequences and proteins in processing site choice and efficiency. Splicing was poor with poly(U) tracts less than 9 nt long, and was influenced by an intergenic region secondary structure. A screen for signals resulted in selection of sequences that were on average 45% U and 35% C. Tethering of either the splicing factor SF1, or the cleavage and polyadenylation factor CPSF3 within the intron stimulated processing in the correct positions, while tethering of two possible homologues of Opisthokont PTB inhibited processing. In contrast, tethering of SR-domain proteins RBSR1, RBSR2, or TSR1 or its interaction partner TSR1IP, promoted use of alternative signals upstream of the tethering sites. RBSR1 interacts predominantly with proteins implicated in splicing, whereas the interactome of RBSR2 is more diverse. Conclusions Our selectable constructs are suitable for screens of both sequences, and proteins that affect mRNA processing in T. brucei. Our results suggest that the functions of PTB and SR-domain proteins in splice site definition may already have been present in the last eukaryotic common ancestor tract binding protein, SR-domain protein.