The EIF4E1-4EIP cap-binding complex of Trypanosoma brucei interacts with the terminal uridylyl transferase TUT3

Abstract

Most transcription in Trypanosoma brucei is constitutive and polycistronic. Consequently, the parasite relies on post-transcriptional mechanisms, especially affecting translation initiation and mRNA decay, to control gene expression both at steady-state and for adaptation to different environments. The parasite has six isoforms of the cap-binding protein EIF4E as well as five EIF4Gs. EIF4E1 does not bind to any EIF4G, instead being associated with a 4E-binding protein, 4EIP. 4EIP represses translation and reduces the stability of a reporter mRNA when artificially tethered to the 3’-UTR, whether or not EIF4E1 is present. 4EIP is essential during the transition from the mammalian bloodstream form to the procyclic form that lives in the Tsetse vector. In contrast, EIF4E1 is dispensable during differentiation, but is required for establishment of growing procyclic forms. In Leishmania, there is some evidence that EIF4E1 might be active in translation initiation, via direct recruitment of EIF3. However in T. brucei, EIF4E1 showed no detectable association with other translation initiation factors, even in the complete absence of 4EIP. There was some evidence for interactions with NOT complex components, but if these occur they must be weak and transient. We found that EIF4E1is less abundant in the absence of 4EIP, and RNA pull-down results suggested this might occur through co-translational complex assembly. We also report that 4EIP directly recruits the cytosolic terminal uridylyl transferase TUT3 to EIF4E1/4EIP complexes. There was, however, no evidence that TUT3 is essential for 4EIP function.

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

“…The results with EIF4E3 and EIF4E6 suggested that we were detecting mainly non-specific associations. Indeed, the binding of EIF4E3 to mRNAs in bloodstream forms correlated suspiciously well with the binding of EIF4E1 to mRNAs in procyclic forms (Falk et al ., 2021) (Figure 5A; Supplementary Table S3). The only exceptions to this were the specifically EIF4E1-associated mRNAs encoding histone H3 and NOT1, and the mRNA encoding 4EIP, which may be associated with EIF4E1 mRNA because of co-translational protein complex formation (Falk et al ., 2021).…”

“…Indeed, the binding of EIF4E3 to mRNAs in bloodstream forms correlated suspiciously well with the binding of EIF4E1 to mRNAs in procyclic forms (Falk et al ., 2021) (Figure 5A; Supplementary Table S3). The only exceptions to this were the specifically EIF4E1-associated mRNAs encoding histone H3 and NOT1, and the mRNA encoding 4EIP, which may be associated with EIF4E1 mRNA because of co-translational protein complex formation (Falk et al ., 2021). Moreover, hardly any mRNAs were more than 4-fold enriched with either EIF4E3 or EIF4E6, and there was a moderate correlation between EIF4E3 mRNA binding and mRNA length (Figure 5B).…”

“…Kinetoplastid EIF4E1 has no EIF4G partner, instead binding to two other proteins, 4EIP and 4EIP2. Results so far indicate that T. brucei EIF4E1 has primarily a suppressive function (Terrao et al ., 2018, Falk et al ., 2021), but additional translation-promoting functions have been suggested for the Leishmania homologue (Yoffe et al ., 2004, Zinoviev et al ., 2012, Tupperwar et al ., 2019, Tupperwar et al ., 2020). One way to investigate the actions of RNA-associated proteins independent of their RNA-binding specificities, is known as “tethering” (Coller & Wickens, 2007).…”

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

“…Proteins associated with RBSR1 and RBSR2 were identified exactly as described in (Falk et al 2021). Briefly, RBSR1-TAP, RBSR2-TAP and TAP-GFP were purified from cell extracts over IgG beads, then released using His-tagged TEV protease.…”

The Trypanosoma brucei RNA-binding protein DRBD18 ensures correct mRNA trans splicing and polyadenylation patterns

“…A cell line in which one allele of DRBD18 bears a sequence encoding a C-terminal TAP tag was used to study the interactome of DRBD18. DRBD18-TAP was purified using IgG magnetic beads and the bound protein was eluted with TEV protease, which was then removed using HisPur Ni-NTA magnetic beads (Thermo Scientific) according to the manufacturer's instructions (Falk et al 2021). The proteins from three independent purifications were subjected to denaturing SDS-PAGE and analysed by mass spectrometry.…”