Falk, F. | Search bioRxiv

bioRxiv2022

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

Trypanosoma 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.

molecular biology↗

bioRxiv2021

Mechanisms of translation repression by the EIF4E1-4EIP cap-binding complex of Trypanosoma brucei: potential roles of the NOT complex and a terminal uridylyl transferase

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. There are two competing models for EIF4E1 function: either EIF4E1 has translation initiation activity that is inhibited by 4EIP, or EIF4E1 acts only as an inhibitor. We here provide evidence for the second hypothesis. Even in the complete absence of 4EIP, EIF4E1 showed no detectable association with other translation initiation factors, and 4EIP loss caused no detectable change in 4E1-associated mRNAs. We found that 4EIP stabilises EIF4E1, probably through co-translational complex assembly, and 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; instead, some evidence implicated the NOT deadenylase complex.

molecular biology↗