The suppressive cap-binding-complex factor 4EIP is required for normal differentiation

Abstract

Summary/AbstractTrypanosoma brucei live in mammals as bloodstream forms and in the Tsetse midgut as procyclic forms. Differentiation from one form to the other proceeds via a growth-arrested stumpy form with low mRNA content and translation. The parasites have six eIF4Es and five eIF4Gs. EIF4E1 pairs with the mRNA-binding protein 4EIP but not with any EIF4G. EIF4E1 and 4EIP each inhibit expression when tethered to a reporter mRNA. The 4E-binding motif in 4EIP is required for the interaction with EIF4E1 both in vivo and in a 2-hybrid assay, but not for the suppressive activity of 4EIP when tethered. However, the suppressive activity of EIF4E1 when tethered requires 4EIP. Correspondingly, in growing bloodstream forms, 4EIP is preferentially associated with unstable mRNAs. Trypanosomes lacking 4EIP have a marginal growth disadvantage as cultured bloodstream or procyclic forms. Bloodstream forms without 4EIP cannot make differentiation-competent stumpy forms, but the defect can be complemented by a truncated 4EIP that does not interact with EIF4E1. Bloodstream forms lacking EIF4E1 have a growth defect but can differentiate. We suggest that 4EIP and EIF4E1 fine-tune mRNA levels in growing cells, and that 4EIP is required for mRNA suppression during differentiation to the stumpy form.

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

“…Pull-down results in Leishmania suggested that about a tenth of the EIF4E1 was 4EIP2-associated. Since 4EIP2 was not detected in our previous 4EIP pull-downs (Terrao et al 2018), and the interaction most likely occurs via the same motif, 4EIP2 and 4EIP probably compete for EIF4E1 binding. It is possible that when 4EIP1 is absent, it is replaced by 4EIP2; this will depend on whether other EIF4Es are also competing for 4EIP2, and on the relative abundances of the different proteins.…”

“…Although tethering of EIF4E1 causes repression when 4EIP is present, in the absence of 4EIP tethering of EIF4E1 has no effect at all. One observation, however, suggests that EIF4E1 has an independent function; unlike 4EIP, we were unable to obtain procyclic trypanosomes lacking EIF4E1 (Terrao et al 2018). Apart from this, Mabille et al highlighted that 4EIP is essential for in vivo infectivity in the vertebrate host, as evidenced by a failure to detect 4EIP KO parasites after one week of infection (Mabille et al 2021); immunosuppressive treatment could not rescue the defect.…”

“…Results with T. brucei have confirmed the interaction between EIF4E1 and 4EIP, but have also suggested that EIF4E1 and 4EIP might have some independent activities. When tethered via a λN peptide to a boxB-bearing reporter mRNA, T. brucei 4EIP strongly represses reporter expression whether or not EIF4E1 is present (Erben et al 2014; Terrao et al 2018). This could mean that the tethering is merely replacing EIF4E1 in recruiting 4EIP to RNA, since similar activities have also been shown for metazoan 4E-BPs (Igreja and Izaurralde 2011; Kamenska et al 2014a).…”

“…Although bloodstream forms lacking either EIF4E1 or 4EIP have only a mild growth defect, 4EIP is essential for full differentiation to the stumpy form; without 4EIP, the translational repression that is intrinsic to this process is delayed and the cells are no longer able to differentiate fully to procyclic forms (Terrao et al 2018). Notably, a truncated form of 4EIP that cannot bind to EIF4E1 can restore stumpy formation in the 4EIP-deficient cells (Terrao et al 2018). Meanwhile, initial differentiation of EIF4E1-deficient cells proceeds normally.…”

Roles of the Pumilio domain protein PUF3 inTrypanosoma bruceigrowth and differentiation

“…These results suggest that the main role of PUF3 is in modulating gene expression during differentiation – presumably in order to suppress the expression of particular proteins. Differentiation to the stumpy form is accompanied by a general decrease in translation, and even a minor delay in this can impair subsequent differentiation (Terrao et al ., 2018), so it is quite feasible that abnormal translation or degradation of a particular subset of mRNAs right at the beginning of the process could prevent subsequent steps, even in monomorphic trypanosomes. Which of the PUF3 targets might be relevant is not obvious.…”