Characterization of an Atypical eIF4E Ortholog in Leishmania, LeishIF4E-6

Abstract

Leishmania parasites are digenetic protists that shuffle between sand fly vectors and mammalian hosts, transforming from flagellated extracellular promastigotes that reside within the intestinal tract of female sand flies to the obligatory intracellular and non-motile amastigotes within mammalian macrophages. Stage differentiation is regulated mainly by post-transcriptional mechanisms, including translation regulation. Leishmania parasites encode six different cap-binding proteins, LeishIF4E1-6, that show poor conservation with their counterparts from higher eukaryotes and among themselves. In view of the changing host milieu encountered throughout their life cycle, we propose that each LeishIF4E has a unique role, although these functions may be difficult to determine. Here we characterize LeishIF4E-6, a unique eIF4E ortholog that does not readily associate with m7GTP cap in either of the tested life forms of the parasite. We discuss the potential effect of substituting two essential tryptophan residues in the cap-binding pocket, expected to be involved in the cap-binding activity, as judged from structural studies in the mammalian eIF4E. LeishIF4E-6 binds to LeishIF4G-5, one of the five eIF4G candidates in Leishmania. However, despite this binding, LeishIF4E-6 does not appear to function as a translation factor. Its episomal overexpression causes a general reduction in the global activity of protein synthesis, which was not observed in the hemizygous deletion mutant generated by CRISPR-Cas9. This genetic profile suggests that LeishIF4E-6 has a repressive role. The interactome of LeishIF4E-6 highlights proteins involved in RNA metabolism such as the P-body marker DHH1, PUF1 and an mRNA-decapping enzyme that is homologous to the TbALPH1.

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

“… Note : This table shows the effects of depleting EIF4Es, EIF4Gs, and other direct interaction partners in either Trypanosoma brucei or Leishmania. References are as follows: (1) (Freire et al, 2011); (2) (Terrao et al, 2018); (3) (Falk et al, 2021); (4) (Tupperwar et al, 2019); (5) (Baron et al, 2021); (6) (Alsford et al, 2011); (7) (Freire et al, 2018); (8) (Archer et al, 2009); (9) (Shrivastava et al, 2019); (10) (Moura et al, 2015); (11) (Freire, Vashisht, et al, 2014); (12) (Freire, Malvezzi, et al, 2014); (13) (Tupperwar et al, 2021); *results from this paper. Reference 6 reports results from a high‐throughput screen, which are less reliable than the tests on individual proteins for which depletion of protein or mRNA was monitored. …”

“…So far, however, there is no evidence for an association between the MKT complex and EIF4E6/EIF4G5 in procyclic forms. Recent evidence suggests that Leishmania EIF4E6 may even inhibit translation (Tupperwar et al, 2021). Although Leishmania EIF4E6 also associates with EIF4G5 and G5‐IP, the MKT1 and PBP1 homologs did not copurify, at least from promastigotes (Tupperwar et al, 2021).…”

“…Recent evidence suggests that Leishmania EIF4E6 may even inhibit translation (Tupperwar et al, 2021). Although Leishmania EIF4E6 also associates with EIF4G5 and G5‐IP, the MKT1 and PBP1 homologs did not copurify, at least from promastigotes (Tupperwar et al, 2021). Overexpression of Leishmania EIF4E6‐SBP inhibited protein synthesis by an unknown mechanism; a hemizygous mutant had morphological defects but normal protein synthesis rates.…”

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

“…EIF4G5 was also weakly co-purified with VSG mRNA, suggesting a role in its translation. Recent evidence suggests that Leishmania EIF4E6 may have a different role and might even inhibit translation (Tupperwar et al ., 2021). Although Leishmania EIF4E6 also associates with EIF4G5 and G5-IP, the MKT1 and PBP1 homologues did not co-purify (Tupperwar et al ., 2021).…”

“…Recent evidence suggests that Leishmania EIF4E6 may have a different role and might even inhibit translation (Tupperwar et al ., 2021). Although Leishmania EIF4E6 also associates with EIF4G5 and G5-IP, the MKT1 and PBP1 homologues did not co-purify (Tupperwar et al ., 2021). Over-expression of Leishmania EIF4E6-SBP inhibited protein synthesis by an unknown mechanism; a hemizygous mutant had morphological defects but normal protein synthesis rates.…”