Beyond translation initiation: dual regulatory interactions with phosphorylated and non-phosphorylated LeishIF4E1

Abstract

Translation initiation in eukaryotes begins with the assembly of the cap-binding eIF4F complex at the 5 ends of mRNAs, through eIF4E. Among the six orthologs of eIF4E in Leishmania, LeishIF4E1 is intriguing, as it does not bind any Leishmania eIF4G. It is expressed in both life-forms, maintaining efficient cap-binding activity for translation initiation, unlike other LeishIF4Es. We identified two novel phosphorylation sites specifically S108, and S112, along with S203, which is conserved with the mammalian phosphorylation site. A non-phosphorylatable, tagged, mutant of LeishIF4E1 created by substituting the phosphorylated serine residues with alanine [S(108,112,203)A] was generated. The phosphorylation status did not affect the binding of LeishIF4E1 to translation initiation factors. However, the phosphorylated LeishIF4E1 interacted with proteins involved in DNA and chromatin structure, while non-phosphorylated LeishIF4E1 interacted with proteins that assist cells under stress and unfavorable conditions, particularly in gluconeogenesis. This suggests secondary regulatory roles for LeishIF4E1. RNA sequencing of the LeishIF4E1-associated transcripts revealed a tenfold reduction in transcripts binding to non-phosphorylated LeishIF4E1. GO enrichment analysis indicated distinct phosphorylation-dependent transcript specificities for each form. This study highlights the critical role of LeishIF4E1 phosphorylation in modulating the selectivity of protein interactome and transcript associations, extending the role of LeishIF4E1 beyond its well-established function in translation initiation. Author SummaryLeishmania are digenetic parasites that transition from sand fly vector to mammalian hosts, undergoing a developmental program of gene expression regulated at the level of translation. Protein synthesis is initiated by the assembly of multi-subunit complexes at the mRNA 5 cap structure and is recruited onto the ribosomes. There are six cap-binding proteins in Leishmania denoted LeishIF4Es1-6, and five LeishIF4G candidates. Among these, the function of LeishIF4E1 is intriguing, as it does not bind any LeishIF4G paralogs, and it is highly expressed in both parasite life forms. We examined how LeishIF4E1 phosphorylation modulates its interaction with proteins and transcripts, given that eIF4E phosphorylation in higher eukaryotes is known to regulate gene expression during malignancy. We identified three phosphorylation sites (S108, S112 and S203) and mutated them to A residues [S(108,112,203)A]. The mutated genes were transfected back into parasites for expression. Using pull-down assays, we identified the interacting proteins and associated transcripts for both the phosphorylated and non-phosphorylated LeishIF4E1 forms. The phosphorylated LeishIF4E1 pulled down proteins involved in DNA and chromatin structure, while the non-phosphorylated LeishIF4E1 interacted with proteins known to assist cells during stress and unfavorable conditions, especially gluconeogenesis. The transcript specificities differed markedly in the number of transcripts bound to both forms, shedding light on the importance of LeishIF4E1 phosphorylation. Furthermore, the phosphorylated form bound more transcripts encoding enzymes related to protein phosphorylation. We also showed that phosphorylation of LeishIF4E1 did not influence the cap-binding ability, as both the forms bind the m7GTP cap with parallel efficiencies. Our data suggest that the different cap-binding proteins in Leishmania are responsible for different processes and have a transcript-specific effect on gene expression.