Baron, N. | Search bioRxiv

bioRxiv2025

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

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.

microbiology↗

bioRxiv2020

Distinct features of the Leishmania cap-binding protein LeishIF4E2 revealed by CRISPR-Cas9 mediated heterozygous deletion

Leishmania parasites cycle between sand-fly vectors and mammalian hosts, adapting to alternating environments by stage-differentiation, accompanied by changes in the proteome profiles. Translation regulation plays a central role in driving the differential program of gene expression, since control of gene regulation in Leishmania is mostly post-transcriptional. The Leishmania genome encodes six eIF4E candidates, each assumed to have specific functions, although overlaps are expected. It is noted that some of them can bind to a dedicated eIF4G candidate partners, and LeishIF4E2 does not bind any known eIF4G ortholog. LeishIF4E2 was previously shown to comigrate in the polysomal fractions of sucrose gradients, whereas initiation factors usually comigrate with pre-initiation and initiation complexes. Using the CRISPR-Cas9 methodology, we deleted one of the two LeishIF4E2 gene copies. The deletion caused severe alterations in the morphology of mutant cells that turned round and equipped with a very short flagellum, but their growth rate and general translation remained unaffected. Proteomic analysis of the LeishIF4E2(+/-) mutant cells compared to wild type controls showed that the number of proteins that were upregulated exceeded the number of downregulated proteins, possibly indicating that a repressor function was eliminated. The upregulated proteins were related mainly to general metabolic processes, DNA repair and replication, signaling, and cellular motor activity. The downregulated proteins included several groups, including cytoskeletal and ribosomal proteins. Despite the fact that only one of the two LeishIF4E2 alleles was deleted, the mutant cells were impaired in their ability to infect cultured macrophages. LeishIF4E2 does not behave like a general translation factor and its function remains elusive. Although it could have a repressive function, we cannot exclude the possibility that it is responsible for translation of a specific set of transcripts. Overall, our results are in line with the possibility that the different LeishIF4Es are assigned unique functions. Author summaryLeishmania parasites cause a broad spectrum of diseases that lead to different pathological symptoms. During their life cycle, the parasites shuffle between sand-fly vectors and mammalian hosts, while adapting to changing environments via a stage specific program of gene expression that assists the survival of Leishmania under the changing conditions. Translation initiation plays a key role in control of gene expression, in Leishmania this is exemplified by the presence of multiple cap-binding complexes that interact with mRNAs. The parasites encode multiple paralogs of the cap-binding translation initiation factor eIF4E, and of its corresponding binding partner eIF4G, forming complexes with different potential functions. Using the CRISPR-Cas9 methodology, we generated a heterozygous mutant of the least studied cap-binding paralog, LeishIF4E2, eliminating one of its two alleles. The mutation caused morphological defects, resulting in short and rounded up cells with a significant reduction in their flagellar length. Although general translation rates and growth of the mutant parasite were not affected, total proteome analysis and translation assay suggested that LeishIF4E2 could possibly function as a translation repressor. Alternatively, LeishIF4E-2 could be responsible for promoting translation of a specific set of transcripts.

microbiology↗