Analysis of eIF4E-family members in fungi contributes to their classification in eukaryotes

Abstract

The kingdom of fungi contains highly diverse species. However, fundamental processes sustaining life such as RNA metabolism are much less comparatively studied in Fungi than in other kingdoms. A key factor in the regulation of mRNA expression is the cap-binding protein eIF4E, which plays roles in mRNA nuclear export, storage, and translation. The advent of massive genomics has unveiled a constellation of eIF4E-family members across eukaryotes. However, how this protein diverged into fungal species remains largely unexplored. Here, we studied the genome of 538 species from six evolutionarily distant phyla and retrieved 1462 eIF4E cognates. The analyzed species contained 1 to 7 paralogs. We sorted all cognates in six phylogenetically coherent clades, that we termed Class I to VII (mammalian Class III was absent in Fungi). Proteins from Classes IV to VII did not match the current eIF4Es classification that is based on variations in the residues equivalent to W43 and W56 of the human protein. eIF4Es from other eukaryotes do not fit into this classification either. Thus, we have updated the eIF4E categorization based on cladistics and the presence of cap-binding amino acids to better fit eIF4E's diversity across eukaryotes. Finally, we predicted the structure of the global protein and the cap-binding pocket and experimentally tested the ability to rescue the lack of endogenous eIF4E in Saccharomyces cerevisiae of representative members of each of the six classes of fungal eIF4E.

Keywords: RNA metabolism; eIF4E; fungi; gene expression; mRNA; translation initiation.

Figure 1

Cladogram showing the phylogenetic relationship of fungal eIF4Es. Full-length sequences of selected eIF4E-family members from six phyla were analyzed by the Neighbor-Joining method. The proteins are distributed in six clades of eIF4E. The Fungi-specific classes are indicated with a F prefix. Phylum name is indicated in parenthesis: (As), Ascomycota; (Ro), Rozellomycota; (Ba), Basidiomycota; (Ch), Chytridiomycota; (Zo), Zoopagomycota; (Mu), Mucoromycota.

Figure 2

Scheme representing the core of prototypical eIF4E and the classes of fungal eIF4Es. Class I eIF4E from metazoan, terrestrial plants and yeast is represented. Mammalian Class III and fungal-specific classes of eIF4E are indicated with the prefix M- and F-, respectively. Numbering is from human protein (43). Amino acid residues directly contacting the mRNA cap structure are indicated: π, residues binding the guanine by π – π interactions; G, residue recognizing the guanine ring; P, positively charged residues interacting with the phosphate groups; m, W recognizing the ⁷methyl group (5, 6, 7, 13). Conservative substitutions with respect to the prototypical proteins are in blue. Non-conservative changes are in red. An asterisk indicates W56 used to classify the eIF4E-family members into three classes (9). Stretches of amino acid insertions and extensions are depicted as black boxes (not to scale).

Figure 3

Three-dimensional prediction of eIF4E amino acids involved in cap recognition. eIF4E structures were predicted with Alphafold2. Only close-ups of the cap-binding pocket are shown. The cap is shown in orange and all amino acids involved in cap-interaction are highlighted in color-code. Yellow, pi-pi interaction; Green, guanine binding; Red, phosphate binding; Blue, ⁷methyl binding. A, S. cerevisiae, Class I (PDB structure 6FC1). B, Cryptococcus gattii, F-Subclass IA. C, Mitosporidium daphnia, F-Sublcass IB. D, Nematocida major, Class II. E, Punctularia strigosozonata, Class II. F, Uncinocarpus reesii, F-Class IV. G, Ascochyta rabiei, F-Class V. H, Malassezia globosa, F-Class VI. I, Gilbertella persicaria, F-Class VII. J, Gamisella multidivaricata, F-Class VII.

Figure 4

Fungal classes of eIF4E differentially rescue the lack of S. cerevisiae endogenous eIF4E. Phenotypic rescue was performed using eIF4E-HA from the Class I, S. cerevisiae (positive control); F-Subclass IA, Cryptococcus gattii; F-Subclass IB, Mitosporidium daphnia; Class II, Nematocida major (N. m.); Class II, Punctularia strigosozonata (P. s.); F-Class IV, Uncinocarpus reesii; F-Class V, Ascochyta rabiei; F-Class VI, Malassezia globosa; F-Class VII, Gilbertella persicaria (G. p.); and F-Class VII, Gamisella multidivaricata (G. m.). A, Growth control. B, Phenotypic rescue. S. cerevisiae eIF4E (positive control) and F-Class IB eIF4E showed a robust growth; F-Class IV eIF4E showed less but significant growth. S, yeast nitrogen base; —, no plasmid; A, adenine; L, leucine; H, histidine; U, uracil; 5-FOA; 5-fuoroorotic acid.