Discovery, Phylogenetic Analysis, and Functional Characterization of a Unique Family of Eukaryotic Translation Initiation Factor 4E, eIF4E, From Amphidinium carterae, a Marine Dinoflagellate

Abstract

This study investigates the eIF4E family members in Dinoflagellates. Dinoflagellates are eukaryotic algae with large genomes and a minimal role for transcriptional regulation. All mRNA in dinoflagellates is trans-spliced with a 22-nucleotide 5'-spliced-leader sequence bearing a multi-methylated cap. Like other eukaryotes, dinoflagellates encode multiple eIF4E family members that are anticipated to fulfill a range of functions. Three distinct and novel clades of eIF4E have been recognized in dinoflagellates that are separate from the three metazoan classes of eIF4E. The dinoflagellate Amphidinium carterae encodes eight eIF4E family members while Karlodinium veneficum encodes fifteen eIF4E family members. I assayed six of these family members from A. carterae for expression levels, m7GTP binding, yeast knockout complementation and affinity for three mRNA cap analogs using surface plasmon resonance (SPR). Transcripts of each are expressed through a diel cycle, but only eIF4E-1 family members and eIF4E-2a are expressed at the level of protein. Recombinant eIF4E-1 family members and eIF4E-3a, but not eIF4E-2a, are able to bind to m7GTP-agarose beads. Of the clade 1 eIF4Es, only eIF4E-1a and -1d1 complement a S. cerevisiae strain conditionally deficient in functional eIF4E, consistent with their function as translation initiation factors. However, only eIF4E-1a can be recovered from A. carterae extracts by m7GTP affinity binding. Using SPR analysis, the affinity of A. carterae eIF4E-1a for m7GTP is lower than that of murine eIF4E-1A. By the same analysis, A. carterae eIF4E-1a has a higher affinity for m7GpppG than m7GTP. In addition, K. veneficum eIF4E-1a1 displays many of the same characteristics as A. carterae eIF4E-1a. Four eIF4E-1 and one eIF4E-2 family members from K. veneficum were characterized for m7GTP binding capacity, only the eIF4E-1 family members can be pulled down with m7GTP. Three eIF4E family members were tested for their ability to interact with a putative eIF4E-interacting protein, although none interacted. Overall, the eIF4E-1a sub-clade emerges with characteristics consistent with the role of a prototypical translation initiation factor. These initial analyses will allow for a better understanding of specific translational control of gene expression through mRNA recruitment in the unique dinoflagellate lineage.