Regulated mRNA recruitment in dinoflagellates is reflected in hyper-variable mRNA spliced leaders and novel eIF4Es

Abstract

Dinoflagellates are eukaryotic algae with large genomes that rely heavily on post-transcriptional control for the regulation of gene expression. Dinoflagellate mRNAs are trans -spliced with a conserved 22 base spliced leader sequence (SL) that includes the 5’-cap to which the translation initiation factor 4E (eIF4E) binds to facilitate ribosomal recruitment. The binding of an eIF4E to a specific mRNA SL is a potential regulatory point in controlling dinoflagellate gene expression. Here we show that m 7 G is the 5’-cap base of the 65 bp SL RNA with additional methylations throughout the SL to give a mixture of novel multi-methylated sequences in Amphidinium carterae (CCMP1314). There is also sequence variability in all four bases seen at the first position followed by a variety of polymorphisms. Three novel clades of eIF4E have been shown in dinoflagellates that are distinct from the three metazoan classes of eIF4E. Members of each clade differ significantly from each other, but all bear the distinctive features of a cap-binding protein. Here we show large differences in expression and activity in six of the eight eIF4E family members from A. carterae . Transcripts of each are expressed throughout the diel cycle, but only eIF4E-1 family members and eIF4E-2a show discernable expression at the level of protein. Recombinant eIF4E-1 family members and eIF4E-3a, but not eIF4E-2a, are able to bind to m 7 GTP substrates in vitro . Overall, eIF4E-1a emerges with characteristics consistent with the role of a prototypical initiation factor; eIF4E-1a is the most conserved and highly expressed eIF4E family member, has the highest affinity for m 7 GpppG and m 7 GpppC by surface plasmon resonance, and is able to complement a yeast strain conditionally deficient in eIF4E. The large number of eIF4E family members along with the sequence and methylation state variability in the mRNA SLs underscore the unique nature of the translational machinery in the dinoflagellate lineage and suggest a wide range of possibilities for differential recruitment of mRNAs to the translation machinery. In the dinoflagellate, A. carterae , hyper-variable mRNA spliced leaders and novel eIF4Es reflect the reliance of dinoflagellates on variable mRNA recruitment for the regulation of gene expression .