Bachvaroff, T. R. | bioRxiv Science

bioRxiv2024

The Nuclear and Mitochondrial Genomes of Amoebophrya sp. ex Karlodinium veneficum

Dinoflagellates are a diverse group of microplankton that include free-living, symbiotic, and parasitic species. Amoebophrya, a basal lineage of parasitic dinoflagellates, infects a variety of marine microorganisms, including harmful-bloom-forming algae. Although there are currently three published Amoebophrya genomes, this genus has considerable genomic diversity. We add to the growing genomic data for Amoebophrya with an annotated genome assembly for Amoebophrya sp. ex Karlodinium veneficum. This species appears to translate all three canonical stop codons contextually. Stop codons are present in the open reading frames of about half of the predicted gene models, including genes essential for cellular function. The in-frame stop codons are likely translated by suppressor tRNAs that were identified in the assembly. We also assembled the mitochondrial genome, which has remained elusive in the previous Amoebophrya genome assemblies. The mitochondrial genome assembly consists of many fragments with high sequence identity in the genes but low sequence identity in intergenic regions. Nuclear and mitochondrially-encoded proteins indicate that Amoebophrya sp. ex K. veneficum does not have a bipartite electron transport chain, unlike previously analyzed Amoebophrya species. This study highlights the importance of analyzing multiple genomes from highly diverse genera such as Amoebophrya. SummaryThis new long-read assembly demonstrates the remarkable diversity found within Amoebophrya. Despite being assigned the rank of genus, the available genome assemblies indicate significant variation in gene content, AT content, genetic codes, and potentially mitochondrial biology. Furthermore, this study contributes to the expanding list of organisms that contextually translate all three canonical stop codons. Although the mechanisms underlying such a genetic code remain elusive, the relative ease of culturing Amoebophrya suggests it may be useful as a model organism for future research on this subject.

genomics↗

bioRxiv2024

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

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 m7G 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 m7GTP 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 m7GpppG and m7GpppC 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. Impact StatementIn 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.

molecular biology↗