Title: "Dinoflagellates - Breaking All the Eukaryotic Paradigms"

Speaker: Dr. Allen Place, UMCES-IMET

Abstract:

One of the tenets for life on earth is that it obeys Chargaff's rules which states that DNA from any cell of all organisms should have a 1:1 ratio (base Pair Rule) of pyrimidine and purine bases and, more specifically, that the amount of guanine should be equal to cytosine and the amount of adenine should be equal to thymine. We have known since the 70’s that a major group of primitive eukaryotes, the dinoflagellates, uniformly break this rule. On average for all core dinoflagellate genera, 40.4±13.1 % of thymine bases are modified to 5-hydroxymethyluracil in genomic DNA. This has resulted in single cell large genomes (~5.9 pg/cell and greater) without nucleosomes. The chromosomes are condensed throughout the cell cycle, which requires a very different approach to transcription and cell division. I will review the literature on this “Fifth Base” of life, how it impacts chromatin structure and transcription, how it might be synthesized in dinoflagellates and the role it may play i in dinoflagellates.

Secondly, I will present our work on trans-splicing in which dinoflagellates add a 22 base sequence to the 5′ end of an RNA transcripts. Using RNA that had been size separated into two pools with a 200 base cutoff, a biotinylated oligo complementary to the spliced leader sequence was used to pull out mature spliced mRNAs as well as immature splice leader donor RNAs. The samples enriched for spliced leader sequences were then subjected to RNAse digestion with either a T2 RNAse, which cannot cleave RNA with a methylated ribose, or RNAse A, which preferentially cleaves single stranded RNA. Size separation following T2 digestion shows multiple bands including the full 22 bases as well as 15, 11, and 6 base lengths. This demonstrates that there are modified bases within the 5′ spliced region as well as the possibility of a “family” of spliced leaders which has previously been thought of as ubiquitous. Compositional analysis of the resultant 22 base product following RNAse A degradation shows an enrichment of A, C and G containing methylated riboses as well as pseudouridine in the spliced leader. Relative to the terminal base, a 7-methylguanosine, the total number of modified As, Cs, and Gs appear to be 4-6 times as high with pseudouridine at about twice the the abundance and the only modification of uridine detected. This would indicate a much more extensive number of methylations and base modifications than the trypanosome cap four structure, the most modified structure described to date. These methylations of the cap residues would have an effect on the folding of the spliced leader and may be involved in protein interactions involved in regulating gene expression. These results provide some hypotheses on how dinoflagellates can regulate gene expression without promoters and may lead to biotech innovations for gene expression systems that are controllable post-transciptionally.