Cell RNA Structural Studies of Spliced Leader RNA Transcripts in the Red Tide Dinoflagellate Karenia brevis

Alissa Stranberg, Tina Batoh, Jackie Esquiaqui

Palm Beach Atlantic University

In the state of Florida, occurrences of red tide are caused by Karenia brevis (K. brevis) algal blooms that result in harmful neurotoxic effects to humans, marine life, and the economy. Little is known about molecular and cellular pathways involved in the production of neurotoxic brevetoxins, including an mRNA maturation mechanism called spliced leader (SL) trans-splicing, which splices a 5ʹ exon (the SL) from a small noncoding RNA (snRNA) to the 5ʹ end of pre-mRNA sequences of different genes, including those associated with toxin production. Details regarding how RNA structure and dynamics are impacted by addition of the SL to mRNA transcripts is even more limited and could contribute to a greater understanding of how brevetoxin-associated genes are effectively expressed. To study these cell RNAs, high-yielding extraction, selective enrichment, and visualization strategies need to be developed and optimized. In this work, live K. brevis cells were successfully cultured and total cell RNA was extracted for enrichment studies. Preliminary results show that total cell RNA can be extracted in yields high enough for visualization by denaturing polyacrylamide gels and in quantities sufficient for future mRNA transcript enrichment via RNA polyadenylation purification and capture assay experiments. Ultimately, these latter experiments will provide an approach to study mRNA structural dynamics of K. brevis SL mRNA transcripts that could provide important insight to understanding the critical steps of K. brevis life-cycle and toxin production at the molecular level.