RNA Structural Studies of Wild-Type and Mutant RNA Spliced Leader Sequences of the Red Tide Dinoflagellate Karenia brevis

Danielle Garzon, LesleyMarie Rivera, Tina Batoh, Jackie Esquiaqui

Palm Beach Atlantic University

Florida Red Tide is a harmful algal bloom caused by the marine dinoflagellate Karenia brevis. The mRNA precursors of enzymes involved in brevetoxin synthesisundergo 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. The details of this mechanism, particularly how SL RNA structure and dynamics impact transcription and splicing, are limited and could provide important insight to understanding thecritical steps of K. brevis life-cycle and toxin production at the molecular level. To study the structure and dynamics of K. brevis SL RNA, molecular cloning of the wild-typeand a mutant version of the 22 nucleotide SL exon and the precursor 61 nucleotide exon-intron construct was conducted. These RNAs were successfully cloned, amplified, in vitro transcribed, and purified for structural studies to assess how environmental factors impact spliced leader trans-splicing events. Electrophoretic mobility shift assays have been successfully performed to test for the influence of various environmental factors and preliminary data suggests that salts impact SL RNA folded structures.