Characterization of the Symbiodinium microadriaticum Spliced Leader RNA and its Response to Stress

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Feltman, Peter
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Spliced leader (SL) mediated RNA trans-splicing has been identified in diverse dinoflagellate species, including the coral symbiont, Symbiodinium microadriaticum. During the process of SL trans-splicing, first described in trypanosomes, RNA messages are trans-spliced with an identical, short leader sequence, donated from a small nuclear-encoded RNA known as the SL RNA. Under conditions of severe stress, trypanosome cells shut off transcription of the SL RNA gene, a response termed spliced leader silencing (SLS), resulting in a cellular decrease in mRNA maturation and translation. This study sought to characterize the Symbiodinium SL RNA and determine its response to cellular stress. Our results revealed a non-polyadenylated RNA with numerous characteristics in common with other published dinoflagellate SL RNAs including sequence identity, small size, and a divergent secondary structure. Utilizing this sequence information, a qPCR-based assay was developed to measure the abundance of SL RNA in response to cellular perturbation. This assay was found to be responsive to transcription inhibition by treatment with actinomycin D. Cultures were assayed for SLS following incubation at 34° C, a temperature that causes extreme physiological impairment, and treatment with 4 mM DTT, a known cause of SLS in trypanosomes. SL RNA levels were unaffected by either treatment, suggesting that SLS is not a Symbiodinium stress response. To determine if the trans-splicing machinery was affected by either treatment, independent of SLS; unspliced, immature messages were assayed for by northern blot. No evidence was found for a reduction in trans-splicing in response to either heat shock or DTT treatment. These results, combined with the uncertain origins of the SL trans-splicing mechanism, support the emerging view that functions of SL trans-splicing may differ among the diverse eukaryotic lineages in which it exists.
Thesis (M.S.) College of Charleston, South Carolina-The Graduate School, 2012
Committee members: Frances Van Dolah, Christine Byrum, Giacomo DiTullio, Cheryl Woodley, Amy T McCandless
coral symbiont, dinoflagellate, heat stress, spliced leader, spliced leader silencing, trans-splicing
Biology, Microbiology, Molecular biology