Date of Award

8-2014

Degree Type

Masters Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

Committee Chair

Timothy McLean

Committee Chair Department

Biological Sciences

Committee Member 2

Glenmore Shearer

Committee Member 2 Department

Biological Sciences

Committee Member 3

Chaoyang Zhang

Committee Member 3 Department

Computing

Abstract

Karenia brevis is a mixotrophic, marine dinoflagellate found in the Gulf of Mexico that generates periodic, if not annual, harmful algal blooms (also known as “red tides”) in certain coastal areas. In an effort to better understand the biology of this organism, a functional genomics project has been initiated. As part of that project, it has been determined that a significant number of natural antisense transcripts (NATs) as well as double-stranded RNA (dsRNA) molecules exist within the transcriptome of K. brevis. I hypothesize that the non-coding NATs, similar to microRNAs (miRNAs) in other organisms play a role in regulating gene expression. To test this prediction, I extracted total RNA from cells grown under different culture conditions, isolated and cloned the dsRNAs and miRNAs separately, and sequenced all transcripts from each sample. Bioinformatic analyses were used to assess the relative expression of miRNAs, NATs, and mRNAs. My determination of any differential expression between day and night conditions should either support or falsify the hypothesis of NATs and/or miRNAs regulating the expression of genes via a post-transcriptional mechanism. The miRNA analysis revealed many mature miRNA candidates, but visualization software suggests that the miRNA pathway may not be present in the K. brevis genome. Also, length distribution of the miRNA samples suggests that the small RNAs are too long to be bound be the Argonaute protein, which is a key factor in miRNA synthesis. Cleavage patterns, transcript shape and read alignment patterns resemble a cis-Nat pathway, although it is undetermined whether this leads to siRNAs or an alternate small RNA. The RNA-seq analysis discovered that a large number of transcripts exhibited differential expression between the two time points of the diel cycle.

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