Date of Award
Honors College Thesis
Alex S. Flynt, Ph.D.
MicroRNAs are a heterogeneous group of small regulatory RNAs generated by many pathways. Mirtrons (miR) are a class of microRNAs produced by splicing, and some mirtrons contain a 3’ tail located downstream from the self-complementary hairpin. During RNA splicing, a loop-like “lariat” intermediate structure is created when the 5’ end of the RNA is attached to an adenine called the branch point. The goal of this project is to uncover the contribution of branch point location to the processing of tailed mirtrons into functional gene regulators. This project approaches this issue from two directions. First, branch points were identified by generating all possible lariat signatures from specific intronic samples using LaSSO and aligning the signatures to a genome-wide Drosophila RNA-sequence library using Bowtie2. Second, a polymerase chain reaction (PCR) based branch point mapping strategy was used to experimentally isolate, mutate, and verify the branch point location of miR-1017. Additionally, a synthetic miR-1017 mutant was cloned without any adenine nucleotides in the 3’ tail. We examined the mutation’s effects on Drosophila S2 cells with transfection and Northern analysis. Northern analysis showed that the mutated branch point actually increased the production of mature mirtrons and removing the branch point abrogated expression entirely, indicating that mirtron maturation may be very dependent on branch point and its location.
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Strickland, Britton A., "Functional Significance of Branch Points in Mirtrons" (2016). Honors Theses. 440.