Date of Award

5-2020

Degree Type

Honors College Thesis

Department

Biological Sciences

First Advisor

Alex Flynt, Ph.D.

Advisor Department

Biological Sciences

Abstract

MicroRNAs are 22 nucleotide, non-coding RNAs that serve as substrates for Argonaute proteins to induce RNA interference pathways. Intron-derived miRNA precursors called “mirtrons” have been identified and classified primarily through deep sequencing methods. Unlike most miRNAs, mirtrons are derived from splicing events and also exhibit high levels of post-transcriptional nucleotide addition to hairpin precursors. Most relevant among these modifications is 3’ uridylation as it inhibits mirtron biogenesis in multiple model systems. Mirtrons may also possess additional nucleotides adjacent to the pre-miRNA hairpin at the 3’ and/or 5’ ends. These nucleotide “tails” are removed prior to Dicer cleaving the hairpin. In 2010, Flynt et al. reported that tail trimming activity in the highly conserved 3’-tailed mirtron dme-miR-1017 was performed by the RNA exosome. The insertion of a tract of guanines within the tail would prevent trimming if the activity is performed by an exoribonuclease. To determine if tail removal in 5’-tailed mirtrons is also exosome-mediated, three mutant constructs including a 12 G insert, 20-bp splice site insert and a splice site mutant (negative control) were derived from hsa-miR-5010 and used for comparing target repression levels and base modification. We employed cell culture, transfection, luciferase assay and sequence analysis to determine the effect of differential tail elements on miRNA processing and activity. Our findings confirm selective uridylation at the 3’ end for all miR-5010 variants. The Poly G mutant had greater activity and stability compared to both the wild-type and the splice site mutant. These data imply that 5’-tailed mirtron tail removal is mediated by an endoribonuclease unlike the 3’-tailed variants.

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