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Date of Award

Spring 2024

Document Type

Masters Thesis

Department or Program Affiliation


Degree Name

Master of Science (MS)



First Advisor

Lee, Suzanne R.

Second Advisor

Galati, Nick

Third Advisor

Pollard, Dan A.


RNA interference (RNAi) is a highly conserved cellular mechanism that acts canonically to contribute to centromere organization and to repress gene expression and mobile elements. However, recent literature has shown many different examples of RNAi facilitated activities acting directly or indirectly in the DNA damage response in eukaryotic cells. A ~23-24 nucleotide (nt) small (s)RNA producing RNAi pathway that maintains genome integrity has been discovered by the Lee Lab in Tetrahymena thermophila, a single-celled ciliate. In this previous work, it was found that the absence of RDRC (RNA-dependent RNA polymerase complex) factor Rdn2, RDRC-associated RNA-silencing protein 1 (Rsp1), or Twi protein Twi8 resulted in elevated levels of Rad51, a DNA repair protein, and γH2A.X, a DNA breakage marker, in the nucleus.

The overall purpose of this study was to build on previous work to identify if additional RNAi factors contribute to genome integrity in T. thermophila. This was largely accomplished by generating multiple RNAi gene knockout strains and analyzing their nuclear Rad51 elevation compared to the parental strain SB210. The first goal was to determine whether other RDRCs in addition to the Rdn2-RDRC are needed to maintain genome integrity. Our results described here suggest that the Rdn2-RDRC is in fact the most critical of three RDRCs for genome integrity maintenance. We also observed that Rsp1 has an additional RDRC-independent role in this maintenance. The second goal was to determine whether other Twi proteins in addition to Twi8 are necessary for genome integrity. Our data shows that Twi2, and possibly additional Twis in the TWI2-6 genomic cluster, are critical for suppressing a DNA damage phenotype.




RNA interference, RNAi, Rad51, DNA damage, PIWI, RdRP, sRNA


Western Washington University

OCLC Number


Subject – LCSH

Tetrahymena; RNA interference; DNA ligases; DNA repair; DNA damage; RNA polymerases; Small interfering RNA; Non-coding RNA




masters theses




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Available for download on Friday, May 23, 2025

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