Disruption Of A Similar To 23-24 Nucleotide Small Rna Pathway Elevates Dna Damage Responses In Tetrahymena Thermophila

Endogenous RNA interference (RNAi) pathways regulate a wide range of cellular processes in diverse eukaryotes, yet in the ciliated eukaryote, Tetrahymena thermophila, the cellular purpose of RNAi pathways that generate similar to 23-24 nucleotide (nt) small (s)RNAs has remained unknown. Here, we investigated the phenotypic and gene expression impacts on vegetatively growing cells when genes involved in similar to 23-24 nt sRNA biogenesis are disrupted. We observed slower proliferation and increased expression of genes involved in DNA metabolism and chromosome organization and maintenance in sRNA biogenesis mutants RSP1 Delta, RDN2 Delta, and RDF2 Delta. In addition, RSP1 Delta and RDN2 Delta cells frequently exhibited enlarged chromatin extrusion bodies, which are nonnuclear, DNA-containing structures that may be akin to mammalian micronuclei. Expression of homologous recombination factor Rad51 was specifically elevated in RSP1 Delta and RDN2 Delta strains, with Rad51 and double-stranded DNA break marker gamma-H2A.X localized to discrete macronuclear foci. In addition, an increase in Rad51 and gamma-H2A.X foci was also found in knockouts of TWI8, a macronucleus-localized PIWI protein. Together, our findings suggest that an evolutionarily conserved role for RNAi pathways in maintaining genome integrity may be extended even to the early branching eukaryotic lineage that gave rise to Tetrahymena thermophila.