A novel mitochondrially driven RNA interference mechanism: discovery, evolution and regulatory effects of smithRNAs

Intergenomic communication is essential for eukaryotic life, particularly the dialogue between the nucleus and mitochondria. This thesis investigates a novel class of molecules, small mitochondrial highly-transcribed RNAs (smithRNAs), as a potential mechanism of cellular regulation. I explore their origins, predicted functions, and functional parallels within bacterium-host endosymbiotic systems. To address the lack of standardized methods, I developed and validated a robust bioinformatic workflow to identify candidate smithRNAs and their putative nuclear targets. The pipeline's efficacy was confirmed by reproducing the discovery of smithRNAs in bivalve species, providing a benchmark for broader application. I conducted the first large-scale comparative genomic analysis of smithRNAs across 14 metazoan species. This study suggests that smithRNAs are a widespread feature of Metazoa rather than a bivalve anomaly. In most animals, smithRNAs are predicted to originate preferentially from tRNA and rRNA genes, contrasting with the non-coding regions seen in bivalves. This aligns with the known involvement of tRNA fragments in signaling. Functional predictions for nuclear targets are lineage-specific, suggesting a dynamic, potentially rapidly evolving regulatory system that requires further experimental validation. Building on sRNA-mediated intergenomic control, I inspected analogues in the Wolbachia pipientis infection of Zyginidia pullula. My studies revealed that Wolbachia alters the host transcriptome, shifting male molecular profiles toward a female phenotype. I identified bacterial sRNAs originating from conserved non-coding regions and tRNA fragments, mirroring smithRNA biogenesis. This bacterial-derived RNAi is predicted to target host genes involved in spermatogenesis and immunity, suggesting a candidate molecular mechanism for Wolbachia's manipulation of host biology, pending functional testing. Collectively, these results provide evidence that sRNA-mediated signaling is a conserved candidate layer of biological regulation. While further lab testing is required to confirm these interactions, these molecules appear to be powerful potential intermediaries shaping symbiotic evolution and host phenotypes.