Aim Evidences of mitochondrially driven RNA interference (RNAi) are recently emerging. Recently, we suggested a new class of small non coding RNAs (sncRNAs) of mitochondrial origin called smithRNAs (Small MITochondrial Highly-transcribed RNAs) in the Manila clam, Ruditapes philippinarum, a bivalve species with Doubly Uniparental Inheritance (DUI) of mitochondrial DNA. Among other possible functions, those smithRNAs were predicted to regulate nuclear gene expression in R. philippinarum gonad formation, a possibility which has never been suggested before. However, all evidences for smithRNAs were in silico predictions, up to now.

Methods We experimentally tested for significant modifications in methylation/acetylation levels in specimens treated with smithRNAs predicted to target nuclear enzymes connected with such regulatory functions.

Results SmithRNAs are conserved in the Manila clam and we present significant clues of their functionality in vivo. Moreover, it is conceivable that smithRNAs will be found in other eukaryotes as well, maybe linked to functions other than gonad development: in fact, we present evidences that putative smithRNA genes are present in other metazoansʼ mtDNAs.

Main conclusion SmithRNAs are good candidates to evolve new functions in the compact mtDNA, because hairpin structures in mitochondrial intergenic regions (needed for correct RNA cleavage) could easily be exapted to evolve RNAi. SmithRNAs make mtDNA a much more complex genome than previously thought, and they represent a new mito-nuclear coevolutionary arena. Actually, the possibility that mtDNA may act as a reservoir of RNAi opens a plethora of new ways for it to interact with the nucleus and significantly raises the level of complexity of mito-nuclear coevolution: for instance, smithRNAs may play a role in reproductive isolation between closely related species.