Translin and its partner Trax (Translin-associated factor X) are highly conserved proteins and they have been shown to have a functional relationship in a range of biological processes including tRNA processing, degradation of microRNAs during oncogenesis, mRNA regulation in spermatogenesis, neuronal function and telomere transcript regulation. Translin was first identified in humans as a protein that binds to chromosomal translocation breakpoint junctions in lymphoid malignancies, although it remains unknown if there is a direct requirement for one, or both of these proteins in the DNA damage response and/or chromosomal translocation formation. This led us to ask whether Translin and/or Trax have any role in genome stability regulation. In the current study, the biological function(s) of Translin and Trax were investigated further using the facile fission yeast (Schizosaccharomyces pombe) model system (Tsn1 = Translin; Tfx1 = Trax). Previously, analysis of null mutants of tsn1 and tfx1 did not reveal a genome instability or DNA damage recovery phenotype. Recently, a hetero dimeric complex that influences the removal of passenger strands in the RNA interference (RNAi) pathway, termed C3PO, has been found to consist of Translin and Trax. In addition, the Dcr1 RNAi regulator was shown to have RNAi-independent functions in controlling genome stability via regulating RNA:DNA hybrid levels within the genome of S. pombe. Give these findings, we generated mutants defective in both Dcr1 and Tsn1 and/or Tfx1 to determine whether Tsn1 and/or Tfx1 played a redundant role in genome stability control in the absence of Dcr1. We reveal that in the absence of Dcr1, Tsn1, but not Tfx1, is required for recovery from some types of DNA damage, but not all. By analysing this response in cells without canonical telomeres, we extend this to demonstrate that this is not associated with the alterations in telomeric TERRA transcripts which have elevated levels in tsn1D cells. Furthermore, by employing an inter-molecular genetic recombination assay we demonstrate that Tsn1, but not Tfx1, is required for replication- associated recombination in a polar fashion suggestive of an association with recombination stimulated by replicative pauses caused by RNA polymerase II, not RNA polymerase III, transcription at a tRNA gene. These data led us to reveal that Tsn1 functions in an RNAse H pathway, but we could find no evidence for a direct role in
DNA double-strand break repair. Collectively, our data reveal a function for Tsn1, but not Tfx1, for prevention of genome instability associated with the replicative stresses caused by genomic RNA:DNA hybrids.