1. Department of Laboratory Medicine and Pathobiology, 2. Department of Molecular Genetics, and 3. Canada Research Chairs Program; Faculty of Medicine, University of Toronto, 1 King’s College Circle, Toronto, Ontario, M5S 1A8, CANADA, *Corresponding author.
Intergenic transcription, particularly within repetitive DNA loci, can be deleterious. Consistent with this notion, stability of the ribosomal DNA (rDNA) repeats of budding yeast requires processes that counteract RNA polymerase II (RNAPII) function at intergenic spacers within the repeats. These processes include Sir2-dependent chromatin silencing and early RNAPII termination. We report a process of rDNA repeat stabilization that relies on suppressing intergenic RNA:DNA hybrids. The yeast Ataxin-2 protein Pbp1 binds intergenic RNA, represses RNA:DNA hybrids, and prevents aberrant rDNA recombination/replication. Targeted elimination of RNA:DNA hybrids via various methods restores rDNA stability in Pbp1-deficient cells. In addition, Pbp1 suppresses hybrids at specific non-rDNA sites. Cells lacking Pbp1 have a short replicative lifespan that is extended upon RNA:DNA hybrid suppression. We also found that human Ataxin-2 performs functions similar to those uncovered herein for yeast Pbp1. Overall, our findings reveal intersecting roles for Pbp1/Ataxin2 and the extracellular environment in the suppression of nucleic acid structures threatening genome stability.