Title : Proper control of R-loop homeostasis is required for maintenance of gene expression and 1 neuronal function during aging 2 Running title : R-loop dysregulation in the aging eye

25 Age-related loss of cellular function and increased cell death are characteristic hallmarks of 26 aging. While defects in gene expression and RNA metabolism have been linked with age27 associated human neuropathies, it is not clear how the changes that occur in aging neurons 28 contribute to loss of gene expression homeostasis. R-loops are RNA-DNA hybrids that typically 29 form co-transcriptionally via annealing of the nascent RNA to the template DNA strand, 30 displacing the non-template DNA strand. Dysregulation of R-loop homeostasis has been 31 associated with both transcriptional impairment and genome instability. Importantly, a growing 32 body of evidence links R-loop accumulation with cellular dysfunction, increased cell death and 33 chronic disease onset. Here, we characterized the R-loop landscape in aging Drosophila 34 melanogaster photoreceptor neurons and showed that bulk R-loop levels increased with age. 35 Further, genome-wide mapping of R-loops revealed that transcribed genes accumulated R36 loops over gene bodies during aging, which correlated with decreased expression of long and 37 highly expressed genes. Importantly, while photoreceptor-specific down-regulation of Top3β, a 38 DNA/RNA topoisomerase associated with R-loop resolution, lead to decreased visual function, 39 overexpression of Top3β or nuclear-localized RNase H1, which resolves R-loops, enhanced 40 positive light response during aging. Together, our studies highlight the functional link between 41 dysregulation of R-loop homeostasis, gene expression and visual function during aging. 42