Investigating the Presence of R-loops at the Centromeres of Drosophila melanogaster

This study investigates the presence and localization of R-loops at the centromeres of Drosophila melanogaster. R-loops are non-canonical nucleic acid structures that have been implicated in various cellular processes, including genomic instability and accurate chromosome segregation. Although previous research has found R-loops at Drosophila satellite sequences and LTR retrotransposons, their presence and function at the centromeres of a whole organism remain largely unknown. Using IF-FISH (immunofluorescence-fluorescence in situ hybridization), we stained R-loops with the S9.6 antibody. A UAS-rnh1 overexpression line was used to induce the overexpression of RNase H1, an enzyme that resolves R-loops, to confirm the specificity of the S9.6 antibody signal. Our results showed that while inducing rnh1 overexpression did cause a decrease in the frequency of colocalization between S9.6 and CENP-C at all centromeres, this change was only statistically significant for centromere X. Similarly, the decrease in S9.6 and rDNA colocalization was only statistically significant for the X chromosome. The S9.6 signal intensity did not significantly change after induction of rnh1 overexpression. These findings prevent any definitive conclusions regarding the presence of R-loops at Drosophila centromeres.