Fig. 4.

BRCA1 BRCT enhances as-rRNA production to promote ds-rRNA formation. (A and B) Quantification of as-5.8S rRNA levels normalized to GAPDH mRNA in UWB1 cells with or without ectopic BRCA1 FL expression (A) and with or without ectopic WT or S1655A BRCT expression (B) by RT-qPCR. Y-axis represents fold difference relative to UWB1 cells transfected with vector alone. (C) Quantification of as-5.8S rRNA levels normalized to GAPDH mRNA in UWB1 cells with or without ectopic as-5.8S rRNA expression by RT-qPCR. Y-axis represents fold difference relative to UWB1 cells transfected with vector alone. (D and E) Intact 5.8S rRNA (D) and ACTB (E) post RNase A treatment and normalized to input RNA in UWB1 cells with or without ectopic as-5.8S rRNA expression were quantified by RT-qPCR. Y-axis represents fold difference relative to UWB1 cells transfected with vector alone. (F) Quantification of 5.8S rRNA levels normalized to GAPDH mRNA in UWB1 cells with or without ectopic as-5.8S rRNA expression by RT-qPCR. Y-axis represents fold difference relative to UWB1 cells transfected with vector alone. (G) Representative images showing immunofluorescent staining of R-loops (green) using S9.6 antibody in UWB1 cells with (Right) or without (Left) exogenously expressed as-5.8S rRNA. (H) Quantification of the number of nucleoli per ten cells showing positive nucleolar signals from samples shown in G. At least 150 cells were analyzed per sample. (I) DRIP analysis using S9.6 antibody to measure RNase H-sensitive R-loop levels at 5.8S rDNA loci normalized to the input DNA in UWB1 cells with or without exogenously expressed as-5.8S rRNA or as-5.8S mutant (mut) rRNA. Y-axis represents fold difference relative to the vector-transfected UWB1 cells. (J) DRIP analysis using S9.6 antibody to measure RNase H-sensitive R-loop levels at ACTB EX3 normalized to the input DNA in UWB1 cells with or without exogenously expressed as-5.8S rRNA. Y-axis represents fold difference relative to the vector-transfected UWB1 cells.