rRNA inheritance in neurons is sufficient for neurodevelopment and protein synthesis.
We next asked if inhibition of rRNA synthesis would differentially affect neural progenitors and neurons. To achieve cell type-specific inhibition of rRNA synthesis, we used a previously characterized RNA interference line [7] to knockdown RNA polymerase I subunit B(Polr1B ) in progenitors (insc-Gal4 x UAS-Polr1B{RNAi} ) or neurons (nSyb-Gal4 x UAS- Polr1B{RNAi}) . Polr1Bknockdown using insc-Gal4 limits rRNA inheritance in neurons but does not affect rRNA synthesis in neurons (Figure 4A ).Polr1B knockdown using nSyb-Gal4 limits rRNA synthesis in neurons but does not affect rRNA inheritance (Figure 4A ). As previously described for a RNAi screen in neuroblasts [23], we found that Polr1B knockdown driven by insc-Gal4 limits neurogenesis and causes nearly 100% failure of adult flies to eclose from their pupal case (Figure 4B ). Insc-Gal4 x UAS-Polr1B{RNAi} flies manually dissected from their pupal case were alive but unable to walk or fly, indicative of neurologic defects. In contrast, Polr1B knockdown in neurons had no effect on development: flies eclosed 100% of the time (Figure 4B ) and exhibited normal motor function.
Next we tested if inherited rRNA is sufficient to support bulk protein synthesis in neurons. We used a fluorophore-“clickable” amino acid analog (homopropargylglycine (HPG)) [31] to quantify protein synthesis in neurons of wildtype, insc-Gal4 x UAS- Polr1B{RNAi}, and nSyb-Gal4 x UAS- Polr1B{RNAi} flies. Dissected brains were soaked in HPG for three hours to allow sufficient labeling while limiting the likelihood of proteins passing from GMCs to neurons at cytokinesis (based on the 4.2-hour average GMC cell cycle). As previously shown [23], Polr1B knockdown usinginsc-Gal4 decreases progenitor proliferation, resulting in smaller brain size, but neurons are still produced (Figure 4C ). These neurons, with intact autonomous rRNA synthesis but limited rRNA inheritance, had significantly reduced levels of protein synthesis (Figures 4C and D ). Neurons with intact rRNA inheritance but limited autonomous rRNA synthesis had wildtype levels of protein synthesis (Figures 4C and D ). We interpret these data as evidence that inherited rRNAs significantly contribute to protein synthesis in neurons.