RNA-tagging reveals differential rRNA synthesis in neural progenitors and neurons.
We recently described a cell type-specific biosynthetic RNA tagging method called EC-tagging [20]. EC-tagging allows cell type-specific labeling of nascent RNAs via conversion of 5-ethynylcytosine (EC) to 5-ethynyluridine (EU) monophosphate in cells that express a cytidine deaminase – uracil phosphoribosyltransferase fusion (CD:UPRT). The labeled RNA can be subsequently interrogated using “click chemistry”. One possible application is visualization of RNA via attachment of an azide-coupled fluorophore. This approach has been used for general RNA imaging (not cell type-specific) via direct application of EU [21]. To test EC-tagging-based RNA imaging, we used the Gal4-UAS transgene expression system [22] to express CD:UPRT in progenitors or neurons. We used insc-Gal4 for progenitor tagging since this line expresses Gal4 in type I neuroblasts, type II neuroblasts, and INPs [23]. In addition, Gal4 and CD:UPRT activities likely perdure in GMCs of insc-Gal4 x UAS-CD:UPRT brains. We usednSyb-Gal4 for neuron tagging since this line expresses Gal4 only in neurons [24]. For all EC-tagging experiments, larvae were fed EC for 24 hours. We detected fluorescent RNA when tagging was targeted to neural progenitors but never detected fluorescent RNA when tagging was targeted to neurons (Figure 1A ).
Since related EU-labeling experiments have shown EU incorporation into rRNA and mRNA [21] and rRNA accounts for at least 80% of the RNA in eukaryotic cells, we predicted that the tagged RNA in Drosophilabrains is predominately rRNA. To test this prediction with an imaging-independent approach, we purified EC-tagged RNA from neuroblasts and neurons following a 24-hour EC feeding. Nascent rRNA was quantified by RT-qPCR using primers specific for precursor rRNA [26]. We also measured levels of a neuron-specific transcript, Synaptotagmin 1(Syt1 ). Syt1 was enriched by neuron-specific EC-tagging, confirming the cell type-specificity of the labeling (Figure 1B ). Similar to the EC-tagging RNA imaging results, pre-rRNA levels were highly reduced in neurons compared to neuroblasts (Figure 1B ).
To determine if the fluorescent signal detected by RNA tagging is primarily rRNA, mRNA, or both, we performed EU-based RNA tagging of dissected brains in the presence or absence of pharmacologic inhibitors of RNA polymerases: triptolide to inhibit RNA polymerase II (blocking mRNA synthesis) [25] and actinomycin D to inhibit RNA polymerase I and II (blocking rRNA and mRNA synthesis) [21]. Triptolide treated brains were indistinguishable from controls, while actinomycin D abolished the EU-tagged RNA signal (Figure 1C ). These results confirm that the tagged RNA is predominately ribosomal RNA. We also imaged EU-tagged RNA in combination with antibody staining for Udd (a nucleolus protein) [7] and PCNA (a marker of proliferating cells). As expected for rRNA, the EU-RNA signal is localized to the nucleolus and as predicted by our EC-tagging data, the signal is restricted to proliferating neuroblasts (Figure 1D ). Altogether, these EC-tagging and EU-tagging data suggest that rRNA synthesis is limited in neurons.