Biosynthetically tagged rRNAs are restricted to recently born neurons.
We previously found that EU feeding results in more robust RNA tagging than EC feeding [20] and therefore sought to test if this approach might identify neuronal rRNA synthesis that is below the detection limit for EC-tagging. We initially attempted short EU feedings and found that feeding for a minimum of four hours was necessary to reliably detect tagged rRNA. This constraint likely reflects the time it takes for ingested EU to accumulate within the nucleoside pool of brain cells and for a visible threshold of tagged rRNA molecules to be produced. Following four hours of EU feeding, tagged rRNA was consistently detected in neuroblasts and differentiated progenitors (INPs and GMCs). In these experiments, tagged rRNA was never detected in neurons (Figure 2A and B ).
The absence of tagged rRNA in neurons could be due to a low rate of rRNA synthesis and a signal below the limit of detection. We therefore tested longer EU feeding periods of 6 and 24 hours. After 6 hours, we detected tagged rRNA in neurons but only in a small number of recently born neurons (located near GMCs) and never mature neurons located further from progenitors (Figures 2B and 3C ). After 24 hours the number of neurons containing tagged rRNA increased but the signal was still restricted to recently born neurons (Figures 2C and D ). One interpretation of these results is that recently born neurons have a low rate of rRNA synthesis, revealed only by the longer labeling periods, while mature neurons produce little or no rRNA and tagged RNAs remain below the detection limit. Another possibility is that a period of 6 hours allows time for GMC divisions to generate neurons containing inherited rRNA. The average GMC cell cycle is 4.2 hours [27]. Given this timing and the lag between initiation of EU feeding and detection of tagged rRNA, very few GMCs are expected to divide and pass tagged rRNA to neuronal progeny during a 4-hour feeding. However, during a 6-hour feeding and even more so a 24-hour feeding, there is time for multiple GMCs to divide and generate neurons with inherited rRNA. The results of these experiments do not rule out either explanation (low rate of rRNA synthesis or rRNA inheritance) and both mechanisms may contribute to the rRNA population in neurons. Neuron-specific measurement of pre-rRNA by EC-tagging and RT-qPCR (Figure 1B ) already revealed that some low level rRNA transcription occurs in neurons. We next sought to investigate the possibility that rRNA synthesized in progenitors is passed to neurons.