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.