Discussion
We set out to investigate the possibility that ribosomal rRNA inheritance is a major contributing factor to establishing neuron-specific translation capacity. Using cell type-specific EC-tagging and standard EU-tagging, we found that nascent rRNA synthesis is very limited in neurons. We also found that progenitor rRNA, most likely in pre-rRNA form, associates with mitotic chromosomes and is passed to progeny at cytokinesis. The functional importance of rRNA inheritance was revealed by RNAi experiments targeting RNA polymerase I in either progenitors or neurons. Knockdown of RNA polymerase I in neurons had no effect on neurodevelopment or protein synthesis while knockdown in progenitors caused a severe neurodevelopment defect and significantly reduced protein synthesis in neurons. Our results support a model in which neurons utilize inherited rRNA to meet their protein synthesis needs.
While we primarily focused on rRNA inheritance in neurons, our data also suggest that rRNA is passed from neuroblasts to INPs and GMCs. In 4-hour EU-labeling experiments, tagged rRNA is strongly detected in these differentiated progenitors and this signal may reflect both nascent and inherited rRNA. The average neuroblast cell cycle is about 1.4 hours [27] so multiple INPs and GMCs are produced during a 4-hour EU feeding. Since we could not reliably detect tagged RNA using shorter labeling times, it is unlikely that the strong signal in INPs and GMCs is solely derived from rRNA synthesized in neuroblasts during interphase (no rRNA synthesis occurs during mitosis [28]). Based on this relationship between labeling time and neuroblast cell cycle length, we predict that at least some nascent rRNA synthesis occurs in INPs and GMCs. This raises the question of how rRNA transcription is activated in progenitors that do not express Myc. One possibility is that sufficient rRNA polymerase I components, cofactors, and rRNA processing enzymes are inherited from the neuroblast. These inherited factors may support nucleolus assembly and rRNA synthesis in INPs and GMCs. Nucleolar proteins and pre-rRNA associate with mitotic chromosomes in mammalian cells and are thought to direct nucleolus formation in daughter cells [28]. A similar mechanism could direct nucleolus formation and activity in neuroblast progeny. In this model, the absence of Myc in GMCs would limit production of nucleolar factors and RNA polymerase I components so that little if any of the machinery necessary to trigger rRNA synthesis is inherited by neurons.
This work contributes to a growing body of evidence that the protein synthesis landscape of neurons is distinct from neural progenitors. While proliferating cells have a high demand for growth-supporting translation, the ribosomal properties of neurons might differ for reasons beyond their post-mitotic status. One possibility is that decreased ribosome concentration establishes neuron-specific translation programs. For example, a decrease in ribosome concentration can selectively limit translation of mRNAs whose translation is difficult to initiate [4]. Our findings may also relate to changes in ribosome requirements that occur throughout the lifetime of a neuron. Immature neurons require ribosomes in their axon growth cones to support pathfinding and synapse formation, but ribosomes are essentially absent from the axons of mature neurons. A ubiquitin-proteosome mechanism removes ribosomes from mature axon terminals [14] but this system could be overwhelmed if mature neurons produced large amounts of ribosomes. Inherited rRNA in newly born neurons may help establish ribosome levels appropriate for growth and synapse formation while weak rRNA synthesis in mature neurons may help ensure ribosomes do not accumulate outside of dendrites and the soma. Similar evidence of dynamic changes in rRNA levels comes from EU imaging in cultured hippocampal neurons and larval zebrafish brains [32]. This study identified a general decrease in RNA synthesis upon neuronal depolarization and a restriction of RNA synthesis to the neurogenic regions of the brain. Finally, reduced ribosome abundance may serve a protective function: lower levels of protein synthesis decrease the likelihood of generating misfolded proteins [1] that may be toxic to neurons.
Here we show that rRNA synthesis is restricted along the differentiation axis and that inherited rRNAs are sufficient for neurodevelopment and protein synthesis. Inheritance of ribosomal proteins remains to be investigated. We have previously shown that ribosomal protein mRNAs have decreased stability in neurons compared to somatic cells [33]. Ribosomal protein abundance in progenitors and neurons may also be regulated via mRNA decay and coordination of rRNA and ribosomal protein levels during differentiation is an important avenue for future studies. It will also be interesting to investigate differences in ribosome production among individual neuroblast lineages. A recent study on the effects of nucleolar stress found that neuroblasts that produce mushroom body neurons are less sensitive to nucleolar stress and have greater reserves of nucleolar proteins than other neuroblasts [34]. Studies of ribosome synthesis and inheritance in Drosophila will help identify conserved mechanisms of neural translation and may contribute to our understanding of ribosomopathies that cause multiple human diseases [35].