Friday, April 19, 2024
4/19/2024
Project Summary Post-transcriptional control of mRNAs plays a key role in regulating important developmental events via RNA binding proteins (RBPs) that modulate key aspects of RNA fate, including splicing, stability, and translation. Developmental roles of RBPs are particularly evident in the nervous system, where their loss is linked to an array of diseases. Understanding how individual RBPs contribute to tissue-specific developmental events is complicated by their generally ubiquitous expression and by the insufficiency of consensus RBP-binding sites to predict in vivo occupancy or regulation. These observations imply as yet undefined mechanisms that elevate requirements for RBPs in specific cell types. We co-discovered an inherited neurodevelopmental disease caused by loss of the ubiquitously expressed, zinc finger RBP ZC3H14 and have probed function of this protein in multiple model organisms. The ZC3H14 ortholog in D. melanogaster, Nab2 (nuclear polyadenylated RNA binding protein 2), provides a tractable genetic model to probe molecular and neurodevelopmental roles. Importantly, ZC3H14/Nab2 is expressed in all cells but required specifically in neurons of the developing nervous system to support viability, brain axon guidance, locomotion, and olfactory memory. Critically, human ZC3H14 can substitute for Nab2 in fly neurons, indicating that the two orthologs share molecular roles and target RNAs. However, the identity of ZC3H14/Nab2-regulated RNAs, and mechanisms that elevate the ZC3H14/Nab2 role in neurons, are gaps that limit understanding of how such RBPs play key roles in neurons. Our recent work exploiting Drosophila provides a foundation to fill these gaps by identifying Nab2 as a novel inhibitor of N6- methyladenosine (m6A) on a subset of mRNAs in the developing nervous system. m6A is deposited at specific sites within mRNAs by the m6A ‘writer’ Mettl3. m6A is enriched in the developing brain, where it plays key roles in neurodevelopment. Our analysis of one newly identified Nab2 neuronal target, the pre-mRNA encoding the Sex Lethal (Sxl) sex determination factor, suggests that Nab2 promotes a key developmentally regulated exon- skipping event in Sxl pre-mRNA by binding an A-rich intronic element and inhibiting m6A deposition by Mettl3. Critically, Nab2-regulation of this splicing event is neuron-specific and rescue of multiple Nab2 phenotypes by concurrent Mettl3 loss implies that the Nab2-m6A hypermethylation model extends to additional RNA targets. We hypothesize that the neurodevelopmental requirement for Nab2/ZC3H14 is based on regulating on a set of mRNAs encoding key neuronal proteins, at least in part, via this novel m6A inhibitory role. We pursue this mechanistic hypothesis in three aims: Aim 1) Exploit the Sxl pre-mRNA as a model to dissect Nab2 inhibition of m6A deposition; Aim 2) Use a Sxl splicing reporter to parse direct and direct effects of Nab2 and m6A-regulation of splicing and identify additional factors in the Nab2-m6A pathway; and Aim 3) Identify key neurodevelopmental targets of Nab2/ZC3H14 in both flies and in vertebrates. Taken together, these approaches will provide insight into why loss of a ubiquitously expressed RNA binding protein causes morphogenesis defects in the brain.
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