A Genetic Screen Links the Disease-Associated Nab2 RNA-Binding Protein to the Planar Cell Polarity Pathway inDrosophila melanogaster

Abstract

Running title: Nab2 interacts with planar cell polarity factors in Drosophila 34 35 ABSTRACT 46 Mutations in the gene encoding the ubiquitously expressed RNA-binding protein ZC3H14 result in a 47 non-syndromic form of autosomal recessive intellectual disability. Studies in Drosophila have defined 48 roles for the ZC3H14 ortholog, Nab2 (aka Drosophila Nab2 or dNab2), in axon guidance and memory 49 due in part to interaction with a second RNA-binding protein, the fly Fragile X homolog Fmr1, and 50 coregulation of shared Nab2-Fmr1 target mRNAs. Despite these advances, neurodevelopmental 51 pathways regulated by Nab2 remain poorly defined. Structural defects in Nab2 null brains resemble 52 defects observed upon disruption of the planar cell polarity (PCP) pathway, which regulates planar 53 orientation of static and motile cells. A kinked bristle phenotype in surviving Nab2 mutant adults 54 additionally suggests a defect in F-actin polymerization and bundling, which is also a PCP-regulated 55 processes. To test for Nab2-PCP genetic interactions, a collection of PCP loss-of-function alleles was 56 screened for modification of a rough-eye phenotype produced by Nab2 overexpression in the eye 57 (GMR-Nab2) and subsequently for modification of Nab2 null phenotypes. Multiple PCP alleles 58 dominantly modify GMR-Nab2 eye roughening and a subset of these alleles also rescue low survival 59 and thoracic bristle kinking in Nab2 zygotic nulls. Moreover, alleles of two X-linked PCP factors, 60 dishevelled (dsh) and β amyloid protein precursor-like (Appl), rescue GMR-Nab2 eye roughening in 61 male progeny derived from hemizygous dsh or Appl mutant fathers, suggesting an additional effect 62 inherited through the male germline. These findings demonstrate a consistent pattern of Nab2-PCP 63 genetic interactions that suggest molecular links between Nab2 and the PCP pathway in the developing 64 eye, wing and germline. 65 66 67 68 3 69 70 Mutations in genes that encode RNA-binding proteins often lead to tissue-specific disease pathology, 71 particularly within the brain and nervous system (reviewed in CASTELLO et al. 2013). Inactivating 72 mutations in the human ZC3H14 gene, which encodes a ubiquitously expressed zinc-finger, poly(A) 73 RNA-binding protein, are linked to a monogenic form of non-syndromic autosomal recessive 74 intellectual disability (reviewed in FASKEN et al. 2019). The ZC3H14 protein and its homologs in the 75 budding yeast S. cerevisiae and fruit fly Drosophila melanogaster (Nab2 in both species) interact with 76 A-rich motifs in RNAs and restrict the length of mRNA polyadenosine (poly(A)) tails. Phenotypes 77 associated with loss of Nab2/ZC3H14 are thus predicted to arise due to defects in post-transcriptional 78 control mechanisms that involve poly(A)-dependent mRNA processing and nuclear export, stability, 79 localization, and/or translation.80Studies in Drosophila indicate that Nab2/ZC3H14 share a conserved and necessary function in 81 brain neurons (PAK et al. 2011; KELLY et al. 2014). Neuron-specific Na...

The RNA-binding protein Nab2 regulates the proteome of the developing Drosophila brain

“…Nab2 is decreased relative to control in Nab2 ex3 brains (log 2 (-8.36)) and increased relative to J o u r n a l P r e -p r o o f control in Nab2 oe brains (log 2 (3.94)) (Figure 4B, Nab2 labeled data point). Finally, the Nab2 ex3 differentially expressed and Nab2 oe differentially expressed datasets each have unique proteins that may provide insight into previously observed phenotypes in Nab2 mutants or overexpression systems (15,17,20,28,38,39). There are 152 proteins changed exclusively in Nab2 ex3 brains relative to control, and 311 proteins changed exclusively in the Nab2 oe brains relative to control (Figure 4A).…”

“…As noted above, the planar cell polarity (PCP) component Vang and the Vang murine homolog Vang like-2 (Vangl2) are among a small group of proteins that are differentially expressed in both Drosophila Nab2 ex3 pupal brains and in P0 hippocampi dissected from Zc3h14 knockout mice (21) (Table 1). This finding is particularly significant given the strong genetic interactions detected between an eye-specific Nab2 overexpression system (GMR-Nab2) and multiple PCP alleles, including an allele of Vang (38). The PCP pathway plays a conserved role in regulating axon projection and guidance in multiple higher eukaryotic species (47)(48)(49)(50)(51), including in the Drosophila MBs (52)(53)(54)(55)(56).…”

The RNA binding protein Nab2 regulates the proteome of the developingDrosophilabrain

“…As noted above, the planar cell polarity (PCP) component Vang and its murine homolog Vang like-2 (Vangl2) are among a small group of homologous protein pairs that are differentially expressed in Drosophila Nab2 ex3 pupal brains and in P0 hippocampi dissected from Zc3h14 knockout mice (21) ( Table 1). This finding is particularly significant given the strong genetic interactions detected between an eye-specific Nab2 overexpression system (GMR-Nab2) and multiple PCP alleles, including an allele of vang (41). The PCP pathway plays a conserved role in regulating axon projection and guidance in multiple higher eukaryotic species (42)(43)(44)(45)(46), including in the Drosophila MBs (47)(48)(49)(50)(51).…”