The Drosophila polyadenosine RNA binding protein Nab2, which is orthologous to a human protein lost in a form of inherited intellectual disability, controls adult locomotion, axon projection, ...dendritic arborization, and memory through a largely undefined set of target RNAs. Here, we show a specific role for Nab2 in regulating splicing of ~150 exons/introns in the head transcriptome and focus on retention of a male-specific exon in the sex determination factor Sex-lethal (Sxl) that is enriched in female neurons. Previous studies have revealed that this splicing event is regulated in females by N6-methyladenosine (m6A) modification by the Mettl3 complex. At a molecular level, Nab2 associates with Sxl pre-mRNA in neurons and limits Sxl m6A methylation at specific sites. In parallel, reducing expression of the Mettl3, Mettl3 complex components, or the m6A reader Ythdc1 rescues mutant phenotypes in Nab2 flies. Overall, these data identify Nab2 as an inhibitor of m6A methylation and imply significant overlap between Nab2 and Mettl3 regulated RNAs in neuronal tissue.
The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in ...neurons is undefined. Here, we present a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with the fragile X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory, and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII, but not futsch, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A) tail length, similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. Altogether, these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-specific defects in patients lacking ZC3H14.
Display omitted
•dNab2 is the fly ortholog of a human RBP lost in inherited intellectual disability•A cytoplasmic pool of dNab2 interacts with the fragile X homolog dFMRP•dNab2 regulates the CamKII mRNA and supports memory with dFMRP•dFMRP and dNab2 both restrict poly(A) length of neuronal mRNAs
Drosophila dNab2 is an ortholog of an RNA binding protein lost in an inherited intellectual disability, but its neuronal role is undefined. Bienkowski et al. present evidence that dNab2 interacts with the fly fragile X ortholog (dFMRP), a key regulator of neuronal mRNA translation, and co-regulates neurodevelopment and function with dFMRP.
The
polyadenosine RNA binding protein Nab2, which is orthologous to a human protein lost in a form of inherited intellectual disability, controls adult locomotion, axon projection, dendritic ...arborization, and memory through a largely undefined set of target RNAs. Here, we show a specific role for Nab2 in regulating splicing of ~150 exons/introns in the head transcriptome and focus on retention of a male-specific exon in the sex determination factor
(
) that is enriched in female neurons. Previous studies have revealed that this splicing event is regulated in females by N6-methyladenosine (m
A) modification by the Mettl3 complex. At a molecular level, Nab2 associates with
pre-mRNA in neurons and limits
m
A methylation at specific sites. In parallel, reducing expression of the Mettl3, Mettl3 complex components, or the m
A reader Ythdc1 rescues mutant phenotypes in
flies. Overall, these data identify Nab2 as an inhibitor of m
A methylation and imply significant overlap between Nab2 and Mettl3 regulated RNAs in neuronal tissue.
Excessive opening of undocked Cx26 hemichannels in the plasma membrane is associated with disease pathogenesis in keratitis-ichthyosis-deafness (KID) syndrome. Thus far, excessive opening of KID ...mutant hemichannels has been attributed, almost solely, to aberrant inhibition by extracellular Ca2+. This study presents two new possible contributing factors, pH and Zn2+. Plasma pH levels and micromolar concentrations of Zn2+ inhibit WT Cx26 hemichannels. However, A40V KID mutant hemichannels show substantially reduced inhibition by these factors. Using excised patches, acidification was shown to be effective from either side of the membrane, suggesting a protonation site accessible to H+ flux through the pore. Sensitivity to pH was not dependent on extracellular aminosulfonate pH buffers. Single channel recordings showed that acidification did not affect unitary conductance or block the hemichannel but rather promoted gating to the closed state with transitions characteristic of the intrinsic loop gating mechanism. Examination of two nearby KID mutants in the E1 domain, G45E and D50N, showed no changes in modulation by pH or Zn2+. N-bromo-succinimide, but not thiol-specific reagents, attenuated both pH and Zn2+ responses. Individually mutating each of the five His residues in WT Cx26 did not reveal a key His residue that conferred sensitivity to pH or Zn2+. From these data and the crystal structure of Cx26 that suggests that Ala-40 contributes to an intrasubunit hydrophobic core, the principal effect of the A40V mutation is probably a perturbation in structure that affects loop gating, thereby affecting multiple factors that act to close Cx26 hemichannels via this gating mechanism.
Background: Aberrantly functioning Cx26 hemichannels are a common feature of GJB2 mutations causing syndromic deafness.
Results: pH and Zn2+, factors that inhibit hemichannels, are less effective in the A40V KID syndrome mutant.
Conclusion: Impaired inhibition by pH and Zn2+ can contribute to the pathogenesis of KID syndrome.
Significance: Data provide new insights into Cx26 hemichannel function and possible contributions to tissue function.
ZC3H14 is an evolutionarily conserved, ubiquitously expressed polyadenosine RNA-binding protein that is lost in an inherited form of non-syndromic intellectual disability (ID). Studies of ZC3H14 ...orthologs have revealed a conserved role for ZC3H14 in the restriction of poly(A) tail length, but the molecular function of this protein in neurons has not been defined. To further our understanding of ZC3H14 function in neurons we have utilized Drosophila melanogaster to model ZC3H14-associated ID. The Drosophila melanogaster ortholog of ZC3H14, dNab2, is required for viability in flies, and is critical for normal neuronal function and axon projection. Here we describe a network of physical and genetic interactions between dNab2 and the fragile X protein homolog dFMRP that link dNab2/ZC3H14 to translational repression. The dNab2 and dFMRP proteins co-precipitate from neurons and can be co-localized to cytoplasmic foci distributed along the neurites of cultured brain neurons. Two well-characterized dFMRP mRNA targets, futsch and CamKII, are repressed in a dNab2-dependent manner, providing strong evidence that dNab2 functions as a translational repressor in conjunction with dFMRP. In parallel, we find murine ZC3H14 enriched in axons of cultured primary hippocampal neurons and associated with the translational machinery, implying a conserved role for dNab2/ZC3H14 in the control of gene expression. These data suggest that dNab2/ZC3H14 contributes to dFMRP-mediated translational regulation of mRNAs trafficked to distal neuronal compartments, a process that is critical in neurons and may underlie brain-specific defects in ZC3H14-associated ID patients.
PDF
