Biology subjects

Corbett, A. H.

Publications and source records attributed to Corbett, A. H..

At least 19 recordsLinked to original sources

bioRxiv2026

Sex-specific metabolic regulation by the Drosophila RNA-binding protein Nab2

Conserved RNA binding proteins (RBPs) regulate key steps of gene expression including mRNA processing, export, localization, stability and translation. Human ZC3H14 is a conserved RBP that regulates pre-mRNA processing in neurons and loss of ZC3H14 leads to neurological defects. Studies of Nab2, the Drosophila orthologue of ZC3H14, have identified potential target RNAs involved in metabolism, suggesting Nab2 may influence neurometabolic circuitry. Here, we show a female-specific increase in dilp2 and dilp5 mRNA levels. The dilps encode insulin-like peptides that signal from the brain insulin producing cells (IPCs) to peripheral tissues. Nab2null females have enlarged lipid droplets in the fat body, a tissue analogous to human adipose tissue and liver. Notably, neuronal depletion of Nab2 increases lipid droplet size while neuronal expression of Nab2 in Nab2null female rescues this phenotype supporting a role for Nab2 in a neuronal circuit that regulates dilp levels. Furthermore, depletion of dilp2 or dilp5 from IPCs rescues the enlarged lipid droplet phenotype in Nab2null females indicating that elevated dilp2/dilp5 contributes to enlarged lipid droplets. Together, these data support a female-specific role for Nab2 in brain neurons to support insulin signaling and fat storage, expanding the known functions of RBPs linking neuronal function and metabolic homeostasis.

cell biology↗

bioRxiv2026

Systematic comparison of pathogenic variants of the RNA exosome gene EXOSC3/RRP40 in Saccharomyces cerevisiae reveals variant-specific functional consequences

The RNA exosome is an essential, evolutionarily conserved ribonuclease complex that processes and degrades many classes of RNA. The complex is composed of three structural cap subunits (EXOSC1-3/Csl4, Rrp4, Rrp40; H. sapiens/S. cerevisiae), six structural core subunits (EXOSC4-9/Rrp41,Rrp46,Mtr3,Rrp42,Rrp43,Rrp45), and a catalytic ribonuclease (DIS3 or DIS3L/Dis3). Cofactors that associate with the RNA exosome confer specificity to target specific RNAs for processing and/or decay. Missense mutations in genes encoding structural subunits of the RNA exosome have been linked to neurological diseases. Notably, several pathogenic mutations have been identified in EXOSC3 that are associated with pontocerebellar hypoplasia type 1b (PCH1b). These pathogenic alleles cause a broad spectrum of clinical severity, suggesting variant-specific functional consequences. Given the high degree of conservation between the human and budding yeast RNA exosome complexes, we performed a systematic analysis of eight pathogenic EXOSC3 variants modeled in budding yeast Rrp40. We find that two Rrp40 variants cause growth defects, show distinct negative genetic interactions with RNA exosome cofactor mutants, and impair RNA processing in budding yeast. One of these variants, EXOSC3-Y109N/Rrp40-Y64N, had not been previously characterized in any mechanistic studies. Computational stability predictions and immunoblot analyses indicate that most EXOSC3/Rrp40 variants display reduced steady-state protein levels, but decreased protein levels do not strictly correlate with phenotype or disease severity, suggesting that individual variants disrupt RNA exosome function through distinct mechanisms. Collectively, our studies suggest that pathogenic EXOSC3 variants alter RNA exosome function through distinct mechanisms and provide insight into the specific molecular defects that could underlie pathology.

genetics↗

bioRxiv2026

Overexpression of Eaf1, a subunit of the NuA4 lysine acetyl transferase complex, rescues growth defects in the budding yeast H3K36M oncohistone model via histone H4 tail acetylation

Histone proteins are critical for regulating nucleic acid transactions, including gene expression. Specific missense mutations in histone genes have been linked to oncogenesis, creating oncohistones. One of the first identified oncohistones converts K36 in histone H3 to M (H3K36M). While humans have 15 H3 genes, the Saccharomyces cerevisiae genome contains only two H3 genes. As H3 shares 90% sequence identity between budding yeast and humans, the budding yeast system provides an ideal model for study of H3K36M. We previously identified the catalytic subunit of the NuA4 lysine acetyltransferase complex as a high copy suppressor of growth defects in H3K36 mutant yeast cells. Here, we demonstrate that the NuA4 scaffold Eaf1 suppresses mutant H3K36 growth defects. We show that this suppression by Eaf1 is dependent on the Eaf1 HSA domain. To examine whether the role of NuA4 in acetylating histone H4 is required for suppression of H3K36 growth defects, we altered the four NuA4 target lysines within H4 (K5, 8, 12, and 16) to create quadruple histone mutants with all four of these lysines converted to alanine (H4-4K[->]A) or arginine (H4-4K[->]R). These H4 mutants have reduced viability and disrupted global H4 acetylation. Eaf1 cannot suppress growth defects in these H4 acetylation mutants, suggesting Eaf1-mediated suppression is dependent on acetylation of H4 N-terminal tails. We further show that inhibiting NuA4 activity in H3K36M human cells limits their oncogenic potential. Together, this work defines a mechanistic connection between H3K36 and H4 N-terminal tail acetylation in the context of a cancer-causing mutation. Article SummarySpecific mutations in histone genes have been found to cause various cancers, creating oncohistones, and the exact mechanisms by which they drive oncogenesis remain to be characterized. Budding yeast is a simple yet powerful model for studying oncohistones such as H3K36M. Subunits of the NuA4 lysine acetyltransferase complex were found to suppress growth defects in budding yeast expressing H3K36M. This suppression was dependent on the presence of the NuA4 target lysines in histone H4. Additionally, inhibiting NuA4 activity in human H3K36M cells limited their oncogenic potential. This work characterizes the mechanistic relationship between H3K36M and the NuA4 complex.

genetics↗

bioRxiv2026

Globular domain histone H3R131C mutation remodels chromatin accessibility to promote oncogenic transcriptional programs

Histone mutations, characterized as oncohistones, have emerged as important oncogenic driver events by altering chromatin structure and/or chromatin modifications, thereby dysregulating gene expression. While H3 tail domain oncohistone mutations such as H3K27M and H3K36M are well characterized, it is currently unknown whether mutations within the H3 globular domain represent oncogenic driver events. Using publicly available cancer patient tumor data, here we identify H3R131C as a recurrent histone H3 globular domain mutation. H3R131C mutation is present in diverse human tumors including breast and bladder cancers. Phenotypic assays demonstrate that H3R131C expression does not augment cellular proliferation but enhances cellular migration and invasion. H3R131C frequently co-occurs with mutations in oncogenes including PIK3CA and ESR1, and tumor suppressors TP53 and CDKN2A with an allele frequency consistent with H3R131C representing a subclonal event that enhances tumor fitness rather than initiating transformation. Mechanistically, ATAC-seq reveals that H3R131C expression increases chromatin accessibility, with enrichment of AP-1/bZIP transcription factor motifs at gained accessible regions. Integration of chromatin accessibility and transcriptomic profiling identifies concordant upregulation of pro-oncogenic target genes including PGF, SOX5, and CCDC88C, supporting a model in which H3R131C destabilizes the nucleosome to remodel chromatin and activate transcriptional programs associated with cellular migration, angiogenesis, and epithelial plasticity. Collectively, these findings identify H3R131C as a functionally active globular domain oncohistone that reshapes the epigenome to promote oncogenic gene expression.

cancer biology↗

bioRxiv2024

The RNA binding protein Nab2 regulates splicing of the RhoGEF trio transcript to govern axon and dendrite morphology

The Drosophila RNA binding protein (RBP) Nab2 acts in neurons to regulate neurodevelopment and is orthologous to the human intellectual disability-linked RBP, ZC3H14. Nab2 governs axon projection in mushroom body neurons and limits dendritic arborization of class IV sensory neurons in part by regulating splicing events in ~150 mRNAs. Analysis of the Sex-lethal (Sxl) mRNA revealed that Nab2 promotes an exon-skipping event and regulates m6A methylation on Sxl pre-mRNA by the Mettl3 methyltransferase. Mettl3 heterozygosity broadly rescues Nab2null phenotypes implying that Nab2 acts through similar mechanisms on other RNAs, including unidentified targets involved in neurodevelopment. Here, we show that Nab2 and Mettl3 regulate the removal of a 5UTR intron in the trio pre-mRNA. Trio utilizes two GEF domains to balance Rac and RhoGTPase activity. Intriguingly, an isoform of Trio containing only the RhoGEF domain, GEF2, is depleted in Nab2null nervous tissue. Expression of Trio-GEF2 rescues projection defects in Nab2nullaxons and dendrites, while the GEF1 Rac1-regulatory domain exacerbates these defects, suggesting Nab2-mediated regulation Trio-GEF activities. Collectively, these data identify Nab2-regulated splicing as a key step in balancing Trio GEF1 and GEF2 activity and show that Nab2, Mettl3, and Trio function in a common pathway that shapes axon and dendrite morphology. Significance StatementO_LIDrosophila Nab2, ortholog of the human RBP ZC3H14 mutated in inherited intellectual disability, acts through unknown RNA targets to control axon and dendrite morphology. C_LIO_LIThis study shows that Nab2 and the Mettl3 methyltransferase guide splicing of trio mRNA, which encodes a conserved GEF-domain protein. Intron retention in trio mRNA leads to an imbalance in levels of two Trio GEF domains in Nab2-deficient neurons and restoring this balance rescues neuronal defects. C_LIO_LIThe authors conclude that Nab2 control of trio splicing is required to pattern axon and dendrite growth and suggests that ZC3H14 may play a similar role in the vertebrate brain. C_LI

molecular biology↗

bioRxiv2023

Comparative analyses of disease-linked missense mutations in the RNA exosome modeled in budding yeast reveal distinct functional consequences in translation

The RNA exosome is a multi-subunit, evolutionarily conserved ribonuclease complex that is essential for processing, decay and surveillance of many cellular RNAs. Missense mutations in genes encoding the structural subunits of the RNA exosome complex cause a diverse range of diseases, collectively known as RNA exosomopathies, often involving neurological and developmental defects. The varied symptoms suggest that different mutations lead to distinct in vivo consequences. To investigate these functional consequences and distinguish whether they are unique to each RNA exosomopathy mutation, we generated a collection of in vivo models by introducing pathogenic missense mutations in orthologous S. cerevisiae genes. Comparative RNA-seq analysis assessing broad transcriptomic changes in each mutant model revealed that three yeast mutant models, rrp4-G226D, rrp40-W195R and rrp46-L191H, which model mutations in the genes encoding EXOSC2, EXOSC3 and EXOSC5, respectively, had the largest transcriptomic differences. While some transcriptomic changes, particularly in transcripts related to ribosome biogenesis, were shared among mutant models, each mutation also induced unique transcriptomic changes. Thus, our data suggests that while there are some shared consequences, there are also distinct differences in RNA exosome function by each variant. Assessment of ribosome biogenesis and translation defects in the three models revealed distinct differences in polysome profiles. Collectively, our results provide the first comparative analyses of RNA exosomopathy mutant models and suggest that different RNA exosome gene mutations result in in vivo consequences that are both unique and shared across each variant, providing further insight into the biology underlying each distinct pathology.

molecular biology↗

bioRxiv2023

Histone H3 E50K mutation confers oncogenic activity and supports an EMT phenotype

Sequencing of human patient tumors has identified recurrent missense mutations in genes encoding core histones. We report that mutations that convert histone H3 amino acid 50 from a glutamate to a lysine (H3E50K) support an oncogenic phenotype in human cells. Expression of H3E50K is sufficient to transform human cells as evidenced by a dramatic increase in cell migration and invasion, and a statistically significant increase in proliferation and clonogenicity. H3E50K also increases the invasive phenotype in the context of co-occurring BRAF mutations, which are present in patient tumors characterized by H3E50K. H3E50 lies on the globular domain surface in a region that contacts H4 within the nucleosome. We find that H3E50K perturbs proximal H3 post-translational modifications globally and dysregulates gene expression, activating the epithelial to mesenchymal transition. Functional studies using S. cerevisiae reveal that, while yeast cells that express H3E50K as the sole copy of histone H3 show sensitivity to cellular stressors, including caffeine, H3E50K cells display some genetic interactions that are distinct from the characterized H3K36M oncohistone yeast model. Taken together, these data suggest that additional histone H3 mutations have the potential to be oncogenic drivers and function through distinct mechanisms that dysregulate gene expression. SummaryO_LIRecurrent missense mutation that alter histone H3E50 were identified in patient tumors C_LIO_LIH3E50K expression dysregulates global H3 post-translational modification, gene expression and may govern a transcriptional program associated with metastatic phenotypes C_LIO_LIhht2-E50K yeast cells exhibit growth defects in the presence of DNA damaging agents C_LI

cancer biology↗

bioRxiv2023

Analysis of RNA Exosome Subunit Transcript Abundance Across Tissues Implications for Neurological Disease Pathogenesis

Exosomopathies are a collection of rare diseases caused by mutations in genes that encode structural subunits of a ribonuclease complex termed the RNA exosome. The RNA exosome mediates both RNA processing and degradation of multiple classes of RNA. This complex is evolutionarily conserved and required for fundamental cellular functions, including rRNA processing. Recently, missense mutations in genes encoding structural subunits of the RNA exosome complex have been linked to a variety of distinct neurological diseases, many of them childhood neuronopathies with at least some cerebellar atrophy. Understanding how these missense mutations lead to the disparate clinical presentations that have been reported for this class of diseases necessitates investigation of how these specific changes alter cell-specific RNA exosome function. Although the RNA exosome complex is routinely referred to as ubiquitously expressed, little is known about the tissue- or cell-specific expression of the RNA exosome complex or any individual subunit. Here, we leverage publicly available RNA-sequencing data to analyze RNA exosome subunit transcript levels in healthy human tissues, focusing on those tissues that are impacted in exosomopathy patients described in clinical reports. This analysis provides evidence to support the characterization of the RNA exosome as ubiquitously expressed with transcript levels for the individual subunits that vary in different tissues. However, the cerebellar hemisphere and cerebellum have high levels of nearly all RNA exosome subunit transcripts. These findings could suggest that the cerebellum has a high requirement for RNA exosome function and potentially explain why cerebellar pathology is common in RNA exosomopathies.

molecular biology↗

bioRxiv2023

The RNA helicase DDX1 associates with the nuclear RNA exosome and modulates R-loops

The RNA exosome is a ribonuclease complex that mediates both RNA processing and degradation. This complex is evolutionarily conserved, ubiquitously expressed, and required for fundamental cellular functions, including rRNA processing. The RNA exosome plays roles in regulating gene expression and protecting the genome, including modulating the accumulation of RNA-DNA hybrids (R-loops). The function of the RNA exosome is facilitated by cofactors, such as the RNA helicase MTR4, which binds/remodels RNAs. Recently, missense mutations in RNA exosome subunit genes have been linked to neurological diseases. One possibility to explain why missense mutations in genes encoding RNA exosome subunits lead to neurological diseases is that the complex may interact with cell- or tissue-specific cofactors that are impacted by these changes. To begin addressing this question, we performed immunoprecipitation of the RNA exosome subunit, EXOSC3, in a neuronal cell line (N2A) followed by proteomic analyses to identify novel interactors. We identified the putative RNA helicase, DDX1, as an interactor. DDX1 plays roles in double-strand break repair, rRNA processing, and R-loop modulation. To explore the functional connections between EXOSC3 and DDX1, we examined the interaction following double-strand breaks, and analyzed changes in R-loops in N2A cells depleted for EXOSC3 or DDX1 by DNA/RNA immunoprecipitation followed by sequencing (DRIP-Seq). We find that EXOSC3 interaction with DDX1 is decreased in the presence of DNA damage and that loss of EXOSC3 or DDX1 alters R-loops. These results suggest EXOSC3 and DDX1 interact during events of cellular homeostasis and potentially suppress unscrupulous expression of genes promoting neuronal projection.

biochemistry↗

bioRxiv2022

In vivo Characterization of the Critical Interaction between the RNA Exosome and the Essential RNA Helicase Mtr4 in Saccharomyces cerevisiae

The RNA exosome is a conserved molecular machine that processes/degrades numerous coding and non-coding RNAs. The 10-subunit complex is composed of three S1/KH cap subunits (human EXOSC2/3/1; yeast Rrp4/40/Csl4), a lower ring of six PH-like subunits (human EXOSC4/7/8/9/5/6; (yeast Rrp41/42/43/45/46/Mtr3), and a singular 3-5 exo/endonuclease DIS3/Rrp44. Recently, several disease-linked missense mutations have been identified in genes encoding the structural cap and core subunits of the RNA exosome. In this study, we characterize a rare multiple myeloma patient missense mutation that was identified in the cap subunit gene EXOSC2. This missense mutation results in a single amino acid substitution, p.Met40Thr, in a highly conserved domain of EXOSC2. Structural studies suggest this Met40 residue makes direct contact with the essential RNA helicase, MTR4, and may help stabilize the critical interaction between the RNA exosome complex and this cofactor. To assess this interaction in vivo, we utilized the Saccharomyces cerevisiae system and modeled the EXOSC2 patient mutation into the orthologous yeast gene RRP4, generating the variant rrp4 M68T. The rrp4 M68T cells have accumulation of certain RNA exosome target RNAs and show sensitivity to drugs that impact RNA processing. Additionally, we identified robust negative genetic interactions the rrp4 M68T variant and RNA exosome cofactor mutants, particularly mtr4 mutant variants. This study suggests that the EXOC2 mutation identified in a multiple myeloma patient may impact the function of the RNA exosome and provides an in vivo assessment of a critical interface between the RNA exosome and Mtr4.

genetics↗

bioRxiv2022

A Saccharomyces cerevisiae Model for Overexpression of Ntg1 a Base Excision DNA Repair Protein Reveals Novel Genetic Interactions

The Base Excision Repair (BER) pathway repairs oxidative DNA damage, a common and detrimental form of damage to the genome. Although biochemical steps in BER have been well define, little is understood about how the pathway is regulated. Such regulation is critical, as cells must respond rapidly to DNA damage while avoiding aberrant activation of repair proteins that can produce DNA damage as intermediates in the repair pathway. Indeed, overexpression of the human BER protein, NTHL1, a DNA N-glycosylase, can cause genomic instability and early cellular hallmarks of cancer. We developed a Saccharomyces cerevisiae model to explore how overexpression of NTHL1 may impair cellular function. Overexpression of Ntg1, the budding yeast orthologue of NTHL1, impairs cell growth. To dissect mechanisms underlying this growth defect, we overexpressed either wild-type Ntg1 or a catalytically inactive variant of Ntg1 (ntg1catdead). Consistent with results obtained for NTHL1, both variants of Ntg1 impair cell growth, but only the wild-type protein causes accumulation of double-strand breaks and chromosome loss. We screened a panel of DNA repair mutants for resistance/sensitivity to overexpression of wild-type Ntg1 or ntg1catdead. This analysis identified several cellular pathways that protect cells from Ntg1-induced damage, providing insight into interplay between DNA repair pathways. Finally, we identified a link to sumoylation and probed how this post-translational modification could contribute to regulation of Ntg1 function. This study describes a budding yeast system to understand how cells regulate and respond to dysregulation of the BER pathway. Take AwayO_LIOverexpression of a base excision DNA repair protein impairs cell growth C_LIO_LIOverexpression of a base excision DNA repair protein can cause DNA damage C_LIO_LIMultiple mechanisms cause DNA damage from overexpression of a repair protein C_LIO_LIDNA repair pathways functionally interact to protect cells from DNA damage C_LIO_LIPrecise regulation of the activity of DNA repair proteins is critical C_LI

genetics↗

bioRxiv2022

The RNA binding protein Nab2 genetically interacts with multiple RNA exosome cofactors to regulate target RNAs

RNA binding proteins play important roles in the processing and precise regulation of RNAs. Highlighting the biological importance of RNA binding proteins is the increasing number of human diseases that result from mutations in genes that encode these proteins. We recently discovered that mutations in the ZC3H14 gene, which encodes an evolutionarily conserved polyadenosine RNA-binding protein, cause intellectual disability. Studies of the budding yeast orthologue of ZC3H14, Nuclear Poly(A) Binding protein 2 (Nab2), have provided insight into the functions of this protein. The NAB2 gene is essential in S. cerevisiae, and conditional nab2 mutants cause defects in a number of steps in RNA processing. To explore the critical functions of the Nab2/ZC3H14 protein family, we performed a high-copy suppressor screen on nab2 mutant cells. This screen identified genes encoding two core subunits of the RNA exosome, as well as Nrd1 and Ski7, nuclear and cytoplasmic cofactors of the RNA exosome, respectively. Nrd1 is an RNA binding protein that is part of the Nrd1-Nab3-Sen1 (NNS) complex, which plays an important role in transcription termination of non-coding RNAs. Ski7 is a GTP-binding protein that mediates interaction between the RNA exosome and the Ski complex, which targets RNA transcripts to the exosome for processing and degradation in the cytoplasm. To explore the functional interactions between the RNA exosome and Nab2, we employed RNA-seq analysis to identify the transcripts most impacted by overexpression of these exosome cofactors in nab2 mutant cells. This analysis revealed that many transcripts show small changes in steady-state levels, consistent with a global role of Nab2 in modulating transcript stability. This study uncovers functional interactions between the RNA exosome and Nab2 in both the nucleus and the cytoplasm.

genetics↗

bioRxiv2021

The Drosophila RNA binding protein Nab2 patterns dendritic arbors and axons via the planar cell polarity pathway

RNA binding proteins support neurodevelopment by modulating numerous steps in post-transcriptional regulation, including splicing, export, translation, and turnover of mRNAs that can traffic into axons and dendrites. One such RBP is ZC3H14, which is lost in an inherited intellectual disability. The Drosophila melanogaster ZC3H14 ortholog, Nab2, localizes to neuronal nuclei and cytoplasmic ribonucleoprotein granules, and is required for olfactory memory and proper axon projection into brain mushroom bodies. Nab2 can act as a translational repressor in conjunction with the Fragile-X mental retardation protein homolog Fmr1 and shares target RNAs with the Fmr1-interacting RBP Ataxin-2. However, neuronal signaling pathways regulated by Nab2 and their potential roles outside of mushroom body axons remain undefined. Here, we demonstrate that Nab2 restricts branching and projection of larval sensory dendrites via the planar cell polarity pathway, and that this link may provide a conserved mechanism through which Nab2/ZC3H14 modulates projection of both axons and dendrites. Planar cell polarity proteins are enriched in a Nab2-regulated brain proteomic dataset. Complementary genetic data indicate that Nab2 guides dendrite and axon growth through the planar-cell-polarity pathway. Analysis of the core planar cell polarity protein Vang, which is depleted in the Nab2 mutant whole-brain proteome, uncovers selective and dramatic loss of Vang within axon/dendrite-enriched brain neuropil relative to brain regions containing cell bodies. Collectively, these data demonstrate that Nab2 regulates dendritic arbors and axon projection by a planar-cell-polarity-linked mechanism and identify Nab2 as required for accumulation of the core planar cell polarity factor Vang in distal neuronal projections.

developmental biology↗

bioRxiv2021

A Budding Yeast Model and Screen to Define the Functional Consequences of Oncogenic Histone Missense Mutations

Somatic missense mutations in histone genes turn these essential proteins into oncohistones, which can drive oncogenesis. Understanding how missense mutations alter histone function is challenging in mammals as mutations occur in a single histone gene. For example, described oncohistone mutations predominantly occur in the histone H3.3 gene, despite the human genome encoding 15 H3 genes. To understand how oncogenic histone missense mutations alter histone function, we leveraged the budding yeast model, which contains only two H3 genes, to explore the functional consequences of oncohistones H3K36M, H3G34W, H3G34L, H3G34R, and H3G34V. Analysis of cells that express each of these variants as the sole copy of H3 reveals that H3K36 mutants show different drug sensitivities compared to H3G34 mutants. This finding suggests that changes to proximal amino acids in the H3 N-terminal tail alter distinct biological pathways. We exploited the caffeine sensitive growth of H3K36 mutant cells to perform a high copy suppressor screen. This screen identified genes linked to histone function and transcriptional regulation, including Esa1, a histone H4/H2A acetyltransferase; Tos4, a forkhead-associated domain-containing gene expression regulator; Pho92, an N6-methyladenosine RNA binding protein and Sgv1/Bur1, a cyclin-dependent kinase. We show that the Esa1 lysine acetyltransferase activity is critical for suppression of the caffeine sensitive growth of H3K36R mutant cells while the previously characterized binding interactions of Tos4 and Pho92 are not required for suppression. This screen identifies pathways that could be altered by oncohistone mutations and highlights the value of yeast genetics to identify pathways altered by such mutations.

genetics↗

bioRxiv2021

The Disease-Associated Proteins Drosophila Nab2 and Ataxin-2 Interact with Shared RNAs and Coregulate Neuronal Morphology

Nab2 encodes a conserved polyadenosine RNA-binding protein (RBP) with broad roles in post-transcriptional regulation, including in poly(A) RNA export, poly(A) tail length control, transcription termination, and mRNA splicing. Mutation of the Nab2 human ortholog ZC3H14 gives rise to an autosomal recessive intellectual disability, but understanding of Nab2/ZC3H14 function in metazoan nervous systems is limited, in part because no comprehensive identification of metazoan Nab2/ZC3H14-associated RNA transcripts has yet been conducted. Moreover, many Nab2/ZC3H14 functional protein partnerships likely remain unidentified. Here we present evidence that Drosophila melanogaster Nab2 interacts with the RBP Ataxin-2 (Atx2), a neuronal translational regulator, and implicate these proteins in coordinate regulation of neuronal morphology and adult viability. We then present the first high-throughput identifications of Nab2- and Atx2-associated RNAs in Drosophila brain neurons using an RNA immunoprecipitation-sequencing (RIP-Seq) approach. Critically, the RNA interactomes of each RBP overlap, and Nab2 exhibits high specificity in its RNA associations in neurons in vivo, associating with a small fraction of all polyadenylated RNAs. The identities of shared associated transcripts (e.g. drk, me31B, stai) and of transcripts specific to Nab2 or Atx2 (e.g. Arpc2, tea, respectively) promise insight into neuronal functions of and interactions between each RBP. Significantly, Nab2-associated RNAs are overrepresented for internal A-rich motifs, suggesting these sequences may partially mediate Nab2 target selection. Taken together, these data demonstrate that Nab2 opposingly regulates neuronal morphology and shares associated neuronal RNAs with Atx2, and that Drosophila Nab2 associates with a more specific subset of polyadenylated mRNAs than its polyadenosine affinity alone may suggest.

neuroscience↗

bioRxiv2020

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

The human ZC3H14 gene, which encodes a ubiquitously expressed polyadenosine zinc finger RNA binding protein, is mutated in an inherited form of autosomal recessive, non-syndromic intellectual disability. To gain insight into ZC3H14 neurological functions, we previously developed a Drosophila melanogaster model of ZC3H14 loss by deleting the fly ortholog, Nab2. Studies in this invertebrate model reveal that Nab2 controls final patterns of neuron projection within fully developed adult brains. Here, we examine earlier pupal stages and define roles for Nab2 in controlling the dynamic growth of axons into the developing brain mushroom bodies (MBs), which support olfactory learning and memory, and in regulating abundance of a small fraction of the total brain proteome, a portion of which is rescued by overexpression of Nab2 specifically in brain neurons. The group of Nab2-regulated brain proteins, identified by quantitative proteomic analysis, includes the microtubule binding protein Futsch, the neuronal Ig-family transmembrane protein Turtle, the glial:neuron adhesion protein Contactin, the RacGAP Tumbleweed, and the planar cell polarity factor Van Gogh, which collectively link Nab2 to a the processes of brain morphogenesis, neuroblast proliferation, circadian sleep/wake cycles, and synaptic development. Overall, these data indicate that Nab2 controls abundance of a subset of brain proteins during the active process of wiring the pupal brain mushroom body, and thus provide a window into potentially conserved functions of the Nab2/ZC3H14 RNA binding proteins in neurodevelopment and function.

biochemistry↗

bioRxiv2020

A Budding Yeast Model for Human Disease Mutations in the EXOSC2 Cap Subunit of the RNA Exosome

RNA exosomopathies, a growing family of tissue-specific diseases, are linked to missense mutations in genes encoding the structural subunits of the conserved 10-subunit exoribonuclease complex, the RNA exosome. Such mutations in the cap subunit gene EXOSC2 cause the novel syndrome SHRF (Short stature, Hearing loss, Retinitis pigmentosa and distinctive Facies). In contrast, exosomopathy mutations in the cap subunit gene EXOSC3 cause pontocerebellar hypoplasia type 1b (PCH1b). Though having strikingly different disease pathologies, EXOSC2 and EXOSC3 exosomopathy mutations result in amino acid substitutions in similar, conserved domains of the cap subunits, suggesting that these exosomopathy mutations have distinct consequences for RNA exosome function. We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by introducing the EXOSC2 mutations in the orthologous S. cerevisiae gene RRP4. The resulting rrp4 mutant cells have defects in cell growth and RNA exosome function. We detect significant transcriptomic changes in both coding and non-coding RNAs in the rrp4 variant, rrp4-G226D, which models EXOSC2 p.Gly198Asp. Comparing this rrp4-G226D mutant to the previously studied S. cerevisiae model of EXOSC3 PCH1b mutation, rrp40-W195R, reveals that these mutants have disparate effects on certain RNA targets, providing the first evidence for different mechanistic consequences of these exosomopathy mutations. Congruently, we detect specific negative genetic interactions between RNA exosome cofactor mutants and rrp4-G226D but not rrp40-W195R. These data provide insight into how SHRF mutations could alter the function of the RNA exosome and allow the first direct comparison of exosomopathy mutations that cause distinct pathologies.

genetics↗

bioRxiv2020

The Nab2 RNA binding protein promotes sex-specific splicing of Sex lethal in Drosophila neuronal tissue

The Drosophila polyadenosine RNA binding protein Nab2, which is orthologous to a human protein lost in a form of inherited intellectual disability, controls axon projection, locomotion, and memory. Here we define an unexpectedly specific role for Nab2 in regulating splicing of [~]150 exons/introns in the head transcriptome and link the most prominent of these, female retention of a male-specific exon in the sex determination factor Sex-lethal (Sxl), to a role in m6A-dependent mRNA splicing. Genetic evidence indicates that aberrant Sxl splicing underlies multiple phenotypes in Nab2 mutant females. At a molecular level, Nab2 associates with Sxl pre-mRNA and ensures proper female-specific splicing by preventing m6A hypermethylation by Mettl3 methyltransferase. Consistent with these results, reducing Mettl3 expression rescues developmental, behavioral and neuroanatomical phenotypes in Nab2 mutants. Overall these data identify Nab2 as a required regulator of m6A-regulated Sxl splicing and imply a broader link between Nab2 and Mettl3-regulated brain RNAs.

developmental biology↗