ABSTRACT 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 (ntg1 catdead ). 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 ntg1 catdead . 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 Away Overexpression of a base excision DNA repair protein impairs cell growth Overexpression of a base excision DNA repair protein can cause DNA damage Multiple mechanisms cause DNA damage from overexpression of a repair protein DNA repair pathways functionally interact to protect cells from DNA damage Precise regulation of the activity of DNA repair proteins is critical

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. Expression of H3E50K is sufficient to transform human cells as evidenced by an increase in cell migration and invasion, and an 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 selectively increases chromatin accessibility and perturbs proximal H3 post-translational modifications including H3K27me3; together these changes to chromatin dynamics dysregulate gene expression to support the epithelial-to-mesenchymal transition. Functional studies using Saccharomyces 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 H3 mutations have the potential to support oncogenic activity and function through distinct mechanisms that dysregulate gene expression.

The RNA exosome is a multisubunit, 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 Saccharomyces 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 suggest 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.

The RNA exosome is an evolutionarily conserved, multiprotein complex that is the major RNase in 3' processing and degradation of a wide range of RNAs in eukaryotes. Single amino acid changes in RNA exosome subunits cause rare genetic diseases collectively called exosomopathies. However, distinguishing disease-causing variants from nonpathogenic ones remains challenging, and the mechanism by which these variants cause disease is largely unknown. Previous studies have employed a budding yeast model of RNA exosome-linked diseases that relies on mutating the orthologous yeast genes. Here, we develop a humanized yeast model of exosomopathies that allows us to unambiguously assess damaging effects of the exact patient variant in budding yeast. Individual replacement of the yeast subunits with corresponding mammalian orthologs identified 6 out of 9 noncatalytic core subunits of the budding yeast RNA exosome that can be replaced by a mammalian subunit, with 3 of the replacements supporting close to normal growth. Further analysis of the disease-associated variants utilizing the hybrid yeast/mammalian RNA exosome revealed functional defects caused by both previously characterized and uncharacterized variants of EXOSC2, EXOSC4, EXOSC7, and EXOSC9. Analysis of the protein levels of these variants indicates that a subset of the patient-derived variants causes reduced protein levels, while other variants are defective but are expressed as well as the reference allele, suggesting a more direct contribution of these residues to RNA exosome function. This humanized yeast model of exosomopathies provides a convenient and sensitive genetic tool to help distinguish damaging RNA exosome variants from benign variants. This disease model can be further exploited to uncover the underpinning mechanism of RNA exosome defects.

The RNA exosome is an essential, evolutionarily conserved ribonuclease required for processing and degradation of key cellular RNAs. The complex maintains RNA homeostasis within every cell by ensuring the proper maturation, quality control and turnover of various RNA species including rRNAs. A growing list of diseases, collectively termed RNA exosomopathies, are caused by mutations in genes encoding structural subunits of the RNA exosome complex. RNA exosomopathies often result in tissue-specific defects, particularly manifesting as neurological disorders, which is intriguing given the ubiquitous functions and expression of the RNA exosome. One such ubiquitous, essential function of the RNA exosome is its involvement in ribosome biogenesis. In this review, we discuss the established connections between the RNA exosome and ribosome biogenesis, exploring the potential mechanisms through which RNA exosomopathies could influence ribosome heterogeneity, leading to aberrant translation and pathogenesis. We highlight the critical need for research in this area that can aid in understanding the complex aetiology of RNA exosomopathies and the future development of therapeutic strategies to mitigate pathology.This article is part of the discussion meeting issue 'Ribosome diversity and its impact on protein synthesis, development and disease'.

Dynamic protein post-translational methylation is essential for cellular function, highlighted by the essential role of methylation in transcriptional regulation and its aberrant dysregulation in diseases, including cancer. This underscores the importance of cataloging the cellular methylproteome. However, comprehensive analysis of the methylproteome remains elusive due to limitations in current enrichment and analysis pipelines. Here, we employ an l-methionine analogue, ProSeMet, that is chemoenzymatically converted to the SAM analogue ProSeAM in cells and in vivo to tag proteins with a biorthogonal alkyne that can be directly detected via liquid chromatography and tandem mass spectrometry (LC-MS/MS), or functionalized for subsequent selective enrichment and LC-MS/MS identification. Without enrichment, we identify known and novel lysine mono-, di-, and tripargylation, histidine propargylation, and arginine propargylation with site-specific resolution on proteins including heat shock protein HSPA8, the translational elongation factor eEF1A1, and the metabolic enzyme phosphoglycerate mutase 1, or PGAM1, for which methylation has been implicated in human disease. With enrichment, we identify 486 proteins known to be methylated and 221 proteins with novel propargylation sites encompassing diverse cellular functions. Systemic ProSeMet delivery in mice propargylates proteins across organ systems with blood-brain barrier penetrance and identifies site-specific propargylation in vivo with LC-MS/MS. Leveraging these pipelines to define the cellular methylproteome may have broad applications for understanding the methylproteome in the context of disease.

In recent years, there has been a growing appreciation for how regulatory events that occur either co- or post-transcriptionally contribute to the control of gene expression. Messenger RNAs (mRNAs) are extensively regulated throughout their metabolism in a precise spatiotemporal manner that requires sophisticated molecular mechanisms for cell-type-specific gene expression, which dictates cell function. Moreover, dysfunction at any of these steps can result in a variety of human diseases, including cancers, muscular atrophies, and neurological diseases. This review summarizes the steps of the central dogma of molecular biology, focusing on the post-transcriptional regulation of gene expression.

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.

The RNA exosome is an evolutionarily conserved complex required for both precise RNA processing and decay. Pathogenic variants in EXOSC genes, which encode structural subunits of this complex, are linked to several autosomal recessive disorders. Here, we describe a missense allele of the EXOSC4 gene that causes a collection of clinical features in two affected siblings. This missense variant (NM_019037.3: exon3:c.560T>C) changes a leucine residue within a conserved region of EXOSC4 to proline (p.Leu187Pro). The two affected individuals show prenatal growth restriction, failure to thrive, global developmental delay, intracerebral and basal ganglia calcifications, and kidney failure. Homozygosity for the damaging variant was identified by exome sequencing with Sanger sequencing to confirm segregation. To explore the functional consequences of this amino acid change, we modeled EXOSC4-L187P in the corresponding budding yeast protein, Rrp41 (Rrp41-L187P). Cells that express Rrp41-L187P as the sole copy of the essential Rrp41 protein show growth defects. Steady-state levels of both Rrp41-L187P and EXOSC4-L187P are decreased compared to controls, and EXOSC4-L187P shows decreased copurification with other RNA exosome subunits. RNA exosome target transcripts accumulate in rrp41-L187P cells, including the 7S precursor of 5.8S rRNA. Polysome profiles show a decrease in actively translating ribosomes in rrp41-L187P cells as compared to control cells with the incorporation of 7S pre-rRNA into polysomes. This work adds EXOSC4 to the structural subunits of the RNA exosome that have been linked to human disease and defines foundational molecular defects that could contribute to the adverse phenotypes caused by EXOSC pathogenic variants.

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 similar to 150 mRNAs. Analysis of the Sex-lethal (Sxl) Sxl ) mRNA revealed that Nab2 promotes an exon-skipping event and regulates m6A 6 A methylation on Sxl pre-mRNA by the Mettl3 methyltransferase. Mettl3 heterozygosity broadly rescues Nab2null null 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 5 ' UTR ' UTR (untranslated region) 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 null nervous tissue. Expression of Trio-GEF2 rescues projection defects in Nab2null null axons and dendrites, while the GEF1 Rac1-regulatory domain exacerbates these defects, suggesting Nab2-mediated regulation Trio-GEF activities. Collectively, these data indicate that Nab2-regulated processing of trio is critical for balancing Trio-GEF1 and-GEF2 activity and show that Nab2, Mettl3, and Trio function in a common pathway that shapes axon and dendrite morphology.

ABSTRACTTheDrosophilaRNA 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 theSex-lethal(Sxl) mRNA revealed that Nab2 promotes an exon-skipping event and regulates m6A methylation onSxlpre-mRNA by the Mettl3 methyltransferase. Mettl3 heterozygosity broadly rescuesNab2nullphenotypes 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 5’UTR intron in thetriopre-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 inNab2nullnervous tissue. Expression of Trio-GEF2 rescues projection defects inNab2nullaxons 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 StatementDrosophilaNab2, ortholog of the human RBP ZC3H14 mutated in inherited intellectual disability, acts through unknown RNA targets to control axon and dendrite morphology.This study shows that Nab2 and the Mettl3 methyltransferase guide splicing oftriomRNA, which encodes a conserved GEF-domain protein. Intron retention intriomRNA leads to an imbalance in levels of two Trio GEF domains in Nab2-deficient neurons and restoring this balance rescues neuronal defects.The authors conclude that Nab2 control oftriosplicing is required to pattern axon and dendrite growth and suggests that ZC3H14 may play a similar role in the vertebrate brain.

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.

Histone proteins are essential to package chromatin within the cell nucleus as well as to regulate gene expression. Post-translational modifications (PTMs) on histones, such as methylation of lysine residues, regulates histone functions. Critically, missense mutations in histone genes can convert histones into oncohistones, such as H3K36M, that alter histone PTMs, dyregulate histone function, and drive cancer phenotypes. One potential way that oncohistones can dysregulate gene expression is through altered chromatin structure. Previous studies provide evidence that altered histones, such as H3K36 mutants, can lead to cryptic transcription and increased production of antisense RNAs. Based on our prior observations and the established relationship between chromatin structure and gene expression, we tested the hypothesis that missense mutations in the histone H3 gene, particularly H3K36R and H3K36M, could cause an increase in antisense RNAs. To test this hypothesis, we employed a budding yeast oncohistone model. Budding yeast contains two genes that encode histone H3, HHT1 and HHT2. In this oncohistone model, the HHT2 gene is edited and the HHT1 gene is deleted to produce cells that express the histone variant as the sole histone H3 protein in the cells. Total RNA from these yeast cells was isolated, converted to cDNA via reverse transcriptase, and quantified using qPCR. Specific antisense RNAs previously identified to be altered in yeast H3K36 mutant cells were measured by qPCR. Preliminary results revealed an increase in antisense RNA in H3K36R and H3K36M mutant cells relative to wildtype control, in support of our hypothesis. This study enhances our molecular understanding of oncohistone mutations and offers novel insights into how histone changes may contribute to dysregulation of gene expression. These observations can be extended to human cells to explore the implications of these results in oncohistone-driven cancers. This research is supported and funded by 1)Department of Biology, Emory College of Arts and Sciences, and 2) Department of Radiation Oncology, Emory University School of Medicine, Atlanta, GA 30322.

The RNA exosome is an evolutionarily conserved ribonuclease complex required for processing and degradation of many cellular coding and non-coding RNAs, including ribosomal RNA (rRNA). The multi-subunit RNA exosome consists of a 3-subunit cap, a 6-subunit barrel-like core, and a catalytically active base. Missense mutations in genes encoding several structural subunits of the RNA exosome have been linked to human diseases, collectively termed "RNA exosomopathies". Symptoms of these diseases vary amongst patients and range from neurological defects to developmental disorders. Using biochemical approaches, here, we compare the impact of four disease-causing missense variants in two cap exosome subunits (Rrp4 and Rrp40) and two core exosome subunits (Rrp41 and Rrp46), modeled in the budding yeast, S. cerevisiae, on ribosome biogenesis and translation. Our results show that all four variants cause rRNA processing defects and an overall significant decrease in translation. However, translation is perturbed in distinct ways in cap vs core subunit variants. Our findings suggest that different RNA exosomopathy mutations can result in in vivo consequences that are both unique and shared amongst the variants and could provide new insights into the molecular defects underlying each distinct pathology. This work was supported by NIH awards R35GM138123 to H.G, R01GM130147 to A.H.C and Ambro van Hoof and a Synergy II Nexus Award provided by the Woodruff Health Sciences Center (WHSC), Emory School of Medicine, the Office of the Provost, and Emory College of Arts and Sciences (ECAS) to H.G. and A.H.C. L.A.C. was supported by NIH T32GM149422.

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.

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.