The dramatic growth that occurs during Drosophila larval development requires rapid conversion of nutrients into biomass. Many larval tissues respond to these biosynthetic demands by increasing carbohydrate metabolism and lactate dehydrogenase (dLDH) activity. The resulting metabolic program is ideally suited to synthesize macromolecules and mimics the manner by which cancer cells rely on aerobic glycolysis. To explore the potential role of Drosophila dLDH in promoting biosynthesis, we examined how dLdh mutations influence larval development. Our studies unexpectantly found that dLdh mutants grow at a normal rate, indicating that dLDH is dispensable for larval biomass production. However, subsequent metabolomic analyses suggested that dLdh mutants compensate for the inability to produce lactate by generating excess glycerol-3-phosphate (G3P), the production of which also influences larval redox balance. Consistent with this possibility, larvae lacking both dLDH and G3P dehydrogenase (GPDH1) exhibit growth defects, synthetic lethality, and decreased glycolytic flux. Considering that human cells also generate G3P upon Lactate Dehydrogenase A (LDHA) inhibition, our findings hint at a conserved mechanism in which the coordinate regulation of lactate and G3P synthesis imparts metabolic robustness upon growing animal tissues.
Abstract Hox transcription factors are conserved regulators of neuronal subtype specification on the anteroposterior axis in animals, with disruption of Hox gene expression leading to homeotic transformations of neuronal identities. We have taken advantage of an unusual mutation in the Caenorhabditis elegans Hox gene lin-39, lin-39(ccc16), which transforms neuronal fates in the C. elegans male ventral nerve cord in a manner that depends on a second Hox gene, mab-5. We have performed a genetic analysis centered around this homeotic allele of lin-39 in conjunction with reporters for neuronal target genes and protein interaction assays to explore how LIN-39 and MAB-5 exert both flexibility and specificity in target regulation. We identify cis-regulatory modules in neuronal reporters that are both region-specific and Hox-responsive. Using these reporters of neuronal subtype, we also find that the lin-39(ccc16) mutation disrupts neuronal fates specifically in the region where lin-39 and mab-5 are coexpressed, and that the protein encoded by lin-39(ccc16) is active only in the absence of mab-5. Moreover, the fates of neurons typical to the region of lin-39-mab-5 coexpression depend on both Hox genes. Our genetic analysis, along with evidence from Bimolecular Fluorescence Complementation protein interaction assays, supports a model in which LIN-39 and MAB-5 act at an array of cis-regulatory modules to cooperatively activate and to individually activate or repress neuronal gene expression, resulting in regionally specific neuronal fates.
Abstract The RNA exosome is an evolutionarily-conserved ribonuclease complex critically important for precise processing and/or complete degradation of a variety of cellular RNAs. The recent discovery that mutations in genes encoding structural RNA exosome subunits cause tissue-specific diseases makes defining the role of this complex within specific tissues critically important. Mutations in the RNA exosome component 3 ( EXOSC3 ) gene cause Pontocerebellar Hypoplasia Type 1b (PCH1b), an autosomal recessive neurologic disorder. The majority of disease-linked mutations are missense mutations that alter evolutionarily-conserved regions of EXOSC3. The tissue-specific defects caused by these amino acid changes in EXOSC3 are challenging to understand based on current models of RNA exosome function with only limited analysis of the complex in any multicellular model in vivo . The goal of this study is to provide insight into how mutations in EXOSC3 impact the function of the RNA exosome. To assess the tissue-specific roles and requirements for the Drosophila ortholog of EXOSC3 termed Rrp40, we utilized tissue-specific RNAi drivers. Depletion of Rrp40 in different tissues reveals a general requirement for Rrp40 in the development of many tissues including the brain, but also highlight an agedependent requirement for Rrp40 in neurons. To assess the functional consequences of the specific amino acid substitutions in EXOSC3 that cause PCH1b, we used CRISPR/Cas9 gene editing technology to generate flies that model this RNA exosome-linked disease. These flies show reduced viability; however, the surviving animals exhibit a spectrum of behavioral and morphological phenotypes. RNA-seq analysis of these Drosophila Rrp40 mutants reveals increases in the steady-state levels of specific mRNAs and ncRNAs, some of which are central to neuronal function. In particular, Arc1 mRNA, which encodes a key regulator of synaptic plasticity, is increased in the Drosophila Rrp40 mutants. Taken together, this study defines a requirement for the RNA exosome in specific tissues/cell types and provides insight into how defects in RNA exosome function caused by specific amino acid substitutions that occur in PCH1b can contribute to neuronal dysfunction. Author Summary Pontocerebellar Hypoplasia Type 1b (PCH1b) is a devastating genetic neurological disorder that preferentially affects specific regions of the brain. Typically, children born with PCH1b have structural defects in regions of the brain including those associated with key autonomic functions. Currently, there is no cure or treatment for the disease. PCH1b is caused by mutations in the RNA exosome component 3 ( EXOSC3 ) gene, which encodes a structural component of the ubiquitous and essential multi-subunit RNA exosome complex. The RNA exosome is critical for both precise processing and turnover of multiple classes of RNAs. To elucidate the functional consequences of amino acid changes in EXOSC3 that cause PCH1b, we exploited well-established genetic approaches in Drosophila melanogaster that model EXOSC3 mutations found in individuals with PCH1b. Using this system, we find that the Drosophila EXOSC3 homolog (termed Rrp40) is essential for normal development and has an important function in neurons. Furthermore, PCH1b missense mutations modeled in Rrp40 cause reduced viability and produce neuronal-specific phenotypes that correlate with altered levels of target RNAs that encode factors with key roles in neurons. These results provide a basis for understanding how amino acid changes that occur in the RNA exosome contribute to neuronal dysfunction and disease.
Abstract Despite the fact that all cells in an organism contain the same genetic information, the cells that comprise any organism have vastly distinct form and function. This diversity of form function is achieved because only a subset of the genetic information is transcribed into messenger RNA (mRNA) and translated into protein at any given time in any cell. While this regulation of gene expression occurs at many levels, extensive regulation occurs after an mRNA is produced prior to translation of that mRNA into protein. These regulatory events include nuclear processing to produce a mature mRNA, export to the cytoplasm, and a variety of potential fates in the cytoplasm including translation to protein and, ultimately, decay. All these regulatory events are termed post‐transcriptional regulation. Increasingly mutations in genes that encode components of this carefully orchestrated post‐transcriptional regulatory pathway are being linked to human disease, highlighting the importance of post‐transcriptional processing events. Key Concepts While all cells in an organism contain the same genetic material, these cells have vastly distinct forms and functions. There is extensive post‐transcriptional processing to produce a mature mRNA that occurs post‐transcriptionally. Numerous regulatory events in both the nucleus and the cytoplasm dictate when, where, and if an mRNA is translated into protein. Most mature mRNAs are produced through extensive processing steps that occur in the nucleus prior to export to the cytoplasm. There are many potential fates for an mRNA in the cytoplasm including transport to a specific cellular location, storage in cytoplasmic bodies/granules, translation and destruction. Turnover/decay of mRNA is a key regulatory step that contributes to the regulation of gene expression. Numerous mutations have been identified in genes encoding key post‐transcriptional processing factors linking post‐transcriptional processing to a variety of human diseases. Mutations in genes that encode key post‐transcriptional regulatory factors often cause tissue‐specific pathology. Many years of effort to define key pathways in post‐transcriptional regulation are starting to come to fruition in the realm of targeting these pathways for drug development. Gene expression and post‐transcriptional processing events are now the targets of successful therapies to treat some devastating diseases with many more exciting advances on the horizon.
Abstract The RNA exosome is an essential ribonuclease complex involved in the processing and degradation of both coding and noncoding RNAs. We present three patients with biallelic variants in EXOSC5 , which encodes a structural subunit of the RNA exosome. The common clinical features of these patients comprise failure to thrive, short stature, feeding difficulties, developmental delays that affect motor skills, hypotonia and esotropia. Brain MRI revealed cerebellar hypoplasia and ventriculomegaly. The first patient had a deletion involving exons 5-6 of EXOSC5 and a missense variant, p.Thr114Ile, that were inherited in trans , the second patient was homozygous for p.Leu206His, and the third patient had paternal isodisomy for chromosome 19 and was homozygous for p.Met148Thr. We employed three complementary approaches to explore the requirement for EXOSC5 in brain development and assess the functional consequences of pathogenic variants in EXOSC5 . Loss of function for the zebrafish ortholog results in shortened and curved tails and bodies, reduced eye and head size and edema. We modeled pathogenic EXOSC5 variants in both budding yeast and mammalian cells. Some of these variants show defects in RNA exosome function as well as altered interactions with other RNA exosome subunits. Overall, these findings expand the number of genes encoding RNA exosome components that have been implicated in human disease, while also suggesting that disease mechanism varies depending on the specific pathogenic variant.
Abstract The RNA exosome is an essential ribonuclease complex required for processing and/or degradation of both coding and non-coding RNAs. We identified five patients with biallelic variants in EXOSC5, which encodes a structural subunit of the RNA exosome. The clinical features of these patients include failure to thrive, short stature, feeding difficulties, developmental delays that affect motor skills, hypotonia and esotropia. Brain MRI revealed cerebellar hypoplasia and ventriculomegaly. While we ascertained five patients, three patients with distinct variants of EXOSC5 were studied in detail. The first patient had a deletion involving exons 5–6 of EXOSC5 and a missense variant, p.Thr114Ile, that were inherited in trans, the second patient was homozygous for p.Leu206His and the third patient had paternal isodisomy for chromosome 19 and was homozygous for p.Met148Thr. The additional two patients ascertained are siblings who had an early frameshift mutation in EXOSC5 and the p.Thr114Ile missense variant that were inherited in trans. We employed three complementary approaches to explore the requirement for EXOSC5 in brain development and assess consequences of pathogenic EXOSC5 variants. Loss of function for exosc5 in zebrafish results in shortened and curved tails/bodies, reduced eye/head size and edema. We modeled pathogenic EXOSC5 variants in both budding yeast and mammalian cells. Some of these variants cause defects in RNA exosome function as well as altered interactions with other RNA exosome subunits. These findings expand the number of genes encoding RNA exosome subunits linked to human disease while also suggesting that disease mechanism varies depending on the specific pathogenic variant.
The Estrogen-Related Receptor (ERR) family of nuclear receptors (NRs) serve key roles in coordinating triglyceride (TAG) accumulation with juvenile growth and development. In both insects and mammals, ERR activity promotes TAG storage during the post-embryonic growth phase, with loss-of-function mutations in mouse
RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia, mutations in the cap subunit gene EXOSC2 cause an apparently distinct clinical presentation that has been defined as a novel syndrome SHRF ( s hort stature, h earing loss, r etinitis pigmentosa, and distinctive f acies). We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by modeling pathogenic EXOSC2 missense mutations (p.Gly30Val and p.Gly198Asp) in the orthologous S. cerevisiae gene RRP4 . The resulting rrp4 mutant cells show defects in cell growth and RNA exosome function. Consistent with altered RNA exosome function, we detect significant transcriptomic changes in both coding and noncoding RNAs in rrp4-G226D cells that model EXOSC2 p.Gly198Asp, suggesting defects in nuclear surveillance. Biochemical and genetic analyses suggest that the Rrp4 G226D variant subunit shows impaired interactions with key RNA exosome cofactors that modulate the function of the complex. These results provide the first in vivo evidence that pathogenic missense mutations present in EXOSC2 impair the function of the RNA exosome. This study also sets the stage to compare exosomopathy models to understand how defects in RNA exosome function underlie distinct pathologies.
Abstract 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 ( S hort stature, H earing loss, R etinitis pigmentosa and distinctive F acies). 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.
The RNA exosome complex is a key component of RNA processing and quality control that both degrades and processes many classes of RNA. This complex is highly conserved among eukaryotes and was first identified and studied in budding yeast ( S. cerevisiae ). Mutations in the human EXOSC2 gene, which encodes a cap subunit of the RNA exosome, have been linked to a novel syndrome characterized by retinitis pigmentosa, progressive hearing loss, premature aging, short stature, mild intellectual disability and distinctive gestalt. While the amino acid substitutions in EXOSC2 that cause this syndrome are known, how these amino acid changes impact RNA exosome function is not. The goal of my project is to analyze the functional consequences of retinitis pigmentosa‐linked amino acid substitutions modeled in the budding yeast ortholog of EXOSC2, Rrp4. The two variants I have analyzed, rrp4‐G58V and rrp4‐G226D , correspond to patient mutations G30V and G198D, respectively. I first assessed growth of the mutant strains compared to wildtype yeast cells, which revealed that rrp4‐G226D mutant cells exhibit a growth defect at 37°C, whereas the rrp4‐G58V mutant cells grow normally. To assess whether these amino acid substitutions affect Rrp4 protein levels, I used immunoblotting. Results of this analysis reveal that the rrp4‐G58V and rrp4‐G226D proteins are expressed, but at somewhat reduced level compared to wildtype Rrp4. In the future, I will continue characterization of the mutants by using biochemical approaches to study the assembly of the RNA exosome complex and genetic analysis of rrp4 mutant interactions with RNA exosome cofactors. Support or Funding Information EMORY INITIATIVE TO MAXIMIZE STUDENT DEVELOPMENT Emory Initiative to Maximize Student Development: R25 GM125598 Neurodevelopmental Role of an RNA Binding Protein Required for Cognitive Function: R01 MH107305 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
