Biology subjects

Sterrett, M. C.

Publications and source records attributed to Sterrett, M. C..

7 recordsLinked to original sources

bioRxiv2024

The Drosophila Estrogen-Related Receptor promotes triglyceride storage within the larval fat body

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 Esrra and Drosophila melanogaster dERR inducing a lean phenotype. However, the role of insect ERRs in controlling TAG accumulation within adipose tissue remains poorly understood, as previous transcriptomic and metabolomic studies relied on whole animal analyses. Here we address this shortcoming by using tissue-specific approaches to examine the role of dERR in regulating lipid metabolism within the Drosophila larval fat body. We find that dERR autonomously promotes TAG accumulation within fat body cells and regulates expression of genes involved in glycolysis, {beta}-oxidation, and mevalonate metabolism. As an extension of these results, we not only discovered that dERR mutant fat bodies exhibit decreased expression of known dHNF4 target genes but also found that dHNF4 activity is decreased in dERR mutants. Overall, our findings indicate that dERR plays a multifaceted role in the larval fat body to coordinate lipid storage with developmental growth and hint at a conserved mechanism by which ERR and HNF4 homologs coordinately regulate metabolic gene expression.

developmental 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↗

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↗

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

Biallelic variants in the RNA exosome gene EXOSC5 are associated with developmental delays, short stature, cerebellar hypoplasia and motor weakness

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.

genetics↗

bioRxiv2019

A Drosophila Model of Pontocerebellar Hypoplasia Reveals a Critical Role for the RNA Exosome in Neurons

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 SummaryPontocerebellar 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.

genetics↗

bioRxiv2019

Lactate and glycerol-3-phosphate metabolism cooperatively regulate growth and redox balance during Drosophila melanogaster larval development

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 developmental delays, synthetic lethality, and aberrant carbohydrate metabolism. 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.

developmental biology↗