The newly organized ASBMB Student Chapter at Emory University aims to provide networking and career‐development opportunities coupled with access to research and science outreach. To broaden our impact, we have established a collaboration with the graduate student led organization, E mory Sc ience A dvocacy N etwork (EScAN). EScAN provides a forum to discuss current issues facing the biomedical field and determine how to support robust federal funding of biomedical research. The two organizations together are preparing for the annual EScAN‐sponsored Letter Writing Campaign. We aim to achieve record‐high participation this year of both undergraduate and graduate students from diverse educational and demographic backgrounds. Increases in political awareness and advocacy such as these will enhance the potential for productive legislative action. We will also participate in local (Atlanta) and national (Washington, D.C.) Hill Day in September 2019. In preparation for these events, we have organized focused workshops, including “Communicating Science to Non‐Scientists,” to ensure an effective interface between congress people and young scientists. Aligned with the ASBMB Student Chapter mission statement, our efforts will provide networking and career‐development opportunities for undergraduate students. This collaboration is supported and facilitated by a volunteer graduate student advisor, serving as a liaison between EScAN and the ASBMB Student Chapter. We will assess the outcomes of this collaboration by tracking the number of undergraduate students that participate in the Letter Writing Campaign as an initial metric of engagement. We also are working together with a sociologist to develop pre‐ and post‐surveys to assess the level of understanding of funding for biomedical science and commitment to advocacy for the ASBMB Student Chapter undergraduates. Ultimately, we seek to build a synergistic network of undergraduate and graduate students who are knowledgeable and engaged in scientific outreach and advocacy. Support or Funding Information R01 GM130147 01, R25 GM125598 01, R01 MH107305 04 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
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.
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 Background: PDA will become the 2nd leading cause of cancer-related mortality in the US by 2020. A recent Phase III randomized controlled trial revealed a 4 month overall survival benefit in metastatic PDA with FOLFIRINOX (Folinic acid, 5-Fluorouracil (5FU), Irinotecan, and Oxaliplatin) compared to gemcitabine, the standard of care. However, the long-term clinical efficacy of FOLFIRINOX and other chemotherapy regimens are limited by tumor-associated drug resistance driven by factors in the tumor microenvironment (e.g., hypoxia). We identified a novel drug resistance mechanism driven by the hypoxia-inducible pro-oncogenic kinase PIM1 and regulated by the RNA binding protein HuR. Herein, we launched into developing a strategy to target this tractable mechanism in an effort to optimize current therapeutic treatments for PDA. Methods: To model hypoxia, PDA cells were incubated in 1% O2 and responses to 5FU or oxaliplatin assessed to obtain IC50 doses. Stabilizing interactions between the RNA-binding protein HuR and PIM1 mRNA were quantified in vitro through binding assays, and confirmed in patients by immunohistochemistry in resected PDAs (n = 44). The contribution of HuR-mediated regulation of PIM1 to resistance to 5FU or oxaliplatin in hypoxia was examined using MS-444 (Novartis), a low-molecular-weight HuR inhibitor. Results: In response to hypoxia, HuR translocates from the nucleus to the cytoplasm where it binds and stabilizes the PIM1 mRNA transcript, thus amplifying PIM1 translation and protein expression. Clinically, we identified a positive correlation (p = 0.011) between cytoplasmic HuR and PIM1 protein expression in a cohort of PDA patients from our institution. In vitro mechanistic studies demonstrated that hypoxia-mediated induction of PIM1 overexpression enhanced DNA repair and evaded the apoptotic response elicited by hypoxic stress. Targeted inhibition of HuR by the HuR inhibitor MS-444 abrogated hypoxia-induced PIM1 overexpression, enhancing PDA cell sensitivity to oxaliplatin and 5FU (P<0.001). Conclusion: The mRNA-stability factor HuR post-transcriptionally induces PIM1 expression under hypoxic conditions, and thereby promotes hypoxia-induced chemoresistance. Ongoing pre-clinical studies will evaluate pharmacologic inhibition of HuR's regulation of PIM1 (e.g., MS-444) as a novel modality to enhance the therapeutic value of FOLFIRINOX for the treatment of metastatic PDA. Citation Format: Fernando F. Blanco, Masaya Jimbo, Liz Enyenihi, Nicole Meisner-Kober, Eric Londin, Isidore Rigoutsos, Makarand Risbud, Peter McCue, Charles Yeo, Jordan Winter, Jonathan R. Brody. Targeting tumor-associated hypoxia to overcome chemoresistance in pancreatic ductal adenocarcinoma (PDA). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2113. doi:10.1158/1538-7445.AM2015-2113
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.
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.
Noonan syndrome with multiple lentigines (NSML) is a rare autosomal dominant disorder that presents with cardio-cutaneous-craniofacial defects. Hypertrophic cardiomyopathy (HCM) represents the major life-threatening presentation in NSML. Mutations in the PTPN11 gene that encodes for the protein tyrosine phosphatase (PTP), SHP2, represents the predominant cause of HCM in NSML. NSML-associated PTPN11 mutations render SHP2 catalytically inactive with an "open" conformation. NSML-associated PTPN11 mutations cause hypertyrosyl phosphorylation of the transmembrane glycoprotein, protein zero-related (PZR), resulting in increased SHP2 binding. Here we show that NSML mice harboring a tyrosyl phosphorylation-defective mutant of PZR (NSML/PZRY242F) that is defective for SHP2 binding fail to develop HCM. Enhanced AKT/S6 kinase signaling in heart lysates of NSML mice was reversed in NSML/PZRY242F mice, demonstrating that PZR/SHP2 interactions promote aberrant AKT/S6 kinase activity in NSML. Enhanced PZR tyrosyl phosphorylation in the hearts of NSML mice was found to drive myocardial fibrosis by engaging an Src/NF-κB pathway, resulting in increased activation of IL-6. Increased expression of IL-6 in the hearts of NSML mice was reversed in NSML/PZRY242F mice, and PZRY242F mutant fibroblasts were defective for IL-6 secretion and STAT3-mediated fibrogenesis. These results demonstrate that NSML-associated PTPN11 mutations that induce PZR hypertyrosyl phosphorylation trigger pathophysiological signaling that promotes HCM and cardiac fibrosis.
Abstract Background Alzheimer’s disease (AD), an age‐associated neurodegenerative disorder, is characterized by progressive neuronal loss and the accumulation of misfolded proteins such as amyloid‐β and tau. While neuroinflammation, mediated by microglia and brain‐resident macrophages, plays a pivotal role in AD pathogenesis, the intricate interactions among age, genes, and other risk factors remain elusive. Somatic mutations, known to accumulate with age, instigate clonal expansion across diverse cell types, impacting both cancer and non‐cancerous conditions. Method Utilizing molecular‐barcoded deep panel sequencing, which enables sensitive detection of somatic mutations with allele fractions as low as 0.1%, we profiled clonal somatic mutations among 149 cancer driver genes in 311 prefrontal cortex samples from AD patients and matched controls. Fluorescence‐activated nuclei sorting and single‐nucleus RNA sequencing were further used to study the cell‐type composition and transcriptomic impact of somatic mutations. Result Our study unveiled an elevated occurrence of somatic single‐nucleotide variants and insertions/deletions within cancer driver genes in AD brains. Recurrent somatic mutations, often multiple, were observed in genes associated with clonal hematopoiesis (CH). Remarkably, these somatic mutations were specifically enriched in CSF1R+ microglia and exhibited signals of positive selection, suggesting mutation‐driven microglial clonal expansion (MiCE) in AD brains. Single‐nucleus RNA sequencing of temporal neocortex samples from an additional 62 AD patients and matched controls revealed a nominal increase in mosaic chromosomal alterations (mCAs) associated with CH in AD microglia, with microglia carrying mCA exhibiting upregulated pro‐inflammatory genes, resembling the transcriptomic features of the disease‐associated state in AD. Conclusion Our findings indicate that proliferation‐related somatic mutations in microglia are prevalent in normal aging but further enriched in AD, driving MiCE and promoting inflammatory, disease‐related microglial signatures. This study provides crucial insights into microglial clonal dynamics in AD, potentially paving the way for novel approaches to AD diagnosis and therapy.
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 .
