key: cord-0863853-igvdso37
authors: Mast, Elizabeth; Bieser, Kayla L; Abraham-Villa, Mary; Adams, Vanessa; Akinlehin, Akinwonuola J; Aquino, Lynarose Z; Austin, Joseph L; Austin, Abigail K; Beckham, Carissa N; Bengson, Ethan J; Bieszk, Amanda; Bogard, Brianna L; Brennan, Rowan C; Brnot, Rebecca M; Cirone, Nicholas J; Clark, Mason R; Cooper, Brianna N; Cruz, Dennys; Daprizio, Katlyn A; DeBoe, Jason; Dencker, Michaela M; Donnelly, Laura L; Driscoll, Leanne; DuBeau, Ryan J; Durso, Sirada W; Ejub, Adam; Elgosbi, Waad; Estrada, Melanie; Evins, Kaeli; Fox, Pearl D; France, Jacob M; Franco Hernandez, Maira G; Garcia, Lizbeth A; Garl, Olivia; Gorsuch, Myeerah R; Gorzeman-mohr, Mikayla A; Grothouse, Madison E; Gubbels, Megan E; Hakemiamjad, Romina; Harvey, Chloé V; Hoeppner, Madeline A; Ivanov, Jessica L; Johnson, Veronica M; Johnson, Jessica L; Johnson, Ashton; Johnston, Kaleigh; Keller, Katie R; Kennedy, Breanna T; Killian, Levi R; Klumb, Marissa; Koehn, Olivia L; Koym, Aaron S; Kress, Kari J; Landis, Regan E; Lewis, Kaitlyn N; Lim, Enosh; Lopez, Ilcen K; Lowe, D’Artagnan; Luengo Carretero, Paula; Lunaburg, Grace; Mallinder, Samantha L; Marshall, Natalie A; Mathew, Jessica; Mathew, Jasmine; Mcmanaway, Hailee S; Meegan, Emily N; Meyst, Jacob D; Miller, Meredith J; Minogue, Colin K; Mohr, Alina A; Moran, Cristhian I; Moran, Adrian; Morris, Morgan D; Morrison, Michael D; Moses, Emmily A; Mullins, Cade J; Neri, Citlalli I; Nichols, Jess M; Nickels, Breanna R; Okai, Akosua M; Okonmah, Chiedu; Paramo, Makena; Paramo, Meagan; Parker, Sydney L; Parmar, Neil K; Paschal, Jacob; Patel, Prem; Patel, Deep; Perkins, Erica B; Perry, Madelyn M; Perry, Zachary; Pollock, Amanda A; Portalatin, Oxxyris; Proffitt, Kamron S; Queen, Jason T; Quemeneur, Alexis C; Richardson, Amelia G; Rosenberger, Kaylee; Rutherford, Allison M; Santos-Perez, Itchel X; Sarti, Christy Y; Schouweiler, Lacey J; Sessing, Lauren M; Setaro, Sara O; Silvestri, Christopher F; Smith, Olivia A; Smith, Mackenzie J; Sumner, Jayson C; Sutton, Rachel R; Sweckard, Lindsay; Talbott, Nicholas B; Traxler, Peyton A; Truesdell, Jenna; Valenti, Aaron F; Verace, Leif; Vijayakumar, Pragathi; Wadley, William L; Walter, Katherine E; Williams, Ayanna R; Wilson, Trey J; Witbeck, Makayla A; Wobler, Trinity M; Wright, Lucas J; Zuczkowska, Karolina A; Devergne, Olivier; Hamill, Danielle R; Shah, Hemin P; Siders, Jamie; Taylor, Elizabeth E; Vrailas-Mortimer, Alysia D; Kagey, Jacob D
title: Genetic mapping of Uba3 (O.2.2) , a pupal lethal mutation in Drosophila melanogaster
date: 2022-03-18
journal: microPublication biology
DOI: 10.17912/micropub.biology.000542
sha: db6db211b8abddafe781607e981c6e348edd1562
doc_id: 863853
cord_uid: igvdso37

nan

Induced ethyl methanesulfonate (EMS) mutations were genetically screened utilizing the FLP/FRT recombinase system to study cell overgrowth phenotypes in mosaic eyes of Drosophila melanogaster. EMS is a mutagen typically resulting in single nucleotide substitutions. Homozygosity was induced in the eye utilizing the FLP/FRT mitotic recombination system to study phenotype developments without homozygous lethality throughout the remainder of the specimen. It has been previously reported that genes impacting cell growth or division (such as Dark, Ptc) trigger apoptosis leading to cell death (Akdemir et al. 2006) . In the presence of Dark 82 , apoptosis is blocked, and the overgrowth phenotype persists in an observable manner (Kagey et al. 2012 Figures 1C and 1D ).

To identify the gene locus responsible for the O.2.2 mutation, complementation mapping was conducted by undergraduate researchers at Nevada State College, Northern Illinois University, Albion College, Ohio Northern University, and Ohio Wesleyan University participating in the Fly-CURE consortium. This mapping was conducted during the COVID-19 pandemic which resulted in data collection through in-person, hybrid, and virtual courses. Complementation mapping has been successfully utilized by undergraduates to map the location of mutations driving the phenotypes produced from the FLP/FRT screen (Talley et al. 2021 Figure 1E ). Students then selected seven genes within this region for further study ( Table 1) . Alleles of six of these genes complemented the O.2.2 mutation (Table 1) . We tested two alleles of Uba3: Uba3 G8197 and Uba3 SH2028 , which are both homozygous lethal transgenic insertion mutations and Uba3 SH2028 has previously been identified as a null mutation (Du et al. 2011) . We found that the Uba3 G8197 allele complemented the O.2.2 mutation while the Uba3 SH2028 mutation failed to complement. We further investigated the Uba3 G8197 stock and found that it was no longer homozygous lethal, suggesting that this stock had lost the mutation in Uba3. The data we provided to the BDSC resulted in the removal of the Uba3 G8197 stock. As the Uba3 SH2028 allele failed to complement O.2.2 (Table 1) , students designed primers to different regions of the Uba3 gene and identified a nonsense mutation at 2R:13,859,838 (Gln281Stop at amino acid 281) leading to a premature stop codon.

Based upon the observed phenotype and genetic mapping in Drosophila melanogaster, we conclude that O.2.2 is a novel allele of Uba3 (Uba3 O.2.2 ), resulting in a mosaic pupal lethal phenotype. Uba3 encodes for a ubiquitin-like activating enzyme that adds NEDD8 (neural precursor cell expressed, developmentally downregulated 8; E1 NEDD8 ) to proteins in a process called neddylation. Neddylation and the ubiquitin-proteasome system (UPS), which regulate proteolysis in the cell (Du et al. 2011 , Nalepa et al. 2006 , are critical for regulation of many developmental processes including multiple pathways for cell cycle progression (Tateishi et al. 2001 , Nalepa et al. 2006 . Evidence of Uba3 knockdown in Drosophila and mice result in early larval/embryonic lethality (Du et al. 2011 , Tateishi et al. 2001 . The nonsense mutation we report is predicted to eliminate the E2-binding domain (associated with amino acids 354-443), which is a necessary conjugating enzyme in the NEED8 cascade (Huang et al. 2005) . The elimination of this critical domain is consistent with the lethal phenotype observed in Uba3 O.2.2 .

Due to its role in disrupting the cell cycle, Uba3 is a plausible early target for disrupting the cell cycle of the E1 activating enzyme in cancer cells. An inhibitor of E1 NEDD8 , MLN4924, has been utilized in vitro to target cancer cells but its effectiveness was reduced in cells with Uba3 mutations leading to resistance of MLN4924 (Xu et al. 2014 ). More recently, MLN4924, has shown promise in clinical trials for inhibiting growth and migration of cancer cells but a greater mechanistic understanding of the role of Uba3 in neddylation is still required as there appears to be an interplay between high glucose levels and the upregulation of Uba3 (Du et al. 2021) . Future characterizations in Drosophila may help to elucidate these complex relationships.

Reagents w -; FRT42D, Dark 82 /CyO (Akdemir et al., 2006) (Cook et al., 2012) Additional Bloomington Stocks (See Table 1 

Funding: Fly-CURE (K. Bieser, J. Kagey, and A. Vrailas-Mortimer) is funded by a National Science Foundation IUSE Award (NSF 2021146).

Author Contributions: Elizabeth Mast: data curation, formal analysis, writing -original draft, investigation, writing -review editing. Kayla L Bieser: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project, supervision, validation, writing -original draft, writing -review editing, visualization. Mary Abraham-Villa: data curation, formal analysis, investigation, writing -review editing. Vanessa Adams: data curation, formal analysis, investigation, writing -review editing. Akinwonuola J Akinlehin: data curation, formal analysis, investigation, writing -review editing. Lynarose Z Aquino: data curation, formal analysis, investigation, writing -review editing. Joseph L Austin: data curation, formal analysis, investigation, writing -review editing. Abigail K Austin: data curation, formal analysis, investigation, writing -review editing. Carissa N Beckham: data curation, formal analysis, investigation, writing -review editing. Ethan J Bengson: data curation, formal analysis, investigation, writing -review editing. Amanda Bieszk: data curation, formal analysis, investigation, writing -review editing. Brianna L Bogard: data curation, formal analysis, investigation, writing -review editing. Rowan C Brennan: data curation, formal analysis, investigation, writing -review editing. Rebecca M Brnot: data curation, formal analysis, investigation, writing -review editing. Nicholas J Cirone: data curation, formal analysis, investigation, writing -review editing. Mason R Clark: data curation, formal analysis, investigation, writing -review editing. Brianna N Cooper: data curation, formal analysis, investigation, writing -review editing. Dennys Cruz: data curation, formal analysis, investigation, writing -review editing. Katlyn A Daprizio: data curation, formal analysis, investigation, writingreview editing. Jason DeBoe: data curation, formal analysis, investigation, writing -review editing. Michaela M Dencker: data curation, formal analysis, investigation, writing -review editing. Laura L Donnelly: data curation, formal analysis, investigation, writing -review editing. Leanne Driscoll: data curation, formal analysis, investigation, writing -review editing. Ryan J DuBeau: data curation, formal analysis, investigation, writing -review editing. Sirada W Durso: data curation, formal analysis, investigation, writing -review editing. Adam Ejub: data curation, formal analysis, investigation, writing -review editing. Waad Elgosbi: data curation, formal analysis, investigation, writing -review editing. Melanie Estrada: data curation, formal analysis, investigation, writing -review editing. Kaeli Evins: data curation, formal analysis, investigation, writingreview editing. Pearl D Fox: data curation, formal analysis, investigation, writing -review editing. Jacob M France: data curation, formal analysis, investigation, writing -review editing. Maira G Franco Hernandez: data curation, formal analysis, investigation, writing -review editing. Lizbeth A Garcia: data curation, formal analysis, investigation, writing -review editing. Olivia Garl: data curation, formal analysis, investigation, writing -review editing. Myeerah R Gorsuch: data curation, formal analysis, investigation, writing -review editing. Mikayla A Gorzeman-mohr: data curation, formal analysis, investigation, writing -review editing. Madison E Grothouse: data curation, formal analysis, investigation, writing -review editing. Megan E Gubbels: data curation, formal analysis, investigation, writing -review editing. Romina Hakemiamjad: data curation, formal analysis, investigation, writing -review editing. Chloé V Harvey: data curation, formal analysis, investigation, writingreview editing. Madeline A Hoeppner: data curation, formal analysis, investigation, writing -review editing. Jessica L Ivanov: data curation, formal analysis, investigation, writing -review editing. Veronica M Johnson: data curation, formal analysis, investigation, writing -review editing. Jessica L Johnson: data curation, formal analysis, investigation, writing -review editing. Ashton Johnson: data curation, formal analysis, investigation, writing -review editing. Kaleigh Johnston: data curation, formal analysis, investigation, writing -review editing. Katie R Keller: data curation, formal analysis, investigation, writing -review editing. Breanna T Kennedy: data curation, formal analysis, investigation, writing -review editing. Levi R Killian: data curation, formal analysis, investigation, writing -review editing. Marissa Klumb: data curation, formal analysis, investigation, writing -review editing. Olivia L Koehn: data curation, formal analysis, investigation, writing -review editing. Aaron S Koym: data curation, formal analysis, investigation, writing -review editing. Kari J Kress: data curation, formal analysis, investigation, writing -review editing. Regan E Landis: data curation, formal analysis, investigation, writing -review editing. Kaitlyn N Lewis: data curation, formal analysis, investigation, writing -review editing. Enosh Lim: data curation, formal analysis, investigation, writing -review editing. Ilcen K Lopez: data curation, formal analysis, investigation, writingreview editing. D'Artagnan Lowe: data curation, formal analysis, investigation, writing -review editing. Paula Luengo Carretero: data curation, formal analysis, investigation, writing -review editing. Grace Lunaburg: data curation, formal analysis, investigation, writing -review editing. Samantha L Mallinder: data curation, formal analysis, investigation, writingreview editing. Natalie A Marshall: data curation, formal analysis, investigation, writing -review editing. Jessica Mathew: data curation, formal analysis, investigation, writing -review editing. Jasmine Mathew: data curation, formal analysis, investigation, writing -review editing. Hailee S Mcmanaway : data curation, formal analysis, investigation, writing -review editing. Emily N Meegan: data curation, formal analysis, investigation, writing -review editing. Jacob D Meyst: data curation, formal analysis, investigation, writing -review editing. Meredith J Miller: data curation, formal analysis, investigation, writing -review editing. Colin K Minogue: data curation, formal analysis, investigation, writing -review editing. Alina A Mohr: data curation, formal analysis, investigation, writing -review editing. Cristhian I Moran: data curation, formal analysis, investigation, writing -review editing. Adrian Moran: data curation, formal analysis, investigation, writing -review editing. Morgan D Morris: data curation, formal analysis, investigation, writing -review editing. Michael D Morrison: data curation, formal analysis, investigation, writing -review editing. Emmily A Moses: data curation, formal analysis, investigation, writing -review editing. Cade J Mullins: data curation, formal analysis, investigation, writing -review editing. Citlalli I Neri: data curation, formal analysis, investigation, writing -review editing. Jess M Nichols: data curation, formal analysis, investigation, writing -review editing. Breanna R Nickels: data curation, formal analysis, investigation, writing -review editing. Akosua M Okai: data curation, formal analysis, investigation, writing -review editing. Chiedu Okonmah: data curation, formal analysis, investigation, writing -review editing. Makena Paramo: data curation, formal analysis, investigation, writing -review editing. Meagan Paramo: data curation, formal analysis, investigation, writing -review editing. Sydney L Parker: data curation, formal analysis, investigation, writing -review editing. Neil K Parmar: data curation, formal analysis, investigation, writing -review editing. Jacob Paschal: data curation, formal analysis, investigation, writing -review editing. Prem Patel: data curation, formal analysis, investigation, writing -review editing. Deep Patel: data curation, formal analysis, investigation, writing -review editing. Erica B Perkins: data curation, formal analysis, investigation, writing -review editing. Madelyn M Perry: data curation, formal analysis, investigation, writing -review editing. Zachary Perry: data curation, formal analysis, investigation, writing -review editing. Amanda A Pollock: data curation, formal analysis, investigation, writing -review editing. Oxxyris Portalatin: data curation, formal analysis, investigation, writing -review editing. Kamron S Proffitt: data curation, formal analysis, investigation, writing -review editing. Jason T Queen: data curation, formal analysis, investigation, writing -review editing. Alexis C Quemeneur: data curation, formal analysis, investigation, writing -review editing. Amelia G Richardson: data curation, formal analysis, investigation, writing -review editing. Kaylee Rosenberger: data curation, formal analysis, investigation, writing -review editing. Allison M Rutherford: data curation, formal analysis, investigation, writing -review editing. Itchel X Santos-Perez: data curation, formal analysis, investigation, writing -review editing. Christy Y Sarti: data curation, formal analysis, investigation, writing -review editing. Lacey J Schouweiler: data curation, formal analysis, investigation, writing -review editing. Lauren M Sessing: data curation, formal analysis, investigation, writing -review editing. Sara O Setaro: data curation, formal analysis, writing -review editing, investigation. Christopher F Silvestri: data curation, formal analysis, investigation, writing -review editing. Olivia A Smith: data curation, formal analysis, investigation, writing -review editing. Mackenzie J Smith: data curation, formal analysis, investigation, writing -review editing. Jayson C Sumner: data curation, formal analysis, investigation, writing -review editing. Rachel R Sutton: data curation, formal analysis, investigation, writing -review editing. Lindsay Sweckard: data curation, formal analysis, investigation, writing -review editing. Nicholas B Talbott: data curation, formal analysis, investigation, writing -review editing. Peyton A Traxler: data curation, formal analysis, investigation, writing -review editing. Jenna Truesdell: data curation, formal analysis, investigation, writing -review editing. Aaron F Valenti: data curation, formal analysis, investigation, writing -review editing. Leif Verace: data curation, formal analysis, investigation, writing -review editing. Pragathi Vijayakumar: data curation, formal analysis, investigation, writing -review editing. William L Wadley: data curation, formal analysis, investigation, writing -review editing. Katherine E Walter: data curation, formal analysis, investigation, writing -review editing. Ayanna R Williams: data curation, formal analysis, investigation, writing -review editing. Trey J Wilson: data curation, formal analysis, investigation, writing -review editing. Makayla A Witbeck: data curation, formal analysis, investigation, writing -review editing. Trinity M Wobler: data curation, formal analysis, investigation, writing -review editing. Lucas J Wright: data curation, formal analysis, investigation, writing -review editing. Karolina A Zuczkowska: data curation, formal analysis, investigation, writing -review editing. Olivier Devergne: data curation, formal analysis, investigation, project, validation, writing -review editing, supervision. Danielle R Hamill: data curation, formal analysis, investigation, project, validation, writing -review editing, supervision. Hemin P Shah: data curation, formal analysis, investigation, supervision, project, validation, writing -review editing. Jamie Siders: data curation, formal analysis, investigation, project, supervision, validation, writing -review editing. Elizabeth E Taylor : data curation, formal analysis, investigation, writing -review editing, supervision, validation, project. Alysia D Vrailas-Mortimer: data curation, formal analysis, investigation, project, validation, funding acquisition, writingreview editing. Jacob D Kagey: conceptualization, data curation, writing -review editing, validation, resources, project, funding acquisition, methodology, investigation, formal analysis, visualization. 

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The generation of chromosomal deletions to provide extensive coverage and subdivision of the Drosophila melanogaster genome

The Absence of PTEN in Breast Cancer Is a Driver of MLN4924 Resistance

In vivo RNAi screen reveals neddylation genes as novel regulators of Hedgehog signaling

Structural basis for recruitment of Ubc12 by an E2 binding domain in NEDD8's E1

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Drug discovery in the ubiquitin-proteasome system

The NEDD8 system is essential for cell cycle progression and morphogenetic pathway in mice

Genetic mapping and phenotypic analysis of shot H.3.2 in Drosophila melanogaster

Mutations in UBA3 confer resistance to the NEDD8-activating enzyme inhibitor MLN4924 in human leukemic cells

Acknowledgments: Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) and Kyoto DGRC were used in this study.