title: RpL22e, but not RpL22e-like-PA, is SUMOylated and localizes to the nucleoplasm of Drosophila meiotic spermatocytes.
abstract: Duplicated ribosomal protein (Rp) gene families often encode highly similar or identical proteins with redundant or unique roles. Eukaryotic-specific paralogues RpL22e and RpL22e-like-PA are structurally divergent within the N terminus and differentially expressed, suggesting tissue-specific functions. We previously identified RpL22e-like-PA as a testis Rp. Strikingly, RpL22e is detected in immunoblots at its expected molecular mass (m) of 33 kD and at increasing m of ~43-55 kD, suggesting RpL22e post-translational modification (PTM). Numerous PTMs, including N-terminal SUMOylation, are predicted computationally. Based on S2 cell co-immunoprecipitations, bacterial-based SUMOylation assays and in vivo germline-specific RNAi depletion of SUMO, we conclude that RpL22e is a SUMO substrate. Testis-specific PTMs are evident, including a phosphorylated version of SUMOylated RpL22e identified by in vitro phosphatase experiments. In ribosomal profiles from S2 cells, only unconjugated RpL22e co-sediments with active ribosomes, supporting an extra-translational role for SUMOylated RpL22e. Ectopic expression of an RpL22e N-terminal deletion (lacking SUMO motifs) shows that truncated RpL22e co-sediments with polysomes, implying that RpL22e SUMOylation is dispensable for ribosome biogenesis and function. In mitotic germ cells, both paralogues localize within the cytoplasm and nucleolus. However, within meiotic cells, phase contrast microscopy and co-immunohistochemical analysis with nucleolar markers nucleostemin1 and fibrillarin reveals diffuse nucleoplasmic, but not nucleolar RpL22e localization that transitions to a punctate pattern as meiotic cells mature, suggesting an RpL22e role outside of translation. Germline-specific knockdown of SUMO shows that RpL22e nucleoplasmic distribution is sensitive to SUMO levels, as immunostaining becomes more dispersed. Overall, these data suggest distinct male germline roles for RpL22e and RpL22e-like-PA.
authors.affiliation: Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Kearse
authors.forename: Jill A
authors.initials: JA
authors.lastname: Ireland
authors.forename: Smrithi M
authors.initials: SM
authors.lastname: Prem
authors.forename: Alex S
authors.initials: AS
authors.lastname: Chen
authors.forename: Vassie C
authors.initials: VC
authors.lastname: Ware
mesh_terms: D000818:Animals; D001692:Biological Transport; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D042581:Intranuclear Space; D008297:Male; D008540:Meiosis; D010766:Phosphorylation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013090:Spermatocytes / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D058207:Sumoylation; D013737:Testis / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; D020033:Protein Isoforms; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C489368:RpL22 protein, Drosophila; C559091:Rpl22-like protein, Drosophila
keywords: Drosophila; RpL22e; RpL22e-like-PA; SUMOylation; duplicated ribosomal proteins; male germline; phosphorylation; post-translational modification; ribosomal protein paralogues