title: Specific interaction with the nuclear transporter importin α2 can modulate paraspeckle protein 1 delivery to nuclear paraspeckles.
abstract: Importin (IMP) superfamily members mediate regulated nucleocytoplasmic transport, which is central to key cellular processes. Although individual IMPα proteins exhibit dynamic synthesis and subcellular localization during cellular differentiation, including during spermatogenesis, little is known of how this affects cell fate. To investigate how IMPαs control cellular development, we conducted a yeast two-hybrid screen for IMPα2 cargoes in embryonic day 12.5 mouse testis, a site of peak IMPα2 expression coincident with germ-line masculization. We identified paraspeckle protein 1 (PSPC1), the original defining component of nuclear paraspeckles, as an IMPα2-binding partner. PSPC1-IMPα2 binding in testis was confirmed in immunoprecipitations and pull downs, and an enzyme-linked immunosorbent assay-based assay demonstrated direct, high-affinity PSPC1 binding to either IMPα2/IMPβ1 or IMPα6/IMPβ1. Coexpression of full-length PSPC1 and IMPα2 in HeLa cells yielded increased PSPC1 localization in nuclear paraspeckles. High-throughput image analysis of >3500 cells indicated IMPα2 levels can directly determine PSPC1-positive nuclear speckle numbers and size; a transport-deficient IMPα2 isoform or small interfering RNA knockdown of IMPα2 each reduced endogenous PSPC1 accumulation in speckles. This first validation of an IMPα2 nuclear import cargo in fetal testis provides novel evidence that PSPC1 delivery to paraspeckles, and consequently paraspeckle function, may be controlled by modulated synthesis of specific IMPs.
authors.affiliation: Department of Anatomy and Developmental Biology, Monash University, Melbourne, VIC 3800, Australia ARC Centre of Excellence in Biotechnology and Development, Australia.
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Major
authors.affiliation: Center for Reproductive Biology and School of Molecular Biosciences, Washington State University, Pullman, WA 99163.
authors.forename: Cathryn A
authors.initials: CA
authors.lastname: Hogarth
authors.affiliation: ARC Centre of Excellence in Biotechnology and Development, Australia Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC 3800, Australia.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Miyamoto
authors.affiliation: ARC Centre of Excellence in Biotechnology and Development, Australia MIMR-PHI Institute of Medical Research, Monash Health Translation Precinct, Clayton, VIC 3168, Australia.
authors.forename: Mai A
authors.initials: MA
authors.lastname: Sarraj
authors.affiliation: Department of Epidemiology and Preventive Medicine, School of Public Health and Preventive Medicine, Monash University, Melbourne, VIC 3004, Australia.
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Smith
authors.affiliation: ARC Centre of Excellence in Biotechnology and Development, Australia Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Koopman
authors.affiliation: Faculty of Engineering Science, Yokohama National University, Yokohama 2408501, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Kurihara
authors.affiliation: ARC Centre of Excellence in Biotechnology and Development, Australia Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC 3800, Australia.
authors.forename: David A
authors.initials: DA
authors.lastname: Jans
authors.affiliation: Department of Anatomy and Developmental Biology, Monash University, Melbourne, VIC 3800, Australia ARC Centre of Excellence in Biotechnology and Development, Australia Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC 3800, Australia School of Clinical Sciences, Monash Health Translation Precinct, Monash University, Clayton, VIC 3168, Australia.
authors.forename: Kate L
authors.initials: KL
authors.lastname: Loveland
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D047468:Immunoprecipitation; D008297:Male; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D013737:Testis / Q000196:embryology / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D028901:alpha Karyopherins
chemical_list: C490631:Kpna2 protein, mouse; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D028901:alpha Karyopherins; C494389:paraspeckle protein 1, mouse