Specific interaction of the nuclear transporter importin alpha 2 with paraspeckle protein 1 modulates nuclear paraspeckle formation — ASN Events
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  2. Session 2.2: How to build a functional spermatozoon
  3. Specific interaction of the nuclear transporter importin alpha 2 with paraspeckle protein 1 modulates nuclear paraspeckle formation

Specific interaction of the nuclear transporter importin alpha 2 with paraspeckle protein 1 modulates nuclear paraspeckle formation (#11)

Andrew T Major 1 2 , Cathryn A Hogarth 3 , Yoichi Miyamoto 2 4 , Mai A Sarraj 5 , Catherine L Smith 6 , Peter Koopman 2 7 , Yasuyuki Kurihara 8 , David A Jans 2 4 , Kate L Loveland 1 2 4
  1. Department of Anatomy and Developmental Biology, Monash University, Melbourne, VIC, Australia
  2. The ARC Centre of Excellence in Biotechnology and Development, Australia
  3. Center for Reproductive Biology and School of Molecular Biosciences, Washington State University, Pullman, Washington, USA
  4. Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, Australia
  5. Prince Henry’s Institute, Melbourne, VIC, Australia
  6. Biostatistics Unit, The Alfred Centre, Monash University, Melbourne, VIC, Australia
  7. Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia
  8. Faculty of Engineering Science, Yokohama National University, Yokohama, Japan

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, where peak IMPα2 expression coincides with germline masculization. We identified paraspeckle protein 1 (PSPC1), the original defining component of nuclear paraspeckles, as an IMPα2 binding partner. Paraspeckles are one of the many distinct sub-nuclear bodies. They arise from the assembly of PSPC1 and two other core proteins, SFPQ and NONO, around the long non-coding RNA transcript NEAT1. PSPC1-IMPα2 binding in testis was confirmed in immunoprecipitations and pull-downs, and an ELISA-based assay demonstrated direct, high affinity PSPC1 binding to each IMPα2/IMPβ1 and IMPα6/IMPβ1. Co-expression of full length PSPC1 and IMPα2 in HeLa cells yielded increased PSPC1 localization to nuclear paraspeckles. Image quantification indicated IMPα2 levels can directly determine paraspeckle number and size; a transport-deficient IMPα2 reduced PSPC1 accumulation in paraspeckles. The observation that IMPα2 can drive an increase in detectable paraspeckle size and numbers in HeLa cells further suggests that peaks in regulated IMPα2 expression, such as in the germ cells of the E12.5 testis or during post-mitotic spermatogenesis, may reflect its role in directing cellular differentiation. Given current knowledge of paraspeckle function, we hypothesise that modulating paraspeckle number and size will manifest as changes that include the nuclear retention of specific A-to-I edited RNA transcripts, altered RNA processing and impacts on general cellular stress responses. This first validation of an IMPα2 nuclear import cargo in fetal testis provides novel evidence that paraspeckle formation and function may be controlled by modulated synthesis of specific IMPs.