Specific interaction with the nuclear transporter importin alpha2 can modulate paraspeckle protein 1 delivery to nuclear paraspeckles

Andrew T Major, Cathryn A Hogarth, Yoichi Miyamoto, Mai Sarraj, Catherine L Smith, Peter A Koopman, Yasuyuki Kurihara, David A Jans, Katherine A L Loveland

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Importin (IMP) superfamily members mediate regulated nucleocytoplasmic transport, which is central to key cellular processes. Although individual IMPalpha 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 IMPalphas control cellular development, we conducted a yeast-two-hybrid screen for IMPalpha2 cargoes in embryonic day 12.5 mouse testis, a site of peak IMPalpha2 expression coincident with germline masculization. We identified paraspeckle protein 1 (PSPC1), the original defining component of nuclear paraspeckles, as an IMPalpha2 binding partner. PSPC1-IMPalpha2 binding in testis was confirmed in immunoprecipitations and pull-downs, and an ELISA-based assay demonstrated direct, high-affinity PSPC1 binding to either IMPalpha2/IMPbeta1 or IMPalpha6/IMPbeta1. Co-expression of full length PSPC1 and IMPalpha2 in HeLa cells yielded increased PSPC1 localization in nuclear paraspeckles. High throughput image analysis of >3500 cells indicated IMPalpha2 levels can directly determine PSPC1-positive nuclear speckle numbers and size; a transport-deficient IMPalpha2 isoform or siRNA knockdown of IMPalpha2 both reduced endogenous PSPC1 accumulation in speckles. This first validation of an IMPalpha2 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.
Original languageEnglish
Pages (from-to)1543 - 1558
Number of pages16
JournalMolecular Biology of the Cell
Volume26
Issue number8
DOIs
Publication statusPublished - 2015

Cite this

Major, Andrew T ; Hogarth, Cathryn A ; Miyamoto, Yoichi ; Sarraj, Mai ; Smith, Catherine L ; Koopman, Peter A ; Kurihara, Yasuyuki ; Jans, David A ; Loveland, Katherine A L. / Specific interaction with the nuclear transporter importin alpha2 can modulate paraspeckle protein 1 delivery to nuclear paraspeckles. In: Molecular Biology of the Cell. 2015 ; Vol. 26, No. 8. pp. 1543 - 1558.
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title = "Specific interaction with the nuclear transporter importin alpha2 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 IMPalpha 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 IMPalphas control cellular development, we conducted a yeast-two-hybrid screen for IMPalpha2 cargoes in embryonic day 12.5 mouse testis, a site of peak IMPalpha2 expression coincident with germline masculization. We identified paraspeckle protein 1 (PSPC1), the original defining component of nuclear paraspeckles, as an IMPalpha2 binding partner. PSPC1-IMPalpha2 binding in testis was confirmed in immunoprecipitations and pull-downs, and an ELISA-based assay demonstrated direct, high-affinity PSPC1 binding to either IMPalpha2/IMPbeta1 or IMPalpha6/IMPbeta1. Co-expression of full length PSPC1 and IMPalpha2 in HeLa cells yielded increased PSPC1 localization in nuclear paraspeckles. High throughput image analysis of >3500 cells indicated IMPalpha2 levels can directly determine PSPC1-positive nuclear speckle numbers and size; a transport-deficient IMPalpha2 isoform or siRNA knockdown of IMPalpha2 both reduced endogenous PSPC1 accumulation in speckles. This first validation of an IMPalpha2 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.",
author = "Major, {Andrew T} and Hogarth, {Cathryn A} and Yoichi Miyamoto and Mai Sarraj and Smith, {Catherine L} and Koopman, {Peter A} and Yasuyuki Kurihara and Jans, {David A} and Loveland, {Katherine A L}",
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doi = "10.1091/mbc.E14-01-0678",
language = "English",
volume = "26",
pages = "1543 -- 1558",
journal = "Molecular Biology of the Cell",
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publisher = "American Society for Cell Biology",
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Specific interaction with the nuclear transporter importin alpha2 can modulate paraspeckle protein 1 delivery to nuclear paraspeckles. / Major, Andrew T; Hogarth, Cathryn A; Miyamoto, Yoichi; Sarraj, Mai; Smith, Catherine L; Koopman, Peter A; Kurihara, Yasuyuki; Jans, David A; Loveland, Katherine A L.

In: Molecular Biology of the Cell, Vol. 26, No. 8, 2015, p. 1543 - 1558.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Specific interaction with the nuclear transporter importin alpha2 can modulate paraspeckle protein 1 delivery to nuclear paraspeckles

AU - Major, Andrew T

AU - Hogarth, Cathryn A

AU - Miyamoto, Yoichi

AU - Sarraj, Mai

AU - Smith, Catherine L

AU - Koopman, Peter A

AU - Kurihara, Yasuyuki

AU - Jans, David A

AU - Loveland, Katherine A L

PY - 2015

Y1 - 2015

N2 - Importin (IMP) superfamily members mediate regulated nucleocytoplasmic transport, which is central to key cellular processes. Although individual IMPalpha 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 IMPalphas control cellular development, we conducted a yeast-two-hybrid screen for IMPalpha2 cargoes in embryonic day 12.5 mouse testis, a site of peak IMPalpha2 expression coincident with germline masculization. We identified paraspeckle protein 1 (PSPC1), the original defining component of nuclear paraspeckles, as an IMPalpha2 binding partner. PSPC1-IMPalpha2 binding in testis was confirmed in immunoprecipitations and pull-downs, and an ELISA-based assay demonstrated direct, high-affinity PSPC1 binding to either IMPalpha2/IMPbeta1 or IMPalpha6/IMPbeta1. Co-expression of full length PSPC1 and IMPalpha2 in HeLa cells yielded increased PSPC1 localization in nuclear paraspeckles. High throughput image analysis of >3500 cells indicated IMPalpha2 levels can directly determine PSPC1-positive nuclear speckle numbers and size; a transport-deficient IMPalpha2 isoform or siRNA knockdown of IMPalpha2 both reduced endogenous PSPC1 accumulation in speckles. This first validation of an IMPalpha2 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.

AB - Importin (IMP) superfamily members mediate regulated nucleocytoplasmic transport, which is central to key cellular processes. Although individual IMPalpha 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 IMPalphas control cellular development, we conducted a yeast-two-hybrid screen for IMPalpha2 cargoes in embryonic day 12.5 mouse testis, a site of peak IMPalpha2 expression coincident with germline masculization. We identified paraspeckle protein 1 (PSPC1), the original defining component of nuclear paraspeckles, as an IMPalpha2 binding partner. PSPC1-IMPalpha2 binding in testis was confirmed in immunoprecipitations and pull-downs, and an ELISA-based assay demonstrated direct, high-affinity PSPC1 binding to either IMPalpha2/IMPbeta1 or IMPalpha6/IMPbeta1. Co-expression of full length PSPC1 and IMPalpha2 in HeLa cells yielded increased PSPC1 localization in nuclear paraspeckles. High throughput image analysis of >3500 cells indicated IMPalpha2 levels can directly determine PSPC1-positive nuclear speckle numbers and size; a transport-deficient IMPalpha2 isoform or siRNA knockdown of IMPalpha2 both reduced endogenous PSPC1 accumulation in speckles. This first validation of an IMPalpha2 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.

UR - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395133/pdf/1543.pdf

U2 - 10.1091/mbc.E14-01-0678

DO - 10.1091/mbc.E14-01-0678

M3 - Article

VL - 26

SP - 1543

EP - 1558

JO - Molecular Biology of the Cell

JF - Molecular Biology of the Cell

SN - 1059-1524

IS - 8

ER -