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KPNB1 mediates PER/CRY nuclear translocation and circadian clock function.

Lee Y, Jang AR, Francey LJ, Sehgal A, Hogenesch JB - Elife (2015)

Bottom Line: Interestingly, KPNB1 regulates the PER/CRY nuclear entry and repressor function, independently of importin α, its classical partner.Moreover, inducible inhibition of the conserved Drosophila importin β in lateral neurons abolishes behavioral rhythms in flies.Collectively, these data show that KPNB1 is required for timely nuclear import of PER/CRY in the negative feedback regulation of the circadian clock.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Pharmacology and Translational Therapeutics, Institute for Translational Medicine and Therapeutics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States.

ABSTRACT
Regulated nuclear translocation of the PER/CRY repressor complex is critical for negative feedback regulation of the circadian clock of mammals. However, the precise molecular mechanism is not fully understood. Here, we report that KPNB1, an importin β component of the ncRNA repressor of nuclear factor of activated T cells (NRON) ribonucleoprotein complex, mediates nuclear translocation and repressor function of the PER/CRY complex. RNAi depletion of KPNB1 traps the PER/CRY complex in the cytoplasm by blocking nuclear entry of PER proteins in human cells. KPNB1 interacts mainly with PER proteins and directs PER/CRY nuclear transport in a circadian fashion. Interestingly, KPNB1 regulates the PER/CRY nuclear entry and repressor function, independently of importin α, its classical partner. Moreover, inducible inhibition of the conserved Drosophila importin β in lateral neurons abolishes behavioral rhythms in flies. Collectively, these data show that KPNB1 is required for timely nuclear import of PER/CRY in the negative feedback regulation of the circadian clock.

No MeSH data available.


Related in: MedlinePlus

Inhibition of KPNA2 does not affect CRY2 nuclear localization.(A) Immunoblot analysis of nuclear and cytoplasmic extracts of U2 OS cells in the presence of control and KPNA1 siRNA using the indicated antibodies for endogenous CRY2 (αCRY2) and KPNB1 (αKPNB1), Tubulin for cytoplasmic fraction (C) marker (αTubulin), and TATA binding protein (TBP) for nuclear fraction (N) marker (αTBP). (B) Fluorescence microscopic analysis of the effect of KPNA2 depletion on subcellular localization of CRY2. Representative fluorescence images of subcellular localization of endogenous CRY2 (upper panels), or ectopically expressed Flag-CRY2 (middle panels), CRY2-Venus (CRY2-V; lower panels) in U2 OS cells treated with control siRNA (siCTL; left panels) or KPNA2 siRNA (siKPNA2; right panels). The images were taken using specific filter sets for TRITC (Red; αCRY2, Flag-CRY2), FITC (green; PER2-V), and DAPI (Blue; Nuclei). DAPI (Blue) merged images was presented in each of the image panels.DOI:http://dx.doi.org/10.7554/eLife.08647.013
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fig4s2: Inhibition of KPNA2 does not affect CRY2 nuclear localization.(A) Immunoblot analysis of nuclear and cytoplasmic extracts of U2 OS cells in the presence of control and KPNA1 siRNA using the indicated antibodies for endogenous CRY2 (αCRY2) and KPNB1 (αKPNB1), Tubulin for cytoplasmic fraction (C) marker (αTubulin), and TATA binding protein (TBP) for nuclear fraction (N) marker (αTBP). (B) Fluorescence microscopic analysis of the effect of KPNA2 depletion on subcellular localization of CRY2. Representative fluorescence images of subcellular localization of endogenous CRY2 (upper panels), or ectopically expressed Flag-CRY2 (middle panels), CRY2-Venus (CRY2-V; lower panels) in U2 OS cells treated with control siRNA (siCTL; left panels) or KPNA2 siRNA (siKPNA2; right panels). The images were taken using specific filter sets for TRITC (Red; αCRY2, Flag-CRY2), FITC (green; PER2-V), and DAPI (Blue; Nuclei). DAPI (Blue) merged images was presented in each of the image panels.DOI:http://dx.doi.org/10.7554/eLife.08647.013

Mentions: In early models, KPNB1 mediates docking of the importin α/substrate complex in the nuclear pore. More recent evidence shows that KPNB1 can function as nuclear transport receptor independently of importin α by directly recognizing substrates such as CREB, AP-1 transcription factors, and parathyroid hormone-related protein (Lam et al., 1999; Forwood et al., 2001; Ghildyal et al., 2005). Interestingly, a previous report showed that importin α isoforms (RCH, QIP-1, NPI-2) mediate nuclear localization of CRY2 through direct interactions but did not investigate the functional consequence of this interaction (Sakakida et al., 2005). In this regard, our data suggest that in mammals KPNB1 mediates nuclear localization of the PER/CRY complex independently of importin α, as overexpression of a KPNB1 mutant lacking an importin α interaction domain had a similar phenotype (Figure 1, Figure 3—figure supplement 2). Consistent with this, both KPNA2 and KPNA1, the well-known KPNB1 interacting importin alpha proteins (importin α1, importin α5), when depleted in cells, did not affect circadian rhythmicity or subcellular localization of PER2 or CRY2 protein (Figure 4—figure supplement 1, Figure 4—figure supplement 2, Figure 4—figure supplement 3). In a recent report, cytoplasmic retention of PER proteins was accelerated by KPNA2 overexpression in mouse stem cells (Umemura et al., 2014). However, intriguingly we found that KPNA2 overexpression substantially reduced cells expressing PER2-Venus protein in our imaging studies (Figure 4—figure supplement 1E). Moreover, both the endogenous and overexpressed KPNA2 protein localized predominantly in the nucleus, which was confirmed by nuclear cytoplasmic fraction Western analysis (Figure 4—figure supplement 1C,E, Figure 4—figure supplement 2A). This suggests that KPNA2 might regulate PER2 protein level through an unknown mechanism in U2 OS cells. Thus, we speculate that these different results are likely due to the different cell types and species used.


KPNB1 mediates PER/CRY nuclear translocation and circadian clock function.

Lee Y, Jang AR, Francey LJ, Sehgal A, Hogenesch JB - Elife (2015)

Inhibition of KPNA2 does not affect CRY2 nuclear localization.(A) Immunoblot analysis of nuclear and cytoplasmic extracts of U2 OS cells in the presence of control and KPNA1 siRNA using the indicated antibodies for endogenous CRY2 (αCRY2) and KPNB1 (αKPNB1), Tubulin for cytoplasmic fraction (C) marker (αTubulin), and TATA binding protein (TBP) for nuclear fraction (N) marker (αTBP). (B) Fluorescence microscopic analysis of the effect of KPNA2 depletion on subcellular localization of CRY2. Representative fluorescence images of subcellular localization of endogenous CRY2 (upper panels), or ectopically expressed Flag-CRY2 (middle panels), CRY2-Venus (CRY2-V; lower panels) in U2 OS cells treated with control siRNA (siCTL; left panels) or KPNA2 siRNA (siKPNA2; right panels). The images were taken using specific filter sets for TRITC (Red; αCRY2, Flag-CRY2), FITC (green; PER2-V), and DAPI (Blue; Nuclei). DAPI (Blue) merged images was presented in each of the image panels.DOI:http://dx.doi.org/10.7554/eLife.08647.013
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fig4s2: Inhibition of KPNA2 does not affect CRY2 nuclear localization.(A) Immunoblot analysis of nuclear and cytoplasmic extracts of U2 OS cells in the presence of control and KPNA1 siRNA using the indicated antibodies for endogenous CRY2 (αCRY2) and KPNB1 (αKPNB1), Tubulin for cytoplasmic fraction (C) marker (αTubulin), and TATA binding protein (TBP) for nuclear fraction (N) marker (αTBP). (B) Fluorescence microscopic analysis of the effect of KPNA2 depletion on subcellular localization of CRY2. Representative fluorescence images of subcellular localization of endogenous CRY2 (upper panels), or ectopically expressed Flag-CRY2 (middle panels), CRY2-Venus (CRY2-V; lower panels) in U2 OS cells treated with control siRNA (siCTL; left panels) or KPNA2 siRNA (siKPNA2; right panels). The images were taken using specific filter sets for TRITC (Red; αCRY2, Flag-CRY2), FITC (green; PER2-V), and DAPI (Blue; Nuclei). DAPI (Blue) merged images was presented in each of the image panels.DOI:http://dx.doi.org/10.7554/eLife.08647.013
Mentions: In early models, KPNB1 mediates docking of the importin α/substrate complex in the nuclear pore. More recent evidence shows that KPNB1 can function as nuclear transport receptor independently of importin α by directly recognizing substrates such as CREB, AP-1 transcription factors, and parathyroid hormone-related protein (Lam et al., 1999; Forwood et al., 2001; Ghildyal et al., 2005). Interestingly, a previous report showed that importin α isoforms (RCH, QIP-1, NPI-2) mediate nuclear localization of CRY2 through direct interactions but did not investigate the functional consequence of this interaction (Sakakida et al., 2005). In this regard, our data suggest that in mammals KPNB1 mediates nuclear localization of the PER/CRY complex independently of importin α, as overexpression of a KPNB1 mutant lacking an importin α interaction domain had a similar phenotype (Figure 1, Figure 3—figure supplement 2). Consistent with this, both KPNA2 and KPNA1, the well-known KPNB1 interacting importin alpha proteins (importin α1, importin α5), when depleted in cells, did not affect circadian rhythmicity or subcellular localization of PER2 or CRY2 protein (Figure 4—figure supplement 1, Figure 4—figure supplement 2, Figure 4—figure supplement 3). In a recent report, cytoplasmic retention of PER proteins was accelerated by KPNA2 overexpression in mouse stem cells (Umemura et al., 2014). However, intriguingly we found that KPNA2 overexpression substantially reduced cells expressing PER2-Venus protein in our imaging studies (Figure 4—figure supplement 1E). Moreover, both the endogenous and overexpressed KPNA2 protein localized predominantly in the nucleus, which was confirmed by nuclear cytoplasmic fraction Western analysis (Figure 4—figure supplement 1C,E, Figure 4—figure supplement 2A). This suggests that KPNA2 might regulate PER2 protein level through an unknown mechanism in U2 OS cells. Thus, we speculate that these different results are likely due to the different cell types and species used.

Bottom Line: Interestingly, KPNB1 regulates the PER/CRY nuclear entry and repressor function, independently of importin α, its classical partner.Moreover, inducible inhibition of the conserved Drosophila importin β in lateral neurons abolishes behavioral rhythms in flies.Collectively, these data show that KPNB1 is required for timely nuclear import of PER/CRY in the negative feedback regulation of the circadian clock.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Pharmacology and Translational Therapeutics, Institute for Translational Medicine and Therapeutics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States.

ABSTRACT
Regulated nuclear translocation of the PER/CRY repressor complex is critical for negative feedback regulation of the circadian clock of mammals. However, the precise molecular mechanism is not fully understood. Here, we report that KPNB1, an importin β component of the ncRNA repressor of nuclear factor of activated T cells (NRON) ribonucleoprotein complex, mediates nuclear translocation and repressor function of the PER/CRY complex. RNAi depletion of KPNB1 traps the PER/CRY complex in the cytoplasm by blocking nuclear entry of PER proteins in human cells. KPNB1 interacts mainly with PER proteins and directs PER/CRY nuclear transport in a circadian fashion. Interestingly, KPNB1 regulates the PER/CRY nuclear entry and repressor function, independently of importin α, its classical partner. Moreover, inducible inhibition of the conserved Drosophila importin β in lateral neurons abolishes behavioral rhythms in flies. Collectively, these data show that KPNB1 is required for timely nuclear import of PER/CRY in the negative feedback regulation of the circadian clock.

No MeSH data available.


Related in: MedlinePlus