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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 KPNB1 alters nuclear localization of core clock repressor proteins.(A) Quantitative RT-PCR analysis of KPNB1 knockdown efficiency in control (siCTL) and KPNB1 siRNA (siKPNB1)-treated U2 OS cells. The data presented are the means ± S.E. of triplicate samples. (B) Representative images of IF detection of endogenous expression of KPNB1 using anti-KPNB1 (αKPNB1) in control and KPNB1 siRNA-treated U2 OS cells. Scale bar: 10 μm. (C) Fluorescence microscopic analysis of KPNB1 knockdown effect on subcellular localization of PER1, PER2, CRY1, and CRY1 in U2 OS cells. Representative images were captured after immunostaining cells expressing flag-tagged PER1, PER2, CRY1, CRY2, CLOCK, REV-VERBα, CHRONO (PER1Flag, PER2Flag, CRY1Flag, CRY2Flag, CLOCKFlag, REV-VERBαFlag, CHRONOFlag) in the presence of control or KPNB1 siRNA (left panels). The images were taken with fluorescence imaging microscopy using specific filter sets for TRITC (Red; Flag tagged protein) and DAPI (Blue; Nuclei). DAPI (Blue) merged images was presented in each of the image panels. For quantification analysis, subcellular localization is categorized as nuclear (Nuc.; orange), cytoplasmic and nuclear (Cyto./Nuc.; gray), and cytoplasmic (Cyto.; blue-green) (right panels). More than 100 cells for each of the images were evaluated. Scale bar: 50 μm.DOI:http://dx.doi.org/10.7554/eLife.08647.004
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fig1s1: Inhibition of KPNB1 alters nuclear localization of core clock repressor proteins.(A) Quantitative RT-PCR analysis of KPNB1 knockdown efficiency in control (siCTL) and KPNB1 siRNA (siKPNB1)-treated U2 OS cells. The data presented are the means ± S.E. of triplicate samples. (B) Representative images of IF detection of endogenous expression of KPNB1 using anti-KPNB1 (αKPNB1) in control and KPNB1 siRNA-treated U2 OS cells. Scale bar: 10 μm. (C) Fluorescence microscopic analysis of KPNB1 knockdown effect on subcellular localization of PER1, PER2, CRY1, and CRY1 in U2 OS cells. Representative images were captured after immunostaining cells expressing flag-tagged PER1, PER2, CRY1, CRY2, CLOCK, REV-VERBα, CHRONO (PER1Flag, PER2Flag, CRY1Flag, CRY2Flag, CLOCKFlag, REV-VERBαFlag, CHRONOFlag) in the presence of control or KPNB1 siRNA (left panels). The images were taken with fluorescence imaging microscopy using specific filter sets for TRITC (Red; Flag tagged protein) and DAPI (Blue; Nuclei). DAPI (Blue) merged images was presented in each of the image panels. For quantification analysis, subcellular localization is categorized as nuclear (Nuc.; orange), cytoplasmic and nuclear (Cyto./Nuc.; gray), and cytoplasmic (Cyto.; blue-green) (right panels). More than 100 cells for each of the images were evaluated. Scale bar: 50 μm.DOI:http://dx.doi.org/10.7554/eLife.08647.004

Mentions: To test this, first, we explored how KPNB1 knockdown affects cellular localization of the core clock factors. Interestingly, in immunofluorescence (IF) analysis of cells expressing several flag-tagged clock proteins, knockdown of KPNB1 completely blocked (PER1, PER2), partly blocked (CRY1), or didn't significantly block (CRY2, REVERBα, CLOCK, CHRONO [Anafi et al., 2014]) nuclear accumulation (Figure 1—figure supplement 1). Similar to the flag-tagged proteins, microscopy and immunoblot analysis of cells expressing the Venus (V)-tagged clock proteins (PER1-V, PER2-V, CRY1-V, CRY2-V), KPNB1 knockdown markedly blocked PER1 and PER2 localization and slightly or negligibly effected CRY1 and CRY2 nuclear localization (Figure 1A,B). In many previous studies, PER and CRY proteins have been shown to work together (Tamanini et al., 2005). Hence, we looked into the effect of KPNB1 knockdown on the subcellular localization of various PERs/CRYs complexes using bimolecular fluorescence complementation (BiFC) (Shyu et al., 2006). The BiFC assay showed that KPNB1 depletion significantly impaired nuclear localization of various combinations of PER and CRY proteins (Figure 1C). Notably, nuclear accumulation of the PER2/CRY1 complex was totally blocked by KPNB1 knockdown (Figure 1C). In addition to ectopically expressed clock proteins, KPNB1 depletion substantially increased cytoplasmic accumulation of endogenous PER1/2 and CRY1/2 proteins thus reducing their relative levels in nuclei in most cells (Figure 1D, Figure 1—figure supplement 2). Taken together, these evidence show that KPNB1 serves as an integral nuclear transport receptor of the PER/CRY complex.10.7554/eLife.08647.003Figure 1.KPNB1 knockdown blocks nuclear translocation of PERs/CRYs complex.


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

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

Inhibition of KPNB1 alters nuclear localization of core clock repressor proteins.(A) Quantitative RT-PCR analysis of KPNB1 knockdown efficiency in control (siCTL) and KPNB1 siRNA (siKPNB1)-treated U2 OS cells. The data presented are the means ± S.E. of triplicate samples. (B) Representative images of IF detection of endogenous expression of KPNB1 using anti-KPNB1 (αKPNB1) in control and KPNB1 siRNA-treated U2 OS cells. Scale bar: 10 μm. (C) Fluorescence microscopic analysis of KPNB1 knockdown effect on subcellular localization of PER1, PER2, CRY1, and CRY1 in U2 OS cells. Representative images were captured after immunostaining cells expressing flag-tagged PER1, PER2, CRY1, CRY2, CLOCK, REV-VERBα, CHRONO (PER1Flag, PER2Flag, CRY1Flag, CRY2Flag, CLOCKFlag, REV-VERBαFlag, CHRONOFlag) in the presence of control or KPNB1 siRNA (left panels). The images were taken with fluorescence imaging microscopy using specific filter sets for TRITC (Red; Flag tagged protein) and DAPI (Blue; Nuclei). DAPI (Blue) merged images was presented in each of the image panels. For quantification analysis, subcellular localization is categorized as nuclear (Nuc.; orange), cytoplasmic and nuclear (Cyto./Nuc.; gray), and cytoplasmic (Cyto.; blue-green) (right panels). More than 100 cells for each of the images were evaluated. Scale bar: 50 μm.DOI:http://dx.doi.org/10.7554/eLife.08647.004
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fig1s1: Inhibition of KPNB1 alters nuclear localization of core clock repressor proteins.(A) Quantitative RT-PCR analysis of KPNB1 knockdown efficiency in control (siCTL) and KPNB1 siRNA (siKPNB1)-treated U2 OS cells. The data presented are the means ± S.E. of triplicate samples. (B) Representative images of IF detection of endogenous expression of KPNB1 using anti-KPNB1 (αKPNB1) in control and KPNB1 siRNA-treated U2 OS cells. Scale bar: 10 μm. (C) Fluorescence microscopic analysis of KPNB1 knockdown effect on subcellular localization of PER1, PER2, CRY1, and CRY1 in U2 OS cells. Representative images were captured after immunostaining cells expressing flag-tagged PER1, PER2, CRY1, CRY2, CLOCK, REV-VERBα, CHRONO (PER1Flag, PER2Flag, CRY1Flag, CRY2Flag, CLOCKFlag, REV-VERBαFlag, CHRONOFlag) in the presence of control or KPNB1 siRNA (left panels). The images were taken with fluorescence imaging microscopy using specific filter sets for TRITC (Red; Flag tagged protein) and DAPI (Blue; Nuclei). DAPI (Blue) merged images was presented in each of the image panels. For quantification analysis, subcellular localization is categorized as nuclear (Nuc.; orange), cytoplasmic and nuclear (Cyto./Nuc.; gray), and cytoplasmic (Cyto.; blue-green) (right panels). More than 100 cells for each of the images were evaluated. Scale bar: 50 μm.DOI:http://dx.doi.org/10.7554/eLife.08647.004
Mentions: To test this, first, we explored how KPNB1 knockdown affects cellular localization of the core clock factors. Interestingly, in immunofluorescence (IF) analysis of cells expressing several flag-tagged clock proteins, knockdown of KPNB1 completely blocked (PER1, PER2), partly blocked (CRY1), or didn't significantly block (CRY2, REVERBα, CLOCK, CHRONO [Anafi et al., 2014]) nuclear accumulation (Figure 1—figure supplement 1). Similar to the flag-tagged proteins, microscopy and immunoblot analysis of cells expressing the Venus (V)-tagged clock proteins (PER1-V, PER2-V, CRY1-V, CRY2-V), KPNB1 knockdown markedly blocked PER1 and PER2 localization and slightly or negligibly effected CRY1 and CRY2 nuclear localization (Figure 1A,B). In many previous studies, PER and CRY proteins have been shown to work together (Tamanini et al., 2005). Hence, we looked into the effect of KPNB1 knockdown on the subcellular localization of various PERs/CRYs complexes using bimolecular fluorescence complementation (BiFC) (Shyu et al., 2006). The BiFC assay showed that KPNB1 depletion significantly impaired nuclear localization of various combinations of PER and CRY proteins (Figure 1C). Notably, nuclear accumulation of the PER2/CRY1 complex was totally blocked by KPNB1 knockdown (Figure 1C). In addition to ectopically expressed clock proteins, KPNB1 depletion substantially increased cytoplasmic accumulation of endogenous PER1/2 and CRY1/2 proteins thus reducing their relative levels in nuclei in most cells (Figure 1D, Figure 1—figure supplement 2). Taken together, these evidence show that KPNB1 serves as an integral nuclear transport receptor of the PER/CRY complex.10.7554/eLife.08647.003Figure 1.KPNB1 knockdown blocks nuclear translocation of PERs/CRYs complex.

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