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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 conserved importin β (ketel) abolishes circadian behavior in flies.(A) Flies in which ketel was downregulated in PDF+ cells and in all clock neurons were assayed for rest/activity rhythms in constant dark (DD). (B) Summary of the circadian rhythms of various flies under DD. Quantification shows percentage of rhythmic (R, orange) and arrhythmic (AR, gray) flies (n = 16–26). (C) Some rhythmic Pdf-Gal4/+; dicer/+ flies showed weak rhythmicity with lower fast Fourier transform value compared to both ketel RNAi/+; dicer/+ and Pdf-GAL4/+; dicer/+ control flies (**p < 0.001, ***p < 0.0001, by Student's t-test) (See also Supplementary file 1). (D) ketel knockdown in PDF+ cells during adulthoods leads to a long period, which eventually degenerates into arrhythmia in DD. They were fed either 500 mM RU486 or ethanol (EtOH, vehicle control) from the time of entrainment. (E) Rhythm strength of rhythmic RU486-treated flies was lower than that of EtOH-treated control flies after 7 days in DD (*p < 0.05, by Student's t-test) (See also Supplementary file 1). (F, G) PDF expression in ketel knockdown flies was only detected in large lateral ventral neurons (l-LNvs), not in the small lateral ventral neurons (s-LNvs). Downregulation of KETEL in PDF+ cells impairs nuclear translocation of PER in l-LNvs. In RU486-treated flies, PER expression in l-LNvs was detected in cytoplasm at ZT1, a time when PER is nuclear in control flies (***p < 0.0001, by Student's t-test).DOI:http://dx.doi.org/10.7554/eLife.08647.016
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fig5: Inhibition of conserved importin β (ketel) abolishes circadian behavior in flies.(A) Flies in which ketel was downregulated in PDF+ cells and in all clock neurons were assayed for rest/activity rhythms in constant dark (DD). (B) Summary of the circadian rhythms of various flies under DD. Quantification shows percentage of rhythmic (R, orange) and arrhythmic (AR, gray) flies (n = 16–26). (C) Some rhythmic Pdf-Gal4/+; dicer/+ flies showed weak rhythmicity with lower fast Fourier transform value compared to both ketel RNAi/+; dicer/+ and Pdf-GAL4/+; dicer/+ control flies (**p < 0.001, ***p < 0.0001, by Student's t-test) (See also Supplementary file 1). (D) ketel knockdown in PDF+ cells during adulthoods leads to a long period, which eventually degenerates into arrhythmia in DD. They were fed either 500 mM RU486 or ethanol (EtOH, vehicle control) from the time of entrainment. (E) Rhythm strength of rhythmic RU486-treated flies was lower than that of EtOH-treated control flies after 7 days in DD (*p < 0.05, by Student's t-test) (See also Supplementary file 1). (F, G) PDF expression in ketel knockdown flies was only detected in large lateral ventral neurons (l-LNvs), not in the small lateral ventral neurons (s-LNvs). Downregulation of KETEL in PDF+ cells impairs nuclear translocation of PER in l-LNvs. In RU486-treated flies, PER expression in l-LNvs was detected in cytoplasm at ZT1, a time when PER is nuclear in control flies (***p < 0.0001, by Student's t-test).DOI:http://dx.doi.org/10.7554/eLife.08647.016

Mentions: The majority of core clock components are conserved between flies (cycle, clk, per, cry) and humans (BMAL1/BMAL2, CLOCK/NPAS2, PER1/PER2/PER3, CRY1/CRY2), across 600 million years of evolution. Indeed, the kinases that regulate PER/CRY translocation to the nucleus are also conserved (dbt, sg; CKIε/CKIδ, GSK3β). Because KPNB1 was required for rhythmicity in human cells, we investigated the role of the Drosophila ortholog, Fs(2)Ket, ketel, in the fly clock by RNAi followed by locomotor activity monitoring (Mason and Goldfarb, 2009). We used the UAS/Gal4 system to target UAS-ketel RNAi to clock cells in Drosophila (Brand and Perrimon, 1993). dicer was also co-expressed to increase the RNA interference effect (Dietzl et al., 2007). Behavioral analysis of flies expressing UAS ketel RNAi in central clock cells, targeted by the relatively weak Pigment dispersing factor (Pdf)-Gal4 driver, revealed that 62% of flies exhibited no rhythms in constant darkness (DD). Although 38% of flies were rhythmic, their rhythm strength determined by fast Fourier transform (FFT) value was low (Figure 5A–C, Supplementary file 1). With a strong tim-UAS-Gal4 driver that is expressed in all clock cells, 100% of flies were arrhythmic (Figure 5A,B, Supplementary file 1). To exclude possible developmental effects on behavioral phenotypes in ketel knockdown flies, we restricted the ketel dsRNA expression to adult PDF+ neurons using the pdf Gene Switch (pdf-GS). We crossed ketel RNAi transgenic flies with pdf-GS and then drove ketel dsRNAs expression with a drug (RU486) during adulthood (McGuire et al., 2004). After 6–7 days in constant darkness, this conditional knockdown of ketel also resulted in weak to arrhythmic locomotor activity (Figure 5D, Supplementary file 1). 81% of flies were arrhythmic and the rest showed weak rhythmicity with low FFT values (Figure 5E, Supplementary file 1). These results show that importin β in PDF+ neurons is required for normal locomotor rhythms in adult flies.10.7554/eLife.08647.016Figure 5.Inhibition of conserved importin β (ketel) abolishes circadian behavior in flies.


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

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

Inhibition of conserved importin β (ketel) abolishes circadian behavior in flies.(A) Flies in which ketel was downregulated in PDF+ cells and in all clock neurons were assayed for rest/activity rhythms in constant dark (DD). (B) Summary of the circadian rhythms of various flies under DD. Quantification shows percentage of rhythmic (R, orange) and arrhythmic (AR, gray) flies (n = 16–26). (C) Some rhythmic Pdf-Gal4/+; dicer/+ flies showed weak rhythmicity with lower fast Fourier transform value compared to both ketel RNAi/+; dicer/+ and Pdf-GAL4/+; dicer/+ control flies (**p < 0.001, ***p < 0.0001, by Student's t-test) (See also Supplementary file 1). (D) ketel knockdown in PDF+ cells during adulthoods leads to a long period, which eventually degenerates into arrhythmia in DD. They were fed either 500 mM RU486 or ethanol (EtOH, vehicle control) from the time of entrainment. (E) Rhythm strength of rhythmic RU486-treated flies was lower than that of EtOH-treated control flies after 7 days in DD (*p < 0.05, by Student's t-test) (See also Supplementary file 1). (F, G) PDF expression in ketel knockdown flies was only detected in large lateral ventral neurons (l-LNvs), not in the small lateral ventral neurons (s-LNvs). Downregulation of KETEL in PDF+ cells impairs nuclear translocation of PER in l-LNvs. In RU486-treated flies, PER expression in l-LNvs was detected in cytoplasm at ZT1, a time when PER is nuclear in control flies (***p < 0.0001, by Student's t-test).DOI:http://dx.doi.org/10.7554/eLife.08647.016
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fig5: Inhibition of conserved importin β (ketel) abolishes circadian behavior in flies.(A) Flies in which ketel was downregulated in PDF+ cells and in all clock neurons were assayed for rest/activity rhythms in constant dark (DD). (B) Summary of the circadian rhythms of various flies under DD. Quantification shows percentage of rhythmic (R, orange) and arrhythmic (AR, gray) flies (n = 16–26). (C) Some rhythmic Pdf-Gal4/+; dicer/+ flies showed weak rhythmicity with lower fast Fourier transform value compared to both ketel RNAi/+; dicer/+ and Pdf-GAL4/+; dicer/+ control flies (**p < 0.001, ***p < 0.0001, by Student's t-test) (See also Supplementary file 1). (D) ketel knockdown in PDF+ cells during adulthoods leads to a long period, which eventually degenerates into arrhythmia in DD. They were fed either 500 mM RU486 or ethanol (EtOH, vehicle control) from the time of entrainment. (E) Rhythm strength of rhythmic RU486-treated flies was lower than that of EtOH-treated control flies after 7 days in DD (*p < 0.05, by Student's t-test) (See also Supplementary file 1). (F, G) PDF expression in ketel knockdown flies was only detected in large lateral ventral neurons (l-LNvs), not in the small lateral ventral neurons (s-LNvs). Downregulation of KETEL in PDF+ cells impairs nuclear translocation of PER in l-LNvs. In RU486-treated flies, PER expression in l-LNvs was detected in cytoplasm at ZT1, a time when PER is nuclear in control flies (***p < 0.0001, by Student's t-test).DOI:http://dx.doi.org/10.7554/eLife.08647.016
Mentions: The majority of core clock components are conserved between flies (cycle, clk, per, cry) and humans (BMAL1/BMAL2, CLOCK/NPAS2, PER1/PER2/PER3, CRY1/CRY2), across 600 million years of evolution. Indeed, the kinases that regulate PER/CRY translocation to the nucleus are also conserved (dbt, sg; CKIε/CKIδ, GSK3β). Because KPNB1 was required for rhythmicity in human cells, we investigated the role of the Drosophila ortholog, Fs(2)Ket, ketel, in the fly clock by RNAi followed by locomotor activity monitoring (Mason and Goldfarb, 2009). We used the UAS/Gal4 system to target UAS-ketel RNAi to clock cells in Drosophila (Brand and Perrimon, 1993). dicer was also co-expressed to increase the RNA interference effect (Dietzl et al., 2007). Behavioral analysis of flies expressing UAS ketel RNAi in central clock cells, targeted by the relatively weak Pigment dispersing factor (Pdf)-Gal4 driver, revealed that 62% of flies exhibited no rhythms in constant darkness (DD). Although 38% of flies were rhythmic, their rhythm strength determined by fast Fourier transform (FFT) value was low (Figure 5A–C, Supplementary file 1). With a strong tim-UAS-Gal4 driver that is expressed in all clock cells, 100% of flies were arrhythmic (Figure 5A,B, Supplementary file 1). To exclude possible developmental effects on behavioral phenotypes in ketel knockdown flies, we restricted the ketel dsRNA expression to adult PDF+ neurons using the pdf Gene Switch (pdf-GS). We crossed ketel RNAi transgenic flies with pdf-GS and then drove ketel dsRNAs expression with a drug (RU486) during adulthood (McGuire et al., 2004). After 6–7 days in constant darkness, this conditional knockdown of ketel also resulted in weak to arrhythmic locomotor activity (Figure 5D, Supplementary file 1). 81% of flies were arrhythmic and the rest showed weak rhythmicity with low FFT values (Figure 5E, Supplementary file 1). These results show that importin β in PDF+ neurons is required for normal locomotor rhythms in adult flies.10.7554/eLife.08647.016Figure 5.Inhibition of conserved importin β (ketel) abolishes circadian behavior in flies.

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