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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

KPNA isoforms are not critical for cellular rhythmicity.Knockdown of various KPNA isoforms does not significantly affect circadian promotor activity rhythm (Blue = Negative control [Control siRNA], Green = Positive control [CRY2 siRNA], Red = Data [Target KPNA siRNA]) (A) KPNA1 (importin α5), (B) KPNA3 (importin α4), (C) KPNA4 (importin α3), (D) KPNA5 (importin α6), (E) KPNA6 (importin α7), (F) KPNA7 (importin α8). Each of data set (A–F) was retrieved from BioGPS website; http://biogps.org (Wu et al., 2009; Zhang et al., 2009).DOI:http://dx.doi.org/10.7554/eLife.08647.015
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fig4s4: KPNA isoforms are not critical for cellular rhythmicity.Knockdown of various KPNA isoforms does not significantly affect circadian promotor activity rhythm (Blue = Negative control [Control siRNA], Green = Positive control [CRY2 siRNA], Red = Data [Target KPNA siRNA]) (A) KPNA1 (importin α5), (B) KPNA3 (importin α4), (C) KPNA4 (importin α3), (D) KPNA5 (importin α6), (E) KPNA6 (importin α7), (F) KPNA7 (importin α8). Each of data set (A–F) was retrieved from BioGPS website; http://biogps.org (Wu et al., 2009; Zhang et al., 2009).DOI:http://dx.doi.org/10.7554/eLife.08647.015

Mentions: On the other hand, knockdown of KPNB1 had strong (PER1, PER2), modest (CRY1), or no effect (CRY2) on single tagged PER and CRY proteins, while it had strong effects on all combinations of PER/CRY complexes (Figure 1A–C). Similarly, knockdown of KPNB1 increased cytoplasmic retention of endogenous PER1, PER2, CRY1, and CRY2 to varying degrees, probably through complex interactions (Figure 1D, Figure1—figure supplement 2). The immunoprecipitation analysis showed that KPNB1 interacted strongly with PERs compared with CRYs (Figure 2—figure supplement 1A). These data suggest that the PERs mediate interaction with KPNB1 and gate nuclear translocation of PER/CRY complex. This is reminiscent of a previous report indicating that the nuclear entry of CRY is mainly dependent on PER, in vivo (Lee et al., 2001). Finally, knockdown of KPNB1 or its fly ortholog ketel resulted in arrhythmic U2 OS cells or fly locomotor activity rhythms, while knockdown of most of KPNA isoforms had no significant effect on the rhythm (Figure 4, Figure 5, Figure 4—figure supplement 4). These data suggest a model in which KPNB1 plays a necessary role in mediating nuclear entry of the PER/CRY complex in core clock regulation (Figure 6).


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

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

KPNA isoforms are not critical for cellular rhythmicity.Knockdown of various KPNA isoforms does not significantly affect circadian promotor activity rhythm (Blue = Negative control [Control siRNA], Green = Positive control [CRY2 siRNA], Red = Data [Target KPNA siRNA]) (A) KPNA1 (importin α5), (B) KPNA3 (importin α4), (C) KPNA4 (importin α3), (D) KPNA5 (importin α6), (E) KPNA6 (importin α7), (F) KPNA7 (importin α8). Each of data set (A–F) was retrieved from BioGPS website; http://biogps.org (Wu et al., 2009; Zhang et al., 2009).DOI:http://dx.doi.org/10.7554/eLife.08647.015
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4597257&req=5

fig4s4: KPNA isoforms are not critical for cellular rhythmicity.Knockdown of various KPNA isoforms does not significantly affect circadian promotor activity rhythm (Blue = Negative control [Control siRNA], Green = Positive control [CRY2 siRNA], Red = Data [Target KPNA siRNA]) (A) KPNA1 (importin α5), (B) KPNA3 (importin α4), (C) KPNA4 (importin α3), (D) KPNA5 (importin α6), (E) KPNA6 (importin α7), (F) KPNA7 (importin α8). Each of data set (A–F) was retrieved from BioGPS website; http://biogps.org (Wu et al., 2009; Zhang et al., 2009).DOI:http://dx.doi.org/10.7554/eLife.08647.015
Mentions: On the other hand, knockdown of KPNB1 had strong (PER1, PER2), modest (CRY1), or no effect (CRY2) on single tagged PER and CRY proteins, while it had strong effects on all combinations of PER/CRY complexes (Figure 1A–C). Similarly, knockdown of KPNB1 increased cytoplasmic retention of endogenous PER1, PER2, CRY1, and CRY2 to varying degrees, probably through complex interactions (Figure 1D, Figure1—figure supplement 2). The immunoprecipitation analysis showed that KPNB1 interacted strongly with PERs compared with CRYs (Figure 2—figure supplement 1A). These data suggest that the PERs mediate interaction with KPNB1 and gate nuclear translocation of PER/CRY complex. This is reminiscent of a previous report indicating that the nuclear entry of CRY is mainly dependent on PER, in vivo (Lee et al., 2001). Finally, knockdown of KPNB1 or its fly ortholog ketel resulted in arrhythmic U2 OS cells or fly locomotor activity rhythms, while knockdown of most of KPNA isoforms had no significant effect on the rhythm (Figure 4, Figure 5, Figure 4—figure supplement 4). These data suggest a model in which KPNB1 plays a necessary role in mediating nuclear entry of the PER/CRY complex in core clock regulation (Figure 6).

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