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UNC79 and UNC80, putative auxiliary subunits of the NARROW ABDOMEN ion channel, are indispensable for robust circadian locomotor rhythms in Drosophila.

Lear BC, Darrah EJ, Aldrich BT, Gebre S, Scott RL, Nash HA, Allada R - PLoS ONE (2013)

Bottom Line: We observe an interdependent, post-transcriptional regulatory relationship among the three gene products, as loss of na, unc79, or unc80 gene function leads to decreased expression of all three proteins, with minimal effect on transcript levels.Immunoprecipitation experiments also confirm that UNC79 and UNC80 form a complex with NA in the Drosophila brain.Taken together, these data suggest that Drosophila NA, UNC79, and UNC80 function together in circadian clock neurons to promote rhythmic behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
In the fruit fly Drosophila melanogaster, a network of circadian pacemaker neurons drives daily rhythms in rest and activity. The ion channel NARROW ABDOMEN (NA), orthologous to the mammalian sodium leak channel NALCN, functions downstream of the molecular circadian clock in pacemaker neurons to promote behavioral rhythmicity. To better understand the function and regulation of the NA channel, we have characterized two putative auxiliary channel subunits in Drosophila, unc79 (aka dunc79) and unc80 (aka CG18437). We have generated novel unc79 and unc80 mutations that represent strong or complete loss-of-function alleles. These mutants display severe defects in circadian locomotor rhythmicity that are indistinguishable from na mutant phenotypes. Tissue-specific RNA interference and rescue analyses indicate that UNC79 and UNC80 likely function within pacemaker neurons, with similar anatomical requirements to NA. We observe an interdependent, post-transcriptional regulatory relationship among the three gene products, as loss of na, unc79, or unc80 gene function leads to decreased expression of all three proteins, with minimal effect on transcript levels. Yet despite this relationship, we find that the requirement for unc79 and unc80 in circadian rhythmicity cannot be bypassed by increasing NA protein expression, nor can these putative auxiliary subunits substitute for each other. These data indicate functional requirements for UNC79 and UNC80 beyond promoting channel subunit expression. Immunoprecipitation experiments also confirm that UNC79 and UNC80 form a complex with NA in the Drosophila brain. Taken together, these data suggest that Drosophila NA, UNC79, and UNC80 function together in circadian clock neurons to promote rhythmic behavior.

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Transgenic unc79, unc80 or na expression produces increased protein levels in wild-type and mutant backgrounds.(A-C) Western blot analyses were used to label UNC79, UNC80, and NA proteins from adult head extracts; representative blots are shown. For all blots shown, lane 1  =  elavGAL4/+ (no UAS); lanes 3,5,7  =  UAS/+ (no GAL4); lanes 2,4,6,8  =  elavGAL4; UAS/+. (A) Pan-neuronal expression of UAS-unc79MYC 23-24 in wild-type (lane 2), nahar (lane 4), unc79x25 (lane 6), or unc80x42 (lane 8) backgrounds. (B) Expression of UAS-HAunc80 1M in wild-type (lane 2), nahar (lane 4), unc79x25 (lane 6), or unc80x42 (lane 8) backgrounds. (C) Expression of UAS-na U3 in wild-type (lane 2), nahar (lane 4), unc79x25 (lane 6), or unc80x42 (lane 8) backgrounds.
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pone-0078147-g005: Transgenic unc79, unc80 or na expression produces increased protein levels in wild-type and mutant backgrounds.(A-C) Western blot analyses were used to label UNC79, UNC80, and NA proteins from adult head extracts; representative blots are shown. For all blots shown, lane 1  =  elavGAL4/+ (no UAS); lanes 3,5,7  =  UAS/+ (no GAL4); lanes 2,4,6,8  =  elavGAL4; UAS/+. (A) Pan-neuronal expression of UAS-unc79MYC 23-24 in wild-type (lane 2), nahar (lane 4), unc79x25 (lane 6), or unc80x42 (lane 8) backgrounds. (B) Expression of UAS-HAunc80 1M in wild-type (lane 2), nahar (lane 4), unc79x25 (lane 6), or unc80x42 (lane 8) backgrounds. (C) Expression of UAS-na U3 in wild-type (lane 2), nahar (lane 4), unc79x25 (lane 6), or unc80x42 (lane 8) backgrounds.

Mentions: To further assess the functional requirements for unc79 and unc80, we performed tissue-specific transgenic expression experiments. Expression of full-length unc79 cDNA in pacemaker neurons using timGAL4 strongly restores circadian behavior in unc79x25 mutants in both LD and DD conditions (Figure 4A, B, P<0.05; Table 3, P<0.001). Moreover, an intronic insertion in unc79 that contains a UAS element (unc79f01615) also rescues behavior in the presence of timGAL4 (Figure 4C, D, P<0.05; Table 3, P<0.001). Based on the position of this insertion, we predict that this UAS element initiates the production of functional, truncated form(s) of UNC79 (Figure S1A). Indeed, at least two truncated UNC79 protein bands can be detected in GAL4/ unc79f01615 flies via Western blot that are not evident in wild-type flies (data not shown). These data suggest that UNC79 function in pacemaker neurons is sufficient to promote rhythmicity. We also assessed whether transgenic expression of UNC79 restores NA and UNC80 expression in unc79 mutants. For these assays we utilized elavGAL4, as this pan-neuronal driver is likely more representative of endogenous channel complex expression than timGAL4[3]. As described earlier, elavGAL4 driven expression of RNAi to na, unc79, or unc80 leads to strong knockdown of the corresponding proteins in adult head extracts, indicating that this driver encompasses the major sources of expression of all three gene products in the Drosophila head (Figure S2A). We find that pan-neuronal expression of either full-length or truncated UNC79 strongly restores NA and UNC80 protein levels to unc79 mutants (Figure 5A, lanes 5-6, P<0.01; data not shown).


UNC79 and UNC80, putative auxiliary subunits of the NARROW ABDOMEN ion channel, are indispensable for robust circadian locomotor rhythms in Drosophila.

Lear BC, Darrah EJ, Aldrich BT, Gebre S, Scott RL, Nash HA, Allada R - PLoS ONE (2013)

Transgenic unc79, unc80 or na expression produces increased protein levels in wild-type and mutant backgrounds.(A-C) Western blot analyses were used to label UNC79, UNC80, and NA proteins from adult head extracts; representative blots are shown. For all blots shown, lane 1  =  elavGAL4/+ (no UAS); lanes 3,5,7  =  UAS/+ (no GAL4); lanes 2,4,6,8  =  elavGAL4; UAS/+. (A) Pan-neuronal expression of UAS-unc79MYC 23-24 in wild-type (lane 2), nahar (lane 4), unc79x25 (lane 6), or unc80x42 (lane 8) backgrounds. (B) Expression of UAS-HAunc80 1M in wild-type (lane 2), nahar (lane 4), unc79x25 (lane 6), or unc80x42 (lane 8) backgrounds. (C) Expression of UAS-na U3 in wild-type (lane 2), nahar (lane 4), unc79x25 (lane 6), or unc80x42 (lane 8) backgrounds.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818319&req=5

pone-0078147-g005: Transgenic unc79, unc80 or na expression produces increased protein levels in wild-type and mutant backgrounds.(A-C) Western blot analyses were used to label UNC79, UNC80, and NA proteins from adult head extracts; representative blots are shown. For all blots shown, lane 1  =  elavGAL4/+ (no UAS); lanes 3,5,7  =  UAS/+ (no GAL4); lanes 2,4,6,8  =  elavGAL4; UAS/+. (A) Pan-neuronal expression of UAS-unc79MYC 23-24 in wild-type (lane 2), nahar (lane 4), unc79x25 (lane 6), or unc80x42 (lane 8) backgrounds. (B) Expression of UAS-HAunc80 1M in wild-type (lane 2), nahar (lane 4), unc79x25 (lane 6), or unc80x42 (lane 8) backgrounds. (C) Expression of UAS-na U3 in wild-type (lane 2), nahar (lane 4), unc79x25 (lane 6), or unc80x42 (lane 8) backgrounds.
Mentions: To further assess the functional requirements for unc79 and unc80, we performed tissue-specific transgenic expression experiments. Expression of full-length unc79 cDNA in pacemaker neurons using timGAL4 strongly restores circadian behavior in unc79x25 mutants in both LD and DD conditions (Figure 4A, B, P<0.05; Table 3, P<0.001). Moreover, an intronic insertion in unc79 that contains a UAS element (unc79f01615) also rescues behavior in the presence of timGAL4 (Figure 4C, D, P<0.05; Table 3, P<0.001). Based on the position of this insertion, we predict that this UAS element initiates the production of functional, truncated form(s) of UNC79 (Figure S1A). Indeed, at least two truncated UNC79 protein bands can be detected in GAL4/ unc79f01615 flies via Western blot that are not evident in wild-type flies (data not shown). These data suggest that UNC79 function in pacemaker neurons is sufficient to promote rhythmicity. We also assessed whether transgenic expression of UNC79 restores NA and UNC80 expression in unc79 mutants. For these assays we utilized elavGAL4, as this pan-neuronal driver is likely more representative of endogenous channel complex expression than timGAL4[3]. As described earlier, elavGAL4 driven expression of RNAi to na, unc79, or unc80 leads to strong knockdown of the corresponding proteins in adult head extracts, indicating that this driver encompasses the major sources of expression of all three gene products in the Drosophila head (Figure S2A). We find that pan-neuronal expression of either full-length or truncated UNC79 strongly restores NA and UNC80 protein levels to unc79 mutants (Figure 5A, lanes 5-6, P<0.01; data not shown).

Bottom Line: We observe an interdependent, post-transcriptional regulatory relationship among the three gene products, as loss of na, unc79, or unc80 gene function leads to decreased expression of all three proteins, with minimal effect on transcript levels.Immunoprecipitation experiments also confirm that UNC79 and UNC80 form a complex with NA in the Drosophila brain.Taken together, these data suggest that Drosophila NA, UNC79, and UNC80 function together in circadian clock neurons to promote rhythmic behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
In the fruit fly Drosophila melanogaster, a network of circadian pacemaker neurons drives daily rhythms in rest and activity. The ion channel NARROW ABDOMEN (NA), orthologous to the mammalian sodium leak channel NALCN, functions downstream of the molecular circadian clock in pacemaker neurons to promote behavioral rhythmicity. To better understand the function and regulation of the NA channel, we have characterized two putative auxiliary channel subunits in Drosophila, unc79 (aka dunc79) and unc80 (aka CG18437). We have generated novel unc79 and unc80 mutations that represent strong or complete loss-of-function alleles. These mutants display severe defects in circadian locomotor rhythmicity that are indistinguishable from na mutant phenotypes. Tissue-specific RNA interference and rescue analyses indicate that UNC79 and UNC80 likely function within pacemaker neurons, with similar anatomical requirements to NA. We observe an interdependent, post-transcriptional regulatory relationship among the three gene products, as loss of na, unc79, or unc80 gene function leads to decreased expression of all three proteins, with minimal effect on transcript levels. Yet despite this relationship, we find that the requirement for unc79 and unc80 in circadian rhythmicity cannot be bypassed by increasing NA protein expression, nor can these putative auxiliary subunits substitute for each other. These data indicate functional requirements for UNC79 and UNC80 beyond promoting channel subunit expression. Immunoprecipitation experiments also confirm that UNC79 and UNC80 form a complex with NA in the Drosophila brain. Taken together, these data suggest that Drosophila NA, UNC79, and UNC80 function together in circadian clock neurons to promote rhythmic behavior.

Show MeSH