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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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Drosophila NA, UNC79, and UNC80 exhibit an interdependent regulatory relationship.(A) Representative Western blot analyses, performed from adult Drosophila head extracts. The strains assayed were generated by backcrossing nae04385 (Lanes 1–2), unc79x25 (Lanes 3–4), unc80x42 (Lanes 5–6), or unc80GS12792 (Lanes 7–8) to iso31 for 6–8 generations. NS  =  non-specific UNC79 bands; see Materials and Methods for more details. (B) Quantitation of NA, UNC79, and UNC80 protein levels in each mutant strain, as a percentage of the level observed in the corresponding wild-type strain. Black bars indicate NA protein, gray bars UNC79 protein, and white bars UNC80 protein. Error bars indicate standard error of the mean, determined from 3 independent experiments. NA, UNC79, and UNC80 protein levels are significantly lower in each mutant strain compared to the corresponding control strain, as determined by Student’s t-test (all P<0.01, except UNC80 levels in nae04358, P  =  0.012).
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pone-0078147-g003: Drosophila NA, UNC79, and UNC80 exhibit an interdependent regulatory relationship.(A) Representative Western blot analyses, performed from adult Drosophila head extracts. The strains assayed were generated by backcrossing nae04385 (Lanes 1–2), unc79x25 (Lanes 3–4), unc80x42 (Lanes 5–6), or unc80GS12792 (Lanes 7–8) to iso31 for 6–8 generations. NS  =  non-specific UNC79 bands; see Materials and Methods for more details. (B) Quantitation of NA, UNC79, and UNC80 protein levels in each mutant strain, as a percentage of the level observed in the corresponding wild-type strain. Black bars indicate NA protein, gray bars UNC79 protein, and white bars UNC80 protein. Error bars indicate standard error of the mean, determined from 3 independent experiments. NA, UNC79, and UNC80 protein levels are significantly lower in each mutant strain compared to the corresponding control strain, as determined by Student’s t-test (all P<0.01, except UNC80 levels in nae04358, P  =  0.012).

Mentions: To examine the regulatory relationship between NA and the putative channel subunits UNC79 and UNC80, we assessed protein and transcript levels in head extracts obtained from na, unc79, and unc80 mutants. It has previously been shown that unc79f03453 mutants express decreased levels of NA protein, but normal levels of na transcript [7]. Consistent with this finding, we observe that unc79x25 mutants express little or no detectable NA protein (Figure 3A, top panel, lanes 3–4; Figure 3B, black bars, P<0.01 compared to unc79+ control), but only a minor decrease in na transcript (Figure S3A, black bars, P<0.05). We also find that unc79x25 flies express strongly decreased levels of UNC80 protein (Figure 3A bottom panel; Figure 3B, white bars, P<0.01), but no significant change in unc80 transcript (Figure S3A, white bars). These data support a post-transcriptional regulatory relationship among the putative subunits. Similarly, we find that unc80x42 and unc80GS12792 mutants express very little NA or UNC79 protein relative to wild-type control strains (Figure 3A, lanes 5-8; Figure 3B, black and gray bars, P<0.01), while unc80x42 mutants express normal levels of na and unc79 transcript (Figure S3B). In addition, we assessed the dependence of UNC79 and UNC80 on na. UNC79 and UNC80 protein levels are strongly decreased in nae04385 mutants (Figure 3A, lanes 1–2; Figure 3B, gray and white bars, P<0.05). While unc79 and unc80 transcript levels are also somewhat lower in nae04385 mutants than controls (Figure S3C, P<0.05), the observed decreases in transcript levels (∼23%) appear insufficient to explain the decreases in protein expression (> =  86%). Notably, pan-neuronal RNAi knockdown of na, unc79, or unc80 is associated with reduced expression of the other two proteins (Figure S2B). Taken together, these data indicate that the expression of NA, UNC79, and UNC80 proteins is strongly interdependent.


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)

Drosophila NA, UNC79, and UNC80 exhibit an interdependent regulatory relationship.(A) Representative Western blot analyses, performed from adult Drosophila head extracts. The strains assayed were generated by backcrossing nae04385 (Lanes 1–2), unc79x25 (Lanes 3–4), unc80x42 (Lanes 5–6), or unc80GS12792 (Lanes 7–8) to iso31 for 6–8 generations. NS  =  non-specific UNC79 bands; see Materials and Methods for more details. (B) Quantitation of NA, UNC79, and UNC80 protein levels in each mutant strain, as a percentage of the level observed in the corresponding wild-type strain. Black bars indicate NA protein, gray bars UNC79 protein, and white bars UNC80 protein. Error bars indicate standard error of the mean, determined from 3 independent experiments. NA, UNC79, and UNC80 protein levels are significantly lower in each mutant strain compared to the corresponding control strain, as determined by Student’s t-test (all P<0.01, except UNC80 levels in nae04358, P  =  0.012).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818319&req=5

pone-0078147-g003: Drosophila NA, UNC79, and UNC80 exhibit an interdependent regulatory relationship.(A) Representative Western blot analyses, performed from adult Drosophila head extracts. The strains assayed were generated by backcrossing nae04385 (Lanes 1–2), unc79x25 (Lanes 3–4), unc80x42 (Lanes 5–6), or unc80GS12792 (Lanes 7–8) to iso31 for 6–8 generations. NS  =  non-specific UNC79 bands; see Materials and Methods for more details. (B) Quantitation of NA, UNC79, and UNC80 protein levels in each mutant strain, as a percentage of the level observed in the corresponding wild-type strain. Black bars indicate NA protein, gray bars UNC79 protein, and white bars UNC80 protein. Error bars indicate standard error of the mean, determined from 3 independent experiments. NA, UNC79, and UNC80 protein levels are significantly lower in each mutant strain compared to the corresponding control strain, as determined by Student’s t-test (all P<0.01, except UNC80 levels in nae04358, P  =  0.012).
Mentions: To examine the regulatory relationship between NA and the putative channel subunits UNC79 and UNC80, we assessed protein and transcript levels in head extracts obtained from na, unc79, and unc80 mutants. It has previously been shown that unc79f03453 mutants express decreased levels of NA protein, but normal levels of na transcript [7]. Consistent with this finding, we observe that unc79x25 mutants express little or no detectable NA protein (Figure 3A, top panel, lanes 3–4; Figure 3B, black bars, P<0.01 compared to unc79+ control), but only a minor decrease in na transcript (Figure S3A, black bars, P<0.05). We also find that unc79x25 flies express strongly decreased levels of UNC80 protein (Figure 3A bottom panel; Figure 3B, white bars, P<0.01), but no significant change in unc80 transcript (Figure S3A, white bars). These data support a post-transcriptional regulatory relationship among the putative subunits. Similarly, we find that unc80x42 and unc80GS12792 mutants express very little NA or UNC79 protein relative to wild-type control strains (Figure 3A, lanes 5-8; Figure 3B, black and gray bars, P<0.01), while unc80x42 mutants express normal levels of na and unc79 transcript (Figure S3B). In addition, we assessed the dependence of UNC79 and UNC80 on na. UNC79 and UNC80 protein levels are strongly decreased in nae04385 mutants (Figure 3A, lanes 1–2; Figure 3B, gray and white bars, P<0.05). While unc79 and unc80 transcript levels are also somewhat lower in nae04385 mutants than controls (Figure S3C, P<0.05), the observed decreases in transcript levels (∼23%) appear insufficient to explain the decreases in protein expression (> =  86%). Notably, pan-neuronal RNAi knockdown of na, unc79, or unc80 is associated with reduced expression of the other two proteins (Figure S2B). Taken together, these data indicate that the expression of NA, UNC79, and UNC80 proteins is strongly interdependent.

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
Related in: MedlinePlus