Abstract
MicroRNAs (miRNAs) modulate gene expression by degrading or inhibiting translation of messenger RNAs (mRNAs). Here, we demonstrated that chicken microRNA-26a (gga-mir-26a) is a key posttranscriptional regulator of photoreceptor L-type voltage-gated calcium channel alpha1C subunit (L-VGCCalpha1C) expression, and its own expression has a diurnal rhythm, thereby explaining the rhythmic nature of L-VGCCalpha1Cs. Circadian oscillators in retinal photoreceptors provide a mechanism that allows photoreceptors to anticipate daily illumination changes. In photoreceptors, L-VGCC activities are under circadian control, which are higher at night and lower during the day. Interestingly, the mRNA level of VGCCalpha1D oscillates, but those for VGCCalpha1C do not. However, the protein expression of both VGCCalpha1C and alpha1D are higher at night in cone photoreceptors. The underlying mechanism regulating L-VGCCalpha1C protein expression was not clear until now. In vitro targeting reporter assays verified that gga-mir-26a specifically targeted the L-VGCCalpha1C 3'-untranslated region, and gga-mir-26a expression in the retina peaked during the day. After transfection with gga-mir-26a, L-VGCCalpha1C protein expression and L-VGCC current density decreased. Therefore, the rhythmic expression of gga-mir-26a regulated the protein expression of the L-VGCCalpha1C subunit. Additionally, both CLOCK (circadian locomoter output cycles kaput) and CREB (cAMP-response element-binding protein-1) activated gga-mir-26a expression in vitro. This result implies that gga-mir-26a might be a downstream target of circadian oscillators. Our work has uncovered new functional roles for miRNAs in the regulation of circadian rhythms in cone photoreceptors. Circadian regulated miRNAs could serve as the link between the core oscillator and output signaling that further govern biological functions.
Highlights
The intrinsic circadian clocks govern various physiological functions and behaviors in animals, ranging from sleep and wakefulness to oscillations of body temperature, heart rate, hormone secretion, food intake, and locomotor activity to list a few (9 –13)
The apparent affinity of CNGCs to cGMP is higher during the subjective night than the subjective day, whereas the maximum current amplitude does not change throughout the day [18, 53]
A common mechanism underlying the circadian regulation of both channels is the Ras-ERK signal transduction pathway
Summary
Circadian Entrainment—Fertilized eggs (Gallus gallus) were obtained from the Poultry Science Department, Texas A&M University (College Station, TX). All PCR products were purified by a gel extraction kit (Qiagen, Valencia, CA) and cloned into pGEM Teasy (gga-mir-26a precursor and gga-mir26a promoter; Promega, Madison, WI) or pBluescript II (L-VGCC␣1C 3Ј-UTR; Stratagene, La Jolla, Ca) cloning vectors for sequence analysis. For miRNA targeting assays, cells were cultured in 24-well plates and transfected with control or gga-mir-26a expression vector in combination with a reporter construct, TK-Luc4XWT (wild type) or TK-luc4Xmt (mutated) (150 ng each). The plasmid encoding enhanced green fluorescent protein (eGFP) is commercially available (Vitality hrGFPII-1 mammalian expression vector; Stratagene) In these experiments, gga-mir-26a or the empty vector was co-transfected with the GFP plasmid. Transfections of anti-mir-26a were done by the same biolistic particle delivery system as described above with a molecular ratio of 20:1 to the phrGFPII-1 vector In these experiments, chicken retina cells (E11) were cultured and LD entrained for 6 days as described earlier.
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