miR-263b controls circadian locomotor activity and the structural plasticity of small ventral lateral neurons by inhibition of Beadex

Circadian clocks drive rhythmic physiology and behavior to allow adaption to daily environmental changes. In Drosophila, the small ventral lateral neurons (sLNvs) are the master pacemakers that control circadian rhythms. Circadian changes are observed in the dorsal axonal projections of the sLNvs, but their physiological importance and the underlying mechanism are unclear. Here we identified miR-263b as an important regulator of circadian rhythms in Drosophila. Flies depleted of miR-263b (miR-263bKO) exhibited dramatically impaired rhythms under constant darkness. Indeed, miR-263b is rhythmically expressed and controls circadian output by affecting the structural plasticity of sLNvs through inhibition of expression of the LIM-only protein Beadex (Bx). The misexpression of Bx in flies phenocopied miR-263bKO in behavior and molecular characteristics. In addition, the circadian phenotypes of miR-263bKO were recapitulated by mutating the miR-263b binding sites in the Bx 3’UTR. Together, these results establish miR-263b as an important regulator of circadian locomotor behavior.

To identify miRNAs that regulate circadian rhythms, we first used miRNA quantitative 122 real-time PCR to identify rhythmically expressed miRNAs in fly heads. We found that the 123 expression of miR-263b is rhythmic in fly heads, consistent with previous microarray 124 data, ( Figure 1A, (Yang, Lee, Padgett, & Edery, 2008)). Importantly, the oscillation of 125 miR-263b was abolished in the Clk Jrk mutant confirming that the expression of miR-263b 126 is under circadian control ( Figure 1A).

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Because of the high percentage of arrhythmia of the miR-263b KO          163

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To test whether sLNv dorsal projections were present in the absence of miR-263b, a 215 membrane-tethered GFP (mCD8-GFP) was used to mark the PDF projections under the 216 control of a pdf-specific promoter. As observed by PDF staining, this marker revealed 217 that the dorsal axonal branches of sLNvs were dramatically reduced ( Figure S4).

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Interestingly, overexpression of miR-263b caused similar defects in PDF axonal 219 projections ( Figure 2B and 2C). These data indicate that the proper level of miR-263b is 220 required for the circadian structural plasticity of sLNv dorsal projections.

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222 Figure S4 shows the axonal projection of an sLNv labeled by a membrane-tethered GFP.

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One of the potential mRNA targets was Beadex (Bx), which encodes a LIM-only protein.

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A disrupted circadian rhythm was previously observed in flies with mutations in Bx (Tsai,

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To test whether miR-263b directly binds to the Bx 3'UTR and inhibits its expression, the 274 Bx 3'UTR was fused downstream of a luciferase reporter and transfected into S2 cells.

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Luciferase activity was significantly suppressed when miR-263b was co-transfected with 276 the reporter. In contrast, the luciferase activity was observed in the presence of miR-277 263b when the putative miR-263b binding sites within the Bx 3'UTR were mutated 278 ( Figure 4A). Encouraged by these S2 cell results, we further tested whether Bx 279 abundance is suppressed by miR-263b in fly brain. Since the BX antibody we generated 280 in our lab did not work for staining, we decided to use an enhanced GFP (EGFP)

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reporter strategy. We tested whether miR-263b regulates BX levels by expressing an 282 EGFP reporter fused to the wild-type Bx 3'UTR or 3'UTR with miR-263b binding sites 283 mutated (same construct as Figure 4A). We expressed these two reporters using a Bx-

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GAL4 line, which is expressed in the PDF-positive sLNvs and lLNvs, as well as other 285 region of fly brain ( Figure S6). Strikingly, we found that the expression of EGFP under 286 the control of the wild-type Bx 3'UTR is significantly lower than the mutant 3'UTR in both 287 sLNvs and lLNvs ( Figure 4B and 4C) . Thus, our S2 cell and imaging results suggest 288 that Bx is a direct target of miR-263b and is negatively regulated by miR-263b.

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Here we demonstrate that miR-263b is critical for circadian behavior rhythms and

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( Figure 1A). This apparent discrepancy between the expression peak and function peak 409 may be due to the mechanism of miRNA action. Most miRNAs function through 410 translational inhibition or degradation of target mRNAs so it is possible that the peak in 411 protein abundance of miRNA targets is opposite the expression peak of the miRNA. In 412 the future, it will be interesting to see whether Bx has a peak of abundance at early 413 morning that matches its function.

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Evidence suggests that miR-263b regulates Bx expression in the sLNv cell bodies.

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A previous study and data shown here clearly show that Bx is enriched in sLNvs (Fig S5, 416 (Tsai et al., 2004)

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Each experiment was conducted three times.

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The authors declare no competing interests.