A conserved long noncoding RNA affects sleep behavior in Drosophila

Abstract

Metazoan genomes encode an abundant collection of mRNA-like, long noncoding (lnc)RNAs. Although lncRNAs greatly expand the transcriptional repertoire, we have a limited understanding of how these RNAs contribute to developmental regulation. Here, we investigate the function of the Drosophila lncRNA called yellow-achaete intergenic RNA (yar). Comparative sequence analyses show that the yar gene is conserved in Drosophila species representing 40-60 million years of evolution, with one of the conserved sequence motifs encompassing the yar promoter. Further, the timing of yar expression in Drosophila virilis parallels that in D. melanogaster, suggesting that transcriptional regulation of yar is conserved. The function of yar was defined by generating null alleles. Flies lacking yar RNAs are viable and show no overt morphological defects, consistent with maintained transcriptional regulation of the adjacent yellow (y) and achaete (ac) genes. The location of yar within a neural gene cluster led to the investigation of effects of yar in behavioral assays. These studies demonstrated that loss of yar alters sleep regulation in the context of a normal circadian rhythm. Nighttime sleep was reduced and fragmented, with yar mutants displaying diminished sleep rebound following sleep deprivation. Importantly, these defects were rescued by a yar transgene. These data provide the first example of a lncRNA gene involved in Drosophila sleep regulation. We find that yar is a cytoplasmic lncRNA, suggesting that yar may regulate sleep by affecting stabilization or translational regulation of mRNAs. Such functions of lncRNAs may extend to vertebrates, as lncRNAs are abundant in neural tissues.

Figure 1

Conservation and tissue localization of yar RNA in D. melanogaster. (A) Multiple DNA motifs are conserved in the y–ac intergenic region. (A, top) Structure of the D. melanogaster genomic region that includes y, yar, the four AS-C genes, and pepsinogen-like (pcl). Genes are shown as rectangles, with promoters shown as bent arrows. The 1A-2 regulatory element is indicated by black inverted triangle. (A, middle) An expanded view of the 9-kb region separating y and ac, including yar, with a representation of its splicing pattern. The tracks located under the yar gene structure indicate the location of the potential yar ORFs, with the size of the peptide noted. (A, bottom) Aligned with the 9-kb region are the percentage of identity plots obtained from MultiPipMaker analyses of the corresponding regions from nine drosophilid species. Regions of no alignment are indicated in white, regions with significant BLASTZ alignment to D. melanogaster are indicated in green, and regions of nongapped alignments of >100 nucleotides with >70% identity are indicated in yellow. Conserved motifs identified by MEME are indicated on the bottom track. Motif 1.1 identifies the yar promoter. A gap in the genome sequence of D. grimshawi spans the region corresponding to motifs 1.1, 1.4, and 1.3. The dashed line indicates where the intergenic regions were split in two fragments for MEME analyses. (B) Whole mount RNA in situ hybridization of aged D. melanogaster embryos. ac mRNA is detected in the neuroectoderm clusters in the early embryogenesis, yar is globally expressed in midembryogenesis, and y is expressed in late embryogenesis in denticle belts. Df(1) y ac embryos serve as a negative control. (C) Analyses of cellular localization of yar transcripts. Total RNA isolated from equal amounts of unfractionated embryos and nuclear fraction was reverse transcribed and analyzed by semiquantitative PCR. The (−) RT lanes control for genomic DNA contamination. Spliced products were detected with primer pairs flanking the intron; intronic sequences were detected with primer pairs located within the intron. The housekeeping gene Ras64B serves as a positive control.

Figure 2

yar is conserved in D. virilis. (A) Structure of the D. virilis 17-kb y–ac intergenic region. The positions of the y and ac genes are shown by rectangles. The Genscan-predicted gene (blue) and MEME-identified motifs (red) are indicated below. Four cDNAs (A–D) obtained from PCR analyses are shown; the thin line in cDNA D indicates an intron. (B) Semiquantitative PCR analyses of RNAs isolated during the indicated times of D. virilis embryogenesis. Primer pairs corresponding to ac, three of the yar cDNAs, and y were analyzed. RpL32 represents a constitutively expressed RNA and serves as a control. The (−) RT lanes control for genomic DNA contamination. RNAs were isolated from aged embryos, as indicated by hours of development.

Figure 3

Structure of the yar alleles used in the study. (Top) Schematic of the genomic region encompassing the 3′ end of y, the 1A-2 element (inverted triangle), and the first exon of yar. Previously identified alternative start sites are indicated by bent arrows (Soshnev et al. 2008). Motifs 1.1 and 1.4 are colored by darker shading in the yar gene. (Middle) Structure of the yar deletion alleles obtained by homologous recombination. Dashed line in brackets indicates deleted region; solid arrowhead represents the residual loxP site. The extant allele Df(1) y ac removes the region spanning the whole y–yar–ac locus. (Bottom) Structure of the P[yar w] transgene used in the rescue studies.

Figure 4

Quantitative analyses of gene expression in yar null mutants. RNA was isolated from wild type (Canton S) and the yar null mutants [yar^ΔHR1 (ΔHR1) and yar^ΔHR2 (ΔHR2)]. (A) Semiquantitative PCR analyses of RNAs isolated from aged embryos and mixed stage pupae. RpL32 is a constitutively expressed gene and serves as a loading control. The (−) RT lanes control for genomic DNA contamination. (B) Quantitative RT-PCR of y, yar, ac, and sc, the gene downstream of ac. Cycle threshold (Ct) values were normalized to the constitutively expressed Ras64B gene to control for the amount of input cDNA (ΔCt). A higher ΔCt value indicates lower level of RNA accumulation. Error bars indicate standard deviation from two biological replicates. Asterisks indicate statistical significance by Student’s t-test, *P < 0.05, **P < 0.01.

Figure 5

Loss of yar affects sleep behavior in Drosophila. (A) Baseline nighttime sleep in the parental yar^+/+ (y¹ yar⁺ w^67c23, open bar) line and yar mutants (y¹ yar^mutant w^67c23, solid bars). Average amounts of sleep for 3- to 5-day-old virgin females are shown (n ≥ 32). (B and C) Average duration of nighttime sleep bout and number of sleep episodes. (A′) Effect of rescue by P[yar w] on nighttime sleep duration. Shown are data obtained from the parental line (y¹ yar⁺ w^67c23, P[ΔHR2 target], open bar), the yar mutant (ΔHR2, solid bar). and the yar mutant carrying the P[yar w] rescue construct inserted at two independent genomic locations (ΔHR2 R1 and ΔHR2 R2, shaded bars). (B′ and C′) Effects of the rescue P[yar w] transposon on sleep bout duration and number of sleep bouts in yar mutants. Kruskal–Wallis one-way ANOVA, *P < 0.05. Error bars represent SEM.

Figure 6

Loss of yar causes defect in homeostatic regulation of sleep. (A) Diagram of experimental strategy for determining effects of sleep deprivation. Flies were preconditioned in the DAM system for 3 days of 12-hr day and night cycles, and baseline daytime sleep bout duration was established on day 4 (open arrowhead). Flies were sleep deprived for one night (SD) and sleep parameters were measured the following morning (solid arrowhead). (B) Total daytime sleep before (open bars) and after (solid bars) sleep deprivation is shown. (C) The average length of sleep bouts before and after sleep deprivation. (B′ and C′) Response to sleep deprivation in the w⁺ reference and yar mutants carrying the P[yar w] rescue transgene. Kruskal–Wallis one-way ANOVA, *P < 0.05, **P < 0.001. Error bars represent SEM.