An Untranslated RNA Sequence Encoded in the Putative Regulatory Region of fruitless , a Major Courtship Regulator Gene of Drosophila

At about 13 kb upstream from the P1 promoter of the Drosophila melanogaster fruitless (fru) gene, we identified a putative non-coding RNA (ncRNA) gene of ~150 bp (fru-upstream ncRNA: fuR). fuR is transcribed from the DNA strand complementary to that encoding the fru transcripts in both female and male adults. Of the 10 species of the subgenus Sophophora examined, 9 have the highly conserved fuR sequence. The exception is D. willistoni, in which a 38 bp insertion splits fuR into two segments. Three species of subgenus Drosophila examined also have fuR with minor insertions or deletions. It remains to be determined whether or not fuR modulates fru functions and whether or not the diversification in this sequence correlates to species differences in fru functions.


Introduction
It has long been recognized that there exist non-coding RNAs, the best characterized of which are the chromatin regulators for gene dosage compensation, such as Tsix and Xist in mammals and RoX in Drosophila (Straub & Baker, 2007).Recently, enormous efforts have been made to comprehensively characterize transcribed sequences and chromatin dynamics across the entire genome, leading to the discovery of nearly 2000 previously undescribed genes, 90% of which appear to be non-protein-coding RNA (ncRNA) genes (modENCODE Consortium, 2010).These include the major classes of structural RNAs, snoRNAs, and Argonaute-associated small RNAs such as miRNAs, siRNAs, and piwi-associated piRNAs (Sayed & Abdellatif, 2011).Although some of these small RNAs are known to be involved in splicing (snRNAs), rRNA modification (snoRNAs), RNA interference (siRNAs), and transposon silencing (piRNAs), the functions of many other ncRNAs remain to be elucidated (Sayed & Abdellatif, 2011).Recent global run-on-sequencing assays revealed the existence of low-level-bi-directional transcripts in the transcriptionally active regulatory region of many Drosophila genes (Kharchenko et al., 2011).These ncRNAs are analogous to the enhancer RNAs (eRNAs) found to be transcribed from the neuronal activity-regulated enhancers in mice; the level of eRNA expression at neuronal enhancers is positively correlated with the level of mRNA synthesis at nearby genes (Kim et al., 2010).
We have been characterizing the enhancer region of the fruitless (fru) gene that specifies the major part of neuronal circuitry for the production of male courtship behavior in Drosophila (Dickson, 2008;Yamamoto, 2008).The male courtship ritual is highly stereotypic and species-specific (Bastock and Manning, 1955;Spieth, 1952;Yamamoto et al., 1997), and fru was suggested to determine some of its species-specific characteristics by QTL analysis (Gleanson and Ritchie, 2004).The fru gene encodes a group of putative transcription factors with the BTB and zinc-finger motifs, and the transcripts from the most distal promoter, P1, are subjected to sexually dimorphic splicing (Ito et al., 1996;Ryner et al., 1996), leading ultimately to the production of Fru protein in the male but not the female nervous system (Lee at al., 2000;Usui-Aoki et al., 2000).The Fru protein thus produced masculinizes the nervous system (Kimura et al., 2005).The Fru protein structure is well conserved, whereas its expression changes markedly across species, implying the fru cis elements play important roles in courtship behavior diversified among species (Usui-Aoki et al., 2005).To evaluate this possibility, we compared the putative regulatory regions of fru among 13 Drosophila species, including 12 species whose genomic sequences have been reported (Drosophila 12 Genomes Consortium, 2007) and D. subobscura, whose genomic sequence we determined 5' to the fru P1 promoter.In the course of this analysis, we identified, within the putative fru enhancer region, a highly conserved ncRNA sequence, fru-upstream ncRNA (fuR), which is transcribed in the direction opposite that of fru.Here we report the sequence comparisons of fuR across species and discuss the evolutionary implications of the results of their phylogenetic analysis.

Flies
The D. melanogaster Canton-S strain was a gift of R. Ueda (National Institute of Genetics, Mishima, Japan).The transgenic fly lines were generated according to the method described previously (Pfeiffer et al., 2008).D. subobscura was obtained at the San Diego Stock Center.The fly stocks were raised on standard cornmeal yeast medium supplemented with (subobscura) or without (melanogaster) soybean fiber (183g/l) at 18˚C (subobscura) or 25˚C (melanogaster) under a 12:12 light-dark cycle.

PCR primer design for amplification of genomic fru of D. subobscura
As no genomic sequence data were available for D. subobscura, we cloned and sequenced the putative regulatory region upstream from the most distal promoter of fru of this species.We amplified the genomic DNA of this region as seven consecutive segments that cover an entire 31 kb region by PCR.The primers for amplification were designed based on the sequences conserved across three species, D. melanogaster, D. yakuba, and D. pseudoobscura, whose genomic sequences were available (Drosophila 12 Genomes Consortium, 2007).

Genomic DNA preparation, PCR, and sequencing
Genomic DNA extracted from whole adult bodies of D. subobscura was prepared for PCR template according to the method described previously (Pfeiffer et al., 2008).For PCR amplification of the seven genomic segments, the Long Range PCR Kit (Qiagen) was used.In the subsequent cloning of the amplified sequence, the PrimeSTAR GXL enzyme (TaKaRa) or PrimeSTAR MAX (TaKaRa) enzyme was used.Sequencing was performed on the CEQ8000 Genetic Analysis System in conjunction with the GenomeLab DTCS-Quick Start Kit (Beckman Coulter) according to the manufacturer's instructions.

Reverse transcription polymerase chain reaction (RT-PCR)
For RT-PCR, total RNA was prepared from 20 whole adult Canton-S males and females, 3-5 days old, using an RNeasy Mini Kit (Qiagen).The RNA samples were reverse transcribed by the Superscript II reverse transcriptase (Invitrogen) and a primer, Forward: CCAACAGCTTTATTGCATTTATTTGG (forward) or TGTGAAACGCACTGTCGTCGAG (reverse) after DNase I treatment, and the resulting cDNAs were used as templates for PCRs.For PCR, the Ex Taq polymerase (Takara) and the forward and reverse primers as described were used.

Results
In characterizing the genomic region upstream from the most distal promoter of fru in D. subobscura, we found, at about 13 kb upstream from the promoter, an extraordinarily conserved stretch of ~150 bp (i.e., fuR) shared by two other species, D. melanogaster and D. pseudoobscura, used for comparison (Figure 1), yet without any apparent open reading frame.Compared to the melanogaster sequence, the identities of the nucleotide sequences are 95% in D. pesudoobscura and 88% in D. subobscura, although the sequences are not conserved in the regions immediately distal and proximal to this ~150 bp stretch.An extended search of the genome database (Drosophila 12 Genomes Consortium, 2007) for the corresponding block of DNA in the fru upstream region reveals the homologous stretch in seven additional species of subgenus Sophophora (three aforementioned species belong to this subgenus), i.e., D. simulans, D. sechelia, D. erecta, D. yakuba, D. persimilis, D. ananassae, and D. willistoni, and three species of subgenus Drosophila, i.e., D. mojavensis, D. virilis, and D. grimshawi (Figure 1).Among these 13 species, D. willistoni is particularly unique in that a 38 bp insertion splits the conserved block into two pieces (Figure 1).The three species of subgenus Drosophila carry some substitutions and insertions/deletions when compared to the sequences of Sophophora species (Figure 1).Two highly conserved segments are separated from each other by a low-homology region in which most of the above-mentioned variations exist (Figure 1).
The high level of conservation of this sequence block in the potential fru regulatory region might indicate that it serves as the binding sites for some transcription factors.Alternatively, this could be a transcribed sequence without an open reading frame.To determine whether or not this conserved sequence block bears any transcript, we carried out an RT-PCR analysis for total RNA prepared from adult whole bodies of D. melanogaster (Figure 2).The result demonstrates that this conserved DNA stretch yields a transcript, which is thus referred to as fuR (Figure 2).The cDNA template prepared with the forward primer, but not the reverse primer, yielded the product, indicating that fuR is transcribed from the strand opposite to that encoding the fru transcripts.No sex difference is detected in the abundance of fuR between the sexes.
To deduce how the fuR transcription unit has been maintained or modified in evolution, we constructed a phylogenetic tree as illustrated in Figure 3.The overall features of the tree for fuR match the phylogenetic relationships based on other molecular and morphological characteristics, implying that fuR has been maintained in different clades under similar functional constraints.

Discussion
We identified an ncRNA gene in the 5' upstream from the fru transcription start site in 13 species belonging to subgenera Sophophora and Drosophila.The distal limit of the fru regulatory region remains undefined.The fru mRNA expression pattern is altered in fru 1 , which has the mutagenic breakpoint of an inversion at about 10 kb upstream from the P1 promoter (Ryner et al., 1996).This means that the fru regulatory region likely spans across this point.Attempts to recapitulate the fru expression pattern with reporters driven by P1-promoter upstream regions have not been fully successful even using the genomic fragments of 30 kb or longer (K.Matsumoto, unpublished observation).This implies that the complete regulatory region of fru extends beyond this range.Therefore, fuR is judged to sit within the putative regulatory region of fru.
The recent discoveries of eRNAs transcribed from active enhancers of certain "neuronal" genes in mice (Kim et al., 2010) suggest that fuR might be analogous to eRNAs.However, known eRNAs are transcribed bi-directionally, unlike fuR, which is uni-directionally transcribed.Our preliminary experiment indicates that overexpression of fuR in the fru-expressing neurons by means of the GAL4-UAS system does not discernibly affect male courtship behavior (S.Takayanagi, unpublished observation).The overexpression of fru in the developing eye disc as driven by GMR-GAL4 results in the distortion of eye morphogenesis (Goto et al., 2011).This fru-dominant phenotype was also unaffected by simultaneous overexpression of fuR (S.Takayanagi, unpublished observation).Further in vivo studies are required to elucidate the functions of fuR.
Sequence comparisons among 13 Drosophila species revealed that fuR is conserved in all species examined.The fru gene seems to play a central role in organizing the neural circuit for male courtship even in non-Drosophilid insects such as the cockroach and locust (Boerjan et al., 2011;Clynen et al., 2011).The ability of a fru-expressing motoneuron to induce a male-specific muscle is also conserved between Drosophila and the mosquito Anopheles (Gailey et al., 2006).These observations suggest that fru expression is rigidly regulated and exposed to strong selective pressure (Sobrinho & de Brito, 2010).It is tempting to speculate that fuR has a role in modulating fru functions for species-specific behavior, differential selection of which has shaped this ncRNA sequence in species-specific patterns.Functional analysis of fuR in the genetic model D. melanogaster will provide insights into the evolutionary constraint involved in the diversification of this fascinating RNA sequence.
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