Sense-overlapping lncRNA as a decoy of translational repressor protein for dimorphic gene expression

Author summary Long noncoding RNAs are vastly transcribed throughout the genome. Among them, RNAs overlapping the protein-coding RNA in sense orientation have been poorly studied because of the difficulty in differentiating their sequences from their overlapping coding RNAs although this class of RNAs has been reported to comprise the majority of the long noncoding RNAs. In the crustacean Daphnia magna, a long noncoding RNA, called DAPALR, is transcribed from the male determining gene, Doublesex1, and overlaps with the Doublesex1 5 ' UTR. DAPALR activates Doublesex1 but this regulatory mechanism remains unknown. We found the RNA binding protein Shep bound to the Doublesex1 5 ' UTR. In vitro and in vivo experiments indicated that Shep suppresses translation of the mRNA and DAPALR increases Doublesex1 translation efficiency by sequestration of Shep. Since male-specific expression of Doublesex1 is also regulated at the transcriptional level, we propose that Shep cancels the unexpected expression of Doublesex1 and maintains the feminized state for sexual dimorphism but DAPALR suppresses this repression by sequestration of Shep. We infer that this mechanism is not only for binary sex regulation but could function in the binary regulation of other genes in various biological processes. Long noncoding RNAs (lncRNAs) are vastly transcribed and extensively studied but lncRNAs overlapping with the sense orientation of mRNA have been poorly studied. We analyzed the lncRNA DAPALR overlapping with the 5 ' UTR of the Doublesex1 (Dsx1), the male determining gene in Daphnia magna. By affinity purification, we identified an RNA binding protein, Shep as a DAPALR binding protein. Shep also binds to Dsx1 5 ' UTR by recognizing the overlapping sequence and suppresses translation of the mRNA. In vitro and in vivo analyses indicated that DAPALR increased Dsx1 translation efficiency by sequestration of Shep. This regulation was impaired when the Shep binding site in DAPALR was deleted. These results suggest that Shep suppresses the unintentional translation of Dsx1 by setting a threshold; and when the sense lncRNA DAPALR is expressed, DAPALR cancels the suppression caused by Shep. This mechanism may be important to show dimorphic gene expressions such as sex determination and it may account for the binary expression in various developmental processes.

Automated summary

Long noncoding RNAs (lncRNAs) are extensively transcribed and studied, but those overlapping with mRNA in the sense orientation have been less explored. We investigated the lncRNA DAPALR overlapping with the 5' UTR of the male determining gene Doublesex1 (Dsx1) in Daphnia magna. Through affinity purification, we identified an RNA binding protein, Shep, as a DAPALR binding protein. Shep binds to Dsx1 5' UTR by recognizing the overlapping sequence and suppresses mRNA translation. In vitro and in vivo analyses indicated that DAPALR enhances Dsx1 translation efficiency by sequestering Shep. This mechanism may play a significant role in displaying dimorphic gene expressions, such as sex determination, and could explain binary expression in various developmental processes.