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

Abstract

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.

Regulation of Doublesex1 Expression for Environmental Sex Determination in the Cladoceran Crustacean Daphnia

“…The RNA binding protein Alan shepard (Shep) ortholog was proved as a post-transcriptional regulator for Dsx1α mRNA in D. magna ( Perez et al, 2021 ). The relationship between Shep and Dsx1α mRNA was found unexpectedly based on phenotypic analysis of female embryos injected with DsRed2 reporter RNA harboring the Dsx1α or Dsx1β 5′ UTR ( Kato et al, 2018 ).…”

“…Dsx1α 5′ UTR has a sequence like “tra-2 and GLI element (TGE)” ( Figure 3 ), which has been identified as a binding site of Sup-26, an ortholog of Shep in C. elegans . In vitro reporter assay demonstrated that Shep represses translation of Dsx1α mRNA via TGE ( Figure 13A ) ( Perez et al, 2021 ).…”

“…* p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant (Student’s t -test). Adapted from Perez et al (2021) .…”

“…In in vitro translation system including the reporter mRNA harboring Dsx1α 5′ UTR, DAPALR addition canceled the Shep-dependent translational suppression ( Perez et al, 2021 ). Similarly, Dsx1α 5′ UTR had the ability for canceling the Shep function ( Figure 13A ).…”

“…Localization of mCherry fluorescence was consistent with Dsx1 mRNA localization that was analyzed by in situ hybridization ( Kato et al, 2011 ). Therefore, Line B has been used as a Dsx1 reporter line that recapitulates Dsx1 expression ( Mohamad Ishak et al, 2017 ; Kato et al, 2018 ; Perez et al, 2021 ).…”