Identification of gene isoforms and their switching events between male and female embryos of the parthenogenetic crustacean Daphnia magna.

The cladoceran crustacean Daphnia exhibits phenotypic plasticity, a phenomenon that leads to diverse phenotypes from one genome. Alternative usage of gene isoforms has been considered a key gene regulation mechanism for controlling different phenotypes. However, to understand the phenotypic plasticity of Daphnia, gene isoforms have not been comprehensively analyzed. Here we identified 25,654 transcripts derived from the 9710 genes expressed during environmental sex determination of Daphnia magna using the long-read RNA-Seq with PacBio Iso-Seq. We found that 14,924 transcripts were previously unidentified and 5713 genes produced two or more isoforms. By a combination of Illumina short-read RNA-Seq, we detected 824 genes that implemented switching of the highest expressed isoform between females and males. Among the 824 genes, we found isoform switching of an ortholog of CREB-regulated transcription coactivator, a major regulator of carbohydrate metabolism in animals, and a correlation of this switching event with the sexually dimorphic expression of carbohydrate metabolic genes. These results suggest that a comprehensive catalog of isoforms may lead to understanding the molecular basis for environmental sex determination of Daphnia. We also infer the applicability of the full-length isoform analyses to the elucidation of phenotypic plasticity in Daphnia.

CELF1 represses Doublesex1 expression via its 5' UTR in the crustacean Daphnia magna.

In sex determination of the crustacean Daphnia magna, male-specific expression of DM-domain transcription factor Doublesex1 (Dsx1) orchestrates the male developmental program triggered by environmental stimuli. We previously identified the CELF1 ortholog as a candidate of proteins associated with the 5' UTR of the Dsx1α isoform. Here we report the CELF1-dependent suppression of Dsx1 expression in D. magna. During embryogenesis, CELF1 expression was not sexually dimorphic. Silencing of CELF1 led to the activation of Dsx1 expression both in female and male embryos. Overexpression of CELF1 in male embryos resulted in a reduction of Dsx1 expression. By these manipulations of CELF1 expression, the Dsx1 transcript level was not significantly changed. To investigate whether the CELF1 controls Dsx1 expression via its 5' UTR, we injected the GFP reporter mRNA having intact Dsx1α 5' UTR or mutated one lacking the GU-rich element (GRE) that is known as a binding site of the CELF1 ortholog. We found that deletion of the GRE significantly increased the reporter gene expression. These results indicate that CELF1 suppresses Dsx1 expression both in females and males, possibly at the post-transcriptional level. We speculate that CELF1 may avoid unintended Dsx1 expression and generation of sexual ambiguity by setting a threshold of Dsx1 expression.

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

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.