Long noncoding RNAs (lncRNAs) are pervasively transcribed in the eukaryotic genome [1] and are important for the control of master regulatory genes that are involved in cell differentiation and development [2, 3]. Here, we show that a 5' UTR-overlapping lncRNA regulates the male-specific expression of the DM-domain gene doublesex1 (dsx1) in the crustacean Daphnia magna, which produces males in response to environmental stimuli. This lncRNA, named doublesex1 alpha promoter-associated long RNA (DAPALR), is transcribed upstream the transcription start site (TSS) in a sense orientation and subjected to 5' end capping and 3' end processing at a stem-loop structure before the dsx1 coding exon. Similar to dsx1, its expression is only activated in males by the juvenile hormone (JH) and basic-leucine zipper (bZIP) transcription factor Vrille (Vri) and is maintained during embryogenesis. Knockdown of DAPALR in males silenced dsx1 and led to feminization, including egg production, whereas ectopic expression of DAPALR in dsx1-silenced females resulted in the de-repression of dsx1. We further demonstrate that the DAPALR transcript overlaps the dsx1 5'-UTR, and this overlapping region is required for dsx1 activation. Our results suggest that DAPALR can transactivate and possibly maintain dsx1 expression. This might be important for converting transient environmental signals into stable male development, controlled by the continuous expression of dsx1.

Loading of small RNAs into Argonaute, the core protein in RNA silencing, requires the Hsp70/Hsp90 chaperone machinery. This machinery also activates many other clients, including steroid hormone receptors and kinases, but how their structures change during chaperone-dependent activation remains unclear. Here, we utilized single-molecule Förster resonance energy transfer (smFRET) to probe the conformational changes of Drosophila Ago2 mediated by the chaperone machinery. We found that empty Ago2 exists in various closed conformations. The Hsp70 system (Hsp40 and Hsp70) and the Hsp90 system (Hop, Hsp90, and p23) together render Ago2 into an open, active form. The Hsp70 system, but not the Hsp90 system alone, is sufficient for Ago2 to partially populate the open form. Instead, the Hsp90 system is required to extend the dwell time of Ago2 in the open state, which must be transiently primed by the Hsp70 system. Our data uncover distinct and coordinated actions of the chaperone machinery, where the Hsp70 system expands the structural ensembles of Ago2 and the Hsp90 system captures and stabilizes the active form.

The functional coherence between microRNAs (miRNAs) and their host genes remains enigmatic. As host genes and its intronic miRNAs are transcribed as cistronic units, their functional coherence could be beneficial for the organism. However, it is not clear how broad the coherence takes place in the organisms. In this study, we focused on the functions of a miRNA miR-140 and its host gene Wwp2, both of which are reported to be important for the craniofacial development in mouse (Miyaki et al. Gene Dev. 2010, Zou et al. Nat. Cell Biol. 2011). Using CRISPR/Cas9 system, we generated single and double knockout mice for miR-140 and Wwp2 to explore the possible coherence in their functions. Unexpectedly, we found that the miRNA, but not the protein coding gene, is involved in the craniofacial development. Further analysis with compound heterozygous mice suggested that the gene-trap cassette used in the original report of Wwp2 knockout mouse (Zou et al. Nat. Cell Biol. 2011) unintendedly downregulated the expression of miR-140, which lies downstream of the insertion site, and this caused the craniofacial phenotype. Our results dissected the role of a microRNA and its host gene by CRISPR/Cas9 system, and raised a caution for the phenotypic evaluation of knockout mice generated by gene-trap or similar strategy.