Keywords: h. translational regulation; f. eye disc
Drosophilanocte encodes a 250 kDa protein with an N-terminal BAT2 domain, RGG motifs in the middle, and large low-complexity regions. Studies in Drosophila showed that a partial mutation of nocte leads to defects in temperature compensation of the circadian clock. A recent study shows that mouse Nocte orthologs PRRC2A is an m6A reader and its binding can stabilize specific mRNAs; and its inactivation disrupts neural development. However, the molecular functions of Nocte and its orthologs are still largely unknown. Our results from IP-Mass Spec experiments of RNA-binding proteins Top3b and TDRD3 identify Nocte as their strong binding partner. IP-Mass Spec experiment of Nocte shows that it interacts with other RNA-binding proteins and ribosomal proteins, supporting that Nocte is an RNA binding protein. We generated nocte knockouts by CRISPR and found that nocte-KO flies die at larval-pupal stages. Specific knockdown of nocte by RNAi in fly eyes induces rough and small eye phenotypes. The RNA-seq results from WT and nocte-RNAi eye discs indicate that Nocte depletion disrupts chaoptin transcription. Immunostaining, qRT-PCR and RIP-qPCR results show that Nocte is necessary and sufficient to promote the translation of glass mRNA, which encodes a transcription factor regulating chaoptin directly. With reporter assays and CRISPR edited alleles, we demonstrated that Nocte counteracts the suppression effects of the upstream ORF (uORF) at glass 5’UTR to maintain glass translation. We are investigating the detailed mechanisms of how Nocte regulates glass translation and its global effects on mRNA translation by Ribo-seq.