Extended Data Figure 8: DENR is required to express stuORF-containing transcripts in vivo in Drosophila.
From: DENR–MCT-1 promotes translation re-initiation downstream of uORFs to control tissue growth
a, Schematic of the fluorescent reporters introduced into Drosophila. Identical reporters were generated containing a Tubulin promoter, the 5′ UTR of CG43674, which does not contain any uORFs, the GFP open reading frame, and the SV40 poly-A. A 6-nucleotide stuORF (ATGTAA) was introduced by mutagenesis into the 5′ UTR of one construct, generating the stuORF reporter. Both constructs were inserted via phiC31-mediated recombination into the VK33 landing site at 65B2 on chromosome 3L, ensuring identical transcriptional regulation. b, The control reporter is well expressed in both larvae containing (DENR+/+) or lacking DENR (DENRKO). Immunoblotting of larval extracts (c) shows that GFP levels of the control reporter do not drop in DENR knockouts compared to controls, indicating that DENR is not required for translation of transcripts lacking uORFs. Introduction of the 6-nucleotide stuORF causes the reporter to be less expressed in a control DENR+/+ background, consistent with uORFs having a repressive role in translation of the main downstream ORF. In contrast to the control reporter, expression of the stuORF reporter is entirely dependent on DENR in vivo, as no GFP expression could be observed when the stuORF reporter is introduced into a DENRKO genetic background. This was confirmed by immunoblotting (c). c, Immunoblot to detect GFP and a loading control (awd) in larval extracts of animals bearing a control or a stuORF reporter, either in a control DENR+/+ or in a DENR knockout genetic background.
