👤 Thomas Goedert

To understand the relative contributions of 5' UTR elements to translation output, we performed a comprehensive analysis of upstream open reading frames (uORFs) in the 5' UTRs of the BDNF (brain-derived neurotrophic factor) transcripts. Predicted uORFs were identified in 14 out of 17 BDNF RefSeq transcript isoforms, and we experimentally validated five of these transcripts as being uORF-repressed, suggesting that uORF elements play an important role in shaping the protein output from this locus. We explored several approaches to disrupt BDNF uORF function. Deletion of a 5' UTR exon in BDNF v11 (containing eight predicted uORFs), in order to simulate an exon skipping outcome, resulted in pronounced upregulation in a reporter construct system. This effect was found to be partially uORF-dependent but was also dependent on the disruption of an RNA secondary structure element. However, this transcript variant was found to not be expressed in human brain. Conversely, direct disruption of a single uORF start codon in the widely expressed BDNF v4 transcript variant using an adenine base editing approach resulted in a ∼1.8-fold upregulation of endogenous BDNF protein expression in cell culture. This study characterizes uORF-mediated regulation of the BDNF locus and demonstrates the potential for BDNF protein upregulation via base editing-mediated uORF disruption. Show less

Previous work identified RMEL3 as a lncRNA with enriched expression in melanoma. Analysis of The Cancer Genome Atlas (TCGA) data confirmed RMEL3 enriched expression in melanoma and demonstrated its association with the presence of BRAFV600E. RMEL3 siRNA-mediated silencing markedly reduced (95%) colony formation in different BRAFV600E melanoma cell lines. Multiple genes of the MAPK and PI3K pathways found to be correlated with RMEL3 in TCGA samples were experimentally confirmed. RMEL3 knockdown led to downregulation of activators or effectors of these pathways, including FGF2, FGF3, DUSP6, ITGB3 and GNG2. RMEL3 knockdown induces gain of protein levels of tumor suppressor PTEN and the G1/S cyclin-Cdk inhibitors p21 and p27, as well as a decrease of pAKT (T308), BRAF, pRB (S807, S811) and cyclin B1. Consistently, knockdown resulted in an accumulation of cells in G1 phase and subG0/G1 in an asynchronously growing population. Thus, TCGA data and functional experiments demonstrate that RMEL3 is required for MAPK and PI3K signaling, and its knockdown decrease BRAFV600E melanoma cell survival and proliferation. Show less

The Axin-dependent phosphorylation of beta-catenin catalysed by glycogen synthase kinase-3 (GSK3) is inhibited during embryogenesis. This protects beta-catenin against ubiquitin-dependent proteolysis, leading to its accumulation in the nucleus, where it controls the expression of genes important for development. Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) is a mammalian homologue of a GSK3-binding protein (GBP), which appears to play a key role in the correct establishment of the dorsal-ventral axis in Xenopus laevis. Here, we demonstrate that FRATtide (a peptide corresponding to residues 188-226 of FRAT1) binds to GSK3 and prevents GSK3 from interacting with Axin. FRATtide also blocks the GSK3-catalysed phosphorylation of Axin and beta-catenin, suggesting a potential mechanism by which GBP could trigger axis formation. In contrast, FRATtide does not suppress GSK3 activity towards other substrates, such as glycogen synthase and eIF2B, whose phosphorylation is independent of Axin but dependent on a 'priming' phosphorylation. This may explain how the essential cellular functions of GSK3 can continue, despite the suppression of beta-catenin phosphorylation. Show less