👤 Nenad Svrzikapa

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