Cellular function of the GndA small open reading frame-encoded polypeptide during heat shock.

Over the past 15 years, hundreds of previously undiscovered bacterial small open reading frame (sORF)-encoded polypeptides (SEPs) of fewer than fifty amino acids have been identified, and biological functions have been ascribed to an increasing number of SEPs from intergenic regions and small RNAs. However, despite numbering in the dozens in Escherichia coli, and hundreds to thousands in humans, same-strand nested sORFs that overlap protein coding genes in alternative reading frames remain understudied. In order to provide insight into this enigmatic class of unannotated genes, we characterized GndA, a 36-amino acid, heat shock-regulated SEP encoded within the +2 reading frame of the gnd gene in E. coli K-12 MG1655. We show that GndA pulls down components of respiratory complex I (RCI) and is required for proper localization of a RCI subunit during heat shock. At high temperature GndA deletion (ΔGndA) cells exhibit perturbations in cell growth, NADH+/NAD ratio, and expression of a number of genes including several associated with oxidative stress. These findings suggest that GndA may function in maintenance of homeostasis during heat shock. Characterization of GndA therefore supports the nascent but growing consensus that functional, overlapping genes occur in genomes from viruses to humans.

Enhanced eMAGE applied to identify genetic factors of nuclear hormone receptor dysfunction via combinatorial gene editing.

Technologies that generate precise combinatorial genome modifications are well suited to dissect the polygenic basis of complex phenotypes and engineer synthetic genomes. Genome modifications with engineered nucleases can lead to undesirable repair outcomes through imprecise homology-directed repair, requiring non-cleavable gene editing strategies. Eukaryotic multiplex genome engineering (eMAGE) generates precise combinatorial genome modifications in Saccharomyces cerevisiae without generating DNA breaks or using engineered nucleases. Here, we systematically optimize eMAGE to achieve 90% editing frequency, reduce workflow time, and extend editing distance to 20 kb. We further engineer an inducible dominant negative mismatch repair system, allowing for high-efficiency editing via eMAGE while suppressing the elevated background mutation rate 17-fold resulting from mismatch repair inactivation. We apply these advances to construct a library of cancer-associated mutations in the ligand-binding domains of human estrogen receptor alpha and progesterone receptor to understand their impact on ligand-independent autoactivation. We validate that this yeast model captures autoactivation mutations characterized in human breast cancer models and further leads to the discovery of several previously uncharacterized autoactivating mutations. This work demonstrates the development and optimization of a cleavage-free method of genome editing well suited for applications requiring efficient multiplex editing with minimal background mutations.