Cytotoxicity and function of incomplete proteins - PROJECT SUMMARY / ABSTRACT Rationale: All cells produce incomplete proteins, a consequence of surprisingly common errors in gene expression. Cells have evolved multiple mechanisms to suppress expression of incomplete proteins, including the degradation of mRNAs containing premature stop codons (nonsense variants). Such nonsense-mediated mRNA quality control occurs in both prokaryotes and eukaryotes. The bacterial process is grounded in premature termination of transcription by Rho and subsequent RNA degradation, a mechanism reliant on tight coupling of transcription and translation. The existence and apparent universality of nonsense-mediated quality control has been taken to suggest that incomplete proteins are highly cytotoxic. However, this proposition has not been systematically investigated. Furthermore, recent work from the Li lab has revealed that a broad swath of bacteria – notably B. subtilis and other Firmicutes (Bacillota) – altogether lack nonsense-mediated quality control and transcription-translation coupling. In other recent work, I developed an approach for massively parallel measurements of bacterial growth inhibition by complex libraries of protein fragments, derived from array-synthesized oligonucleotides. This methodological innovation provides the previously missing tool needed to systematically investigate cytotoxicity of incomplete proteins. In this proposal I bring together these conceptual and technological breakthroughs, along with computational analyses and other complementary methods, to address fundamental questions about the physiological impacts of incomplete proteins and the importance of their suppression. I hypothesize that most incomplete proteoforms are innocuous, but a subset are cytotoxic, and that B. subtilis protein sequences have evolved to limit truncated variant toxicity. I will also investigate the contributions of incomplete proteins to bactericidal activity of ribosome-inhibitory antibiotics, and the evolutionary pathways enabled by a lack of suppression of premature stop codon mutants. Objective: To address these questions, I will systematically define the cytotoxicity of incomplete proteins by measuring growth rate effects of incomplete proteins from across the B. subtilis and E. coli proteomes, determining protein features associated with cytotoxicity, and further investigating physiological effects of select proteoforms using ribosome profiling, RNA sequencing, and pull-down mass spectrometry. I will also determine how incomplete proteins contribute to bactericidal activity of ribosome-inhibiting antibiotics by combining translatomic and proteomic measurements of drug-induced incomplete protein production with massively parallel cytotoxicity measurements. In a third approach, I will redefine the functions and evolutionary roles of pseudogenes in Firmicutes by using a knockout library to demonstrate premature-stop pseudogene function and employing bioinformatic analyses to determine the prevalence of duplication and truncation as a path to novel genes. Finally, I will use a high-throughput assay to systematically map the landscape of potentially beneficial truncation variants.
