Nsr1, a nitrogen source-regulated microprotein, confers an alternative mechanism of G1/S transcriptional activation in budding yeast

Commitment to cell division at the end of G1 phase, termed Start in the budding yeast Saccharomyces cerevisiae, is strongly influenced by nutrient availability. To identify new dominant activators of Start that might operate under different nutrient conditions, we screened a genome-wide ORF overexpression library for genes that bypass a Start arrest caused by absence of the G1 cyclin Cln3 and the transcriptional activator Bck2. We recovered a hypothetical gene YLR053c, renamed NRS1 for Nitrogen-Responsive Start regulator 1, which encodes a poorly characterized 108 amino acid microprotein. Endogenous Nrs1 was nuclear-localized, restricted to poor nitrogen conditions, induced upon mTORCl inhibition, and cell cycle-regulated with a peak at Start. NRS1 interacted genetically with SWI4 and SWI6, which encode subunits of the main G1/S transcription factor complex SBF. Correspondingly, Nrs1 physically interacted with Swi4 and Swi6 and was localized to G1/S promoter DNA. Nrs1 exhibited inherent transactivation activity and fusion of Nrs1 to the SBF inhibitor Whi5 was sufficient to suppress other Start defects. Nrs1 appears to be a recently evolved microprotein that rewires the G1/S transcriptional machinery under poor nitrogeny conditions. Author SummarUnicellular microorganisms must adapt to ever-changing nutrient conditions and hence must adjust cell growth and proliferation to maximize fitness. In the budding yeast Saccharomyces cerevisiae, commitment to cell division, termed Start, is heavily influenced by nutrient availability. Our understanding of how Start is activated is based mainly on experiments carried out under rich nutrient conditions. To identify potential new Start regulators specific to poor nutrient environments, we screened for genes able to bypass a genetic Start arrest caused by loss of the G1 cyclin Cln3 and the transcriptional activator Bck2. This screen uncovered YLR053c, which we renamed NRS1 for Nitrogen-Responsive Start regulator. Sequence analysis across yeast species indicated that Nrs1 is a recently-evolved microprotein. We showed that NRS1 is nutrient- and cell cycle-regulated, and directly binds the main G1/S transcription factor complex SBF. We demonstrated that Nrs1 has an intrinsic trans-activation activity and provided genetic evidence to suggest that Nrs1 can bypass the requirement for normal Cln3-dependent activation of G1/S transcription. These results uncover a new mechanism of Start activation and illustrate how microproteins can rapidly emerge to rewire fundamental cellular processes.