Fig 1: Characterization of mechanism and molecular function of candidate tsRNAs in pluripotent vs. differentiating mESCs AGO annotation of 109 common tsRNA-interacting proteins.BEffects of candidate 5'-tsRNAs: tsGlnCTG, tsGlyGCC, tsMetCAT, and tsIleGAT on uncapped luciferase mRNA in an in vitro translation system (n = 2–3, error bars represent SEM, and significance was calculated using unpaired t-test).B'Effects of tsGlnCTG on capped GFP mRNA in an in vitro translation system (n = 2–3, error bars represent SD, and significance was calculated using unpaired t-test).CPolysome profiles of different stem vs. differentiating states tested in this study. Gray areas indicate the fractions collected for sequencing.C'tsRNA distribution among the polysome fractions from indicated cell states.DWorkflow to identify proteomic and transcriptomic interactions of tsGlnCTG in pluripotency vs. differentiating mESCs.EFalse discovery rate (FDR) of peptides detected in LC-MS/MS of biotinylated tsGlnCTG pulldown.FWestern blot showing differential interaction of Igf2bp1, Ybx1, and Rpl10 with tsGlnCTG between LIF and RA conditions.GOverlap between transcripts that are associated with tsRNAs in RA condition and the transcripts that are affected upon ASO transfection in RA conditions.HSchematic of the experimental setup to identify protein-mediated and complementarity-based tsRNA target interaction.IqPCR analysis showing the enrichment of transcripts pulled down by tsGlnCTG in lysates devoid of proteins compared with normal lysate (n = 2, error bars represent SEM, and significance was calculated using unpaired t-test).JTop 10 tsGlnCTG-associated proteins in indicated cell states.Data information: *P value < 0.05, **P value < 0.01; ***P value < 0.001; n.s, non-significant.