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.
Fig 2: Proposed mechanistic model for tsRNA cellular and molecular functionsThe dynamic expression of 5'-tsRNAs plays a crucial role in modulating stem cell differentiation. During differentiation, 5'-tsRNAs regulate the translation and/or the stability of several transcripts through its interaction with ribosomes, RNA-binding proteins (ribonucleoproteins), such as IGF2BP1, or through direct sequence complementarity.
Fig 3: Functional characterization of tsGlnCTG in the regulation of IGF2BP1 and c-Myc AMerged triplicate Western immunoblots of Igf2bp1 and ß-actin in mESCs grown under LIF or RA conditions (top) and densitometric quantitation of the same (below) (n = 3, error bars represent SD, and significance was calculated using Student's t-test).BValidation of siRNA-mediated knockdowns of Igf2bp1 in RA-treated mESCs (n = 3, error bars represent SD, and significance was calculated using Student's t-test).B'Expression of pluripotency genes in Igf2bp1 knockdown cells (n = 3, error bars represent SD, and significance was calculated using Student's t-test).CSmall RNAs identified (18–35 nt) from input and Igf2bp1 pulldown in RA-treated mESCs.DqPCR validation of the tsGlnCTG enrichment upon Igf2bp1 pulldown in RA-treated mESCs.EBinding curve showing the interaction of tsGlnCTG and Igf2bp1 (Kd = 33 nM). The intensities of the Igf2bp1 band were normalized to band intensity of the total Igf2bp1 protein used for pulldown.FBiological duplicate for c-Myc quantitation from Igf2bp1 pulldown between LIF vs. RA conditions (n = 2, for duplicate check Fig 5B).Data information: *P value < 0.05, **P value < 0.01; ***P value < 0.001; n.s, non-significant.
Fig 4: 5'-tsRNA-mediated regulation of c-Myc through Igf2bp1 interaction. In vitro binding analysis of tsGlnCTG to Igf2bp1 in the presence and absence of antisense oligo (ASO) against tsGlnCTG. ASOs effectively disrupt the binding of tsGlnCTG to Igf2bp1. (n = 2) error bars represent SEM.Quantification of the Igf2bp1-bound c-Myc mRNA between LIF- vs. RA-treated mESCs (n = 2; see Fig EV5F for duplicate data).Association of c-Myc transcript in different translating pools in RA-treated mESCs as compared to LIF condition. (n = 2), error bars represent SD, and significance was calculated by one-tailed unpaired t-test.Relative enrichment of c-Myc mRNA in translating (80S and polysome) and non-translating (mRNPs) pools (fractionated from polysome profiling) between ASO-treated and mock-treated RA-induced differentiating mESCs. (n = 2), error bars represent SD, and significance was calculated using one-tailed unpaired t-test.Relative levels of c-Myc mRNA in ASO-treated and mock-treated RA-induced differentiating mESCs compared with LIF-treated mESCs. (n = 3), error bars represent SD, and significance was calculated using one-tailed unpaired t-test.Epistatic analysis of 5'-tsRNAs and IGF2BP1 in regulating Myc transcriptional reporter activity (n = 3), error bars represent SD, and significance was calculated using unpaired t-test.Schematic representing tsRNA based c-Myc transcript regulation.Data information: *P value < 0.05, **P value < 0.01; ***P value < 0.001.
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