snRIC2C identified 311 short RNA binding proteins in yeasts. (A) Schematic representation of snRIC2C workflow. (B) Volcano plot displaying log2 fold change of protein abundance versus −log10 P-value for total (left panel) and short (right panel) 2C-RNA, respectively. Orange dots represent statistically significantly enriched proteins in CL versus NoCL samples. Glycolytic enzymes statistically enriched in the CL RNA fractions are highlighted. (C) Comparison of RBPs detected in the short RNA fraction versus the 2C total RNA fraction or any other yeast RIC experiment. (D) Top 10 significantly enriched GO molecular functions terms of short RNA binding proteins identified by snRIC2C compared to RIC2C. (E) Venn diagram classifying the RBPs identified in the snRIC2C experiment. Proteins statistically enriched (logFC higher than 1 and P-value lower than 0.05) in the total RNA fraction and not enriched in the short RNA one, were considered long RNA binders. Proteins statistically enriched in the short RNA fraction were considered short RNA binders. Proteins statistically enriched in the total RNA fraction and candidate hits in the short RNA fraction (logFC between 0.5 and 1 and P-value lower than 0.05) were considered wide range RNA binders (light blue circle). (F) Validation of the classification of RBPs based on the length of RNA target molecules by 2C-western blot. TAP-tagged and an untagged WT strain were UV cross-linked, and 1 mg of lysate was subjected to a 2C extraction for total RNA. TAP-tagged strains were probed with a PAP antibody for Protein A detection. The untagged WT strain was probed with a GAPDH antibody. Pub1-TAP was tested as a wide range RBP. Ssd1-TAP, Pfk2-TAP, Cbf5-TAP, and Gar1-TAP were tested as long RNA binders. GAPDH, in the untagged WT strain, Cdc60-TAP, Grs1-TAP, and Gus1-TAP were tested as short RNA binders.
