Fig 1: Characterization of Dhx30 as a bona-fide mRNA-independent RAP(A) Analysis of GO terms in forebrain MS data. RAPIDASH was performed on E12.5 mouse forebrain samples. These samples were analyzed by LC-MS/MS. The resulting proteins were analyzed by Manteia30 for GOMF terms level 4 or higher. The top 5 GOMF terms are shown.(B) Dhx30 co-fractionates with ribosomes, specifically the 40S fractions. Sucrose gradient fractionation was performed on E14 mESCs. The proteins from each fraction were precipitated and analyzed by western blotting. Dhx30 does not co-fractionate with the free fraction; instead, it largely co-fractionates with the 40S and 80S fractions. Rps26 and Rpl29 are shown as controls for small and large subunits, respectively.(C) E14 mESCs were treated with EDTA and subjected to sucrose gradient fractionation followed by western blotting as in (B).(D) Dhx30 still associates with the pellet fraction after RNase treatment. Top: illustration of sucrose cushion and RNase A treatment of E14 mESC cytoplasmic lysate. Bottom: western blot of control and RNase A-treated E14 mESC subjected to sucrose cushion ultracentrifugation. Pabp1 is a positive control for mRNA-dependent association with ribosomes. Rps19 is a positive control for ribosomes. Sup., supernatant.(E) Knockdown of Dhx30 by siRNA in mESC does not affect global protein synthesis. Protein synthesis was measured by O-propargyl-puromycin (OP-Puro) incorporation into the nascent proteome. Incorporated OP-Puro was fluorescently labeled using a click chemistry reaction. Translation activity was then measured using flow cytometry. Top: there is no change in OP-Puro median fluorescence intensity (MFI) between siFluc and siDhx30 (n=4). Bottom: western blotting of Dhx30 shows that siRNA knockdown is successful.(F) Illustration of Dhx30 constructs for transient transfection in E14 mESCs. Oligosaccharide binding (OB) fold domain; and double-stranded RNA-binding domains (dsRBDs).(G) Top: western blots of E14 mESCs transfected with V5-Dhx30, ΔOB-fold, and R805/8A constructs and subjected to sucrose cushion ultracentrifugation. Bottom: similar samples, but instead transfected with V5-Dhx30, ΔdsRBD-1, and ΔdsRBD-1/2 constructs. Loss of either OB-fold, dsRBD-1, or dsRBD-2 results in the loss of Dhx30 association with the ribosome. However, Dhx30 loss of function due to loss of helicase activity in R805/8A mutant does not affect its association with the ribosome.(H) Quantification of Dhx30 western blots shows a significant shift from pellet to supernatant in ΔOB-fold transiently transfected E14 mESCs but not R805/8A transfected E14 mESCs.