Molecular mechanism of the RNA helicase DHX37 and its activation by UTP14A in ribosome biogenesis

UTP14A stimulates ATPase activity of DHX37 and enhances RNA binding. (A, top) Schematic diagram of human UTP14A and constructs used for pull-down experiment. SSU-N and SSU-C denote the amino- and carboxy-terminal regions corresponding to the regions of Utp14 bound in the yeast SSU processome structure. Red boundary denotes region corresponding to the Dhr1 interacting region in yeast Utp14. Solid red box denotes minimal region in HsUTP14A required for interaction with DHX37. (Bottom) SDS-PAGE analysis of streptactin pull-down experiment using StrepII-tagged MmDHX37^139–1150 as bait and indicated mCherry-tagged HsUTP14A constructs as prey. UTP14A constructs are visualized by mCherry fluorescence. (B, top) Schematic diagram of human UTP14A and constructs used for pull-down experiment. (Bottom) SDS-PAGE analysis of streptactin pull-down experiment using StrepII-tagged MmDHX37^139–1150 as bait and indicated mCherry-tagged HsUTP14A constructs as prey. MIEF denotes the M594A/I595A/E597A/F599A quadruple point mutant; MIDL denotes the M558A/I559A/D560A/L561A mutant. (C) RNA-dependent ATPase activity of MmDHX37^179–1150 or the MmDHX37^179–1150–HsUTP14A^min complex as a function of ATP concentration. The data were subjected to Michaelis–Menten analysis and the calculated k_cat and K_M values are provided in Table 1. Experiments were performed in triplicates, with data points indicating the mean and error bars indicating the standard error of the mean. (D) RNA-dependent ATPase activity of MmDHX37^179–1150 or the MmDHX37^179–1150–HsUTP14A^min complex as a function of U₁₀ RNA concentration. Experiments were performed in triplicates, with data points indicating the mean and error bars indicating the standard error of the mean. EC₅₀ values are provided in Table 1. (E) Fluorescence-polarization equilibrium binding assays of MmDHX37^179–1150 or the MmDHX37^179–1150–HsUTP14A^min complex with ATTO532 fluorophore-labeled U₁₂ RNA in the absence of adenosine nucleotides (left) and in the presence of ADP (center) and the nonhydrolyzable ATP analog AMPPNP (right). K_d values are provided in Table 1.