Figure 4.

Effects of the NTR and the Prp8 Jab1 domain on RNA binding and unwinding by Brr2. (A,B) Assays monitoring RNA binding by hBrr2^FL (A) or hBrr2^T3 (B). Black curves – hBrr2^FL or hBrr2^T3 alone; gray curves – hBrr2^FL or hBrr2^T3 in complex with Jab1; red curves – hBrr2^FL or hBrr2^T3 in complex with Jab^ΔC. Left panels – binding to ssRNA; central panels – binding to dsRNA; right panels – binding to yU4/U6 snRNA. Binding of ssRNA and dsRNA was monitored by FP using 5-FAM-labeled RNAs. Binding of yU4/U6 was monitored via EMSA. Data points and error bars represent means +/− SEMs of at least 2 independent experiments. (C) RNA affinities. Values represent means +/− SEMs of at least 2 independent experiments. K_d values were obtained by fitting the data from (A) and (B) to a single exponential Hill function (fraction bound = A[protein]ⁿ/([protein]ⁿ+K_dⁿ); A – fitted maximum of RNA bound; n – Hill coefficient).⁵⁰ (D) Scheme summarizing the effects of the NTR and the Jab1 domain on different portions of a substrate. Red symbols – inhibition; green double-arrow – reinforcement. (E) U4/U6 unwinding by Brr2 variants in the absence or presence of Jab1 variants. Black curves – Brr2^FL or Brr2^T3 alone; gray curves – Brr2^FL or Brr2^T3 in complex with Jab1; red curves – Brr2^FL or Brr2^T3 in complex with Jab^ΔC. (F) Unwinding parameters. Amplitudes and unwinding rates were obtained by fitting the data to a first-order reaction (fraction unwound = A[1-exp{-k_u t}]; A – amplitude of the reaction; k_u – apparent first-order rate constant of unwinding; t – time). Values represent means +/− SEMs of at least 4 independent experiments.