Fig. 4

The CTD RRM domain is critical for SDN1 binding and enzymatic activities on ssRNA. a EMSA assays of wild-type SDN1 with 22 nt, 16 nt, 10 nt or 6 nt ssRNA substrates. Assays were conducted at a series of protein concentrations: 0.5 µM, 0.75 µM, 1 µM, 2.5 µM, 5 µM, 7.5 µM, 10 µM, 25 µM, and 50 µM (from left to right). No Mg²⁺ was included to prevent catalytic activity. b Plots showing the RNA fractions bound at varying protein concentrations. The fractions bound were calculated from the densities of the free RNA bands in a. Scale bars represent standard deviations calculated from three biological replicates. c Diagram of 5-iodoU-substituted ssRNAs used in SDN1 UV crosslinking assays. 5-iodoUs are shown in red in the sequences. d UV crosslinking assays of wild-type SDN1 or SDN1 ΔC with ssRNAs shown in c. The red and yellow asterisks indicate full-length SDN1 crosslinked to RNA and SDN1 ΔC crosslinked to RNA, respectively. e Diagrams of how SDN1 or SDN1 ΔC was crosslinked to different 5-iodoU ssRNAs. f Enzymatic assays of wild-type SDN1 or SDN1^2FA with or without cold RNA competitor. The labeled RNAs were first incubated with the enzymes to allow for binding but not catalysis, then Mg²⁺ or a mixture of Mg²⁺ and cold RNAs was added to initiate the reactions. The time indicated above each lane represents the elapsed time from the time Mg²⁺ was added