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. 2019 Nov;25(11):1509–1521. doi: 10.1261/rna.072256.119

FIGURE 1.

FIGURE 1.

The carboxy-terminal UBL domain of SF3A1 binds to U1-SL4 RNA. (A) Schematic diagram of human SF3A1 protein depicting the known domain organization and the regions that interact with SF3A2 and SF3A3. (B) Schematic of all amino- and carboxy-terminal deletion constructs. (C) Western blot of 6×His-tagged protein expressed in cell-free expression (CFE) reactions containing the control empty vector (EV), or expression vectors with full-length (FL) protein, carboxy-terminal truncations (ΔC546, ΔC475, ΔC317), and amino-terminal truncations (ΔN547, ΔN676, ΔN703). Proteins were detected using anti-6×His primary antibody. (D) 32P-U1-SL4 was added to CFE extracts containing SF3A1 proteins, UV-crosslinked, and then separated on SDS-PAGE gels and visualized by phosphor imaging. (E) Schematic of wild-type (WT) and mutant (M10h) U1-SL4 RNAs. (F) Crosslinking of 32P-U1-SL4-WT and -M10h RNAs in CFE extracts expressing EV or FL SF3A1. (G) Crosslinking of WT or M10h 32P-U1-SL4 in CFE extracts expressing EV or ΔN SF3A1 constructs. Arrows indicate specific crosslinked products while asterisks indicate nonspecific products that were observed in all reactions including the EV control.