Abstract
Several members of the SMAD family of transcription factors have been reported to bind RNA in addition to their canonical double-stranded DNA (dsDNA) ligand. RNA binding by SMAD has the potential to affect numerous cellular functions that involve RNA. However, the affinity and specificity of this RNA binding activity has not been well characterized, which limits the ability to validate and extrapolate functional implications of this activity. Here we perform quantitative binding experiments in vitro to determine the ligand requirements for RNA binding by SMAD3. We find that SMAD3 binds poorly to single- and double-stranded RNA, regardless of sequence. However, SMAD3 binds RNA with large internal loops or bulges with high apparent affinity. This apparent affinity matches that for its canonical dsDNA ligand, suggesting a biological role for RNA binding by SMAD3.