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. 2017 Nov 16;45(20):11980-11988.
doi: 10.1093/nar/gkx846.

The SMAD3 Transcription Factor Binds Complex RNA Structures With High Affinity

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Free PMC article

The SMAD3 Transcription Factor Binds Complex RNA Structures With High Affinity

Thayne H Dickey et al. Nucleic Acids Res. .
Free PMC article

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.

Figures

Figure 1.
Figure 1.
SMAD3 does not specifically bind to SBE-containing pri-miRNAs. (A) pri-miRNAs in blue font were selected based on their reported ability to bind SMAD via an SBE motif (10). Pri-miRNAs in red font lack an SBE and are included as negative controls. (B) Constructs derived from the pri-miRNAs in panel A were designed to test the effect of the SBE on SMAD binding. Closing basepairs and UUCG tetraloops (cyan) were added to the predicted pri-miRNA secondary structures to stabilize the RNA secondary structure in vitro. The local architecture surrounding the SBE (green) was unaltered in the stabilized designs. Negative controls (e.g. 1b) were created by mutating the SBE sequence. To confirm that our design features did not impact binding, we also tested longer pri-miRNA hairpins without artificial basepairs (right panel). Again, constructs were tested with (3c) and without (9c) an SBE. (C) Binding affinities for each pri-miRNA construct were estimated by incubating SMAD3 MH1 dilutions with each RNA and separating free and bound RNA by EMSA. Representative EMSAs are shown, illustrating that the presence of the SBE does not affect SMAD3 binding. (D) Summary of the approximate dissociation constants measured for all pri-miRNA constructs tested. Constructs with putative SBEs are represented by blue circles and those without SBEs are represented by red squares.
Figure 2.
Figure 2.
Complex structural features promote SMAD3 binding. (A) RNA secondary structures and representative EMSAs designed to test the effect of RNA structure on SMAD3 binding. SMAD3 binds weakly to predominantly single- and double-stranded RNA (12, 16 and 9c). However, complex RNA structures (17 and 18) are bound with high affinity, comparable to a canonical SMAD3 dsDNA ligand (19) containing the SBE sequence (green). Construct 17 misfolds under some conditions and this conformer is marked by an asterisk. (B) Data points and global curve fits for SMAD3 RNA and dsDNA binding.
Figure 3.
Figure 3.
High affinity RNA binding competes with dsDNA binding. (A) Representative EMSA competition experiment in which labeled dsDNA (19) was preincubated with SMAD3 and unlabeled RNA or DNA was titrated in. (B) IC50s were calculated for several RNA and DNA oligonucleotides, demonstrating that high-affinity RNAs compete with DNA binding more efficiently than low-affinity RNAs.
Figure 4.
Figure 4.
SMAD3 recognizes large internal loops via a mechanism more complex than B-form mimicry. (A) Predicted secondary structures and binding affinities of constructs used to determine the minimal structural element recognized by SMAD3. Truncation analysis of 17 (left panel) reveals decreases in affinity upon disruption of each large asymetric internal loop. Either internal loop of 17 is sufficient to confer nanomolar binding in isolation, but not the full affinity conferred by the complete construct 17 (middle panel). The wide-major grooves created by the large internal loops of the NF-κB aptamer (20) and RRE (21) are insufficient to confer high-affinity SMAD3 binding (right panel). (B) Representative EMSAs of SMAD3 binding to constructs containing the isolated internal loops of 17.

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