Certain transcription factors are proposed to form functional interactions with RNA to facilitate proper regulation of gene expression. Sox2, a transcription factor critical for maintenance of pluripotency and neurogenesis, has been found associated with several lncRNAs, although it is unknown whether these interactions are direct or via other proteins. Here we demonstrate that human Sox2 interacts directly with one of these lncRNAs with high affinity through its HMG DNA-binding domain in vitro. These interactions are primarily with double-stranded RNA in a non-sequence specific fashion, mediated by a similar but not identical interaction surface. We further determined that Sox2 directly binds RNA in mouse embryonic stem cells by UV-cross-linked immunoprecipitation of Sox2 and more than a thousand Sox2-RNA interactions in vivo were identified using fRIP-seq. Together, these data reveal that Sox2 employs a high-affinity/low-specificity paradigm for RNA binding in vitro and in vivo.

The glucocorticoid receptor (GR) binds the noncoding RNA Gas5 via its DNA-binding domain (DBD) with functional implications in pro-apoptosis signaling. Here, we report a comprehensive in vitro binding study where we have determined that GR-DBD is a robust structure-specific RNA-binding domain. GR-DBD binds to a diverse range of RNA hairpin motifs, both synthetic and biologically derived, with apparent mid-nanomolar affinity while discriminating against uniform dsRNA. As opposed to dimeric recognition of dsDNA, GR-DBD binds to RNA as a monomer and confers high affinity primarily through electrostatic contacts. GR-DBD adopts a discrete RNA-bound state, as assessed by NMR, distinct from both free and DNA-bound. NMR and alanine mutagenesis suggest a heightened involvement of the C-terminal α-helix of the GR-DBD in RNA-binding. RNA competes for binding with dsDNA and occurs in a similar affinity range as dimer binding to the canonical DNA element. Given the prevalence of RNA hairpins within the transcriptome, our findings strongly suggest that many RNAs have potential to impact GR biology.

SignificanceProteins that bind nucleic acids are frequently categorized as being either specific or nonspecific, with interfaces to match that activity. In this study, we have found that a telomere-binding protein exhibits a degree of specificity for ssDNA that is finely tuned for its function, which includes specificity for G-rich sequences with some tolerance for substitution. Mutations of the protein that dramatically impact its affinity for single-stranded telomeric DNA are lethal, as expected; however, mutations that alter specificity also impact biological function. Unexpectedly, we found mutations that make the protein more specific are also deleterious, suggesting that specificity and nonspecificity in nucleic acid recognition may be achieved through more nuanced mechanisms than currently recognized.