Biochemical and biophysical characterization of the nucleic acid binding properties of the RNA/DNA binding protein EWS

Biopolymers. 2023 May;114(5):e23536. doi: 10.1002/bip.23536. Epub 2023 Mar 17.

Abstract

EWS is a member of the FET family of RNA/DNA binding proteins that regulate crucial phases of nucleic acid metabolism. EWS comprises an N-terminal low-complexity domain (LCD) and a C-terminal RNA-binding domain (RBD). The RBD is further divided into three RG-rich regions, which flank an RNA-recognition motif (RRM) and a zinc finger (ZnF) domain. Recently, EWS was shown to regulate R-loops in Ewing sarcoma, a pediatric bone and soft-tissue cancer in which a chromosomal translocation fuses the N-terminal LCD of EWS to the C-terminal DNA binding domain of the transcription factor FLI1. Though EWS was shown to directly bind R-loops, the binding mechanism was not elucidated. In the current study, the RBD of EWS was divided into several constructs, which were subsequently assayed for binding to various nucleic acid structures expected to form at R-loops, including RNA stem-loops, DNA G-quadruplexes, and RNA:DNA hybrids. EWS interacted with all three nucleic acid structures with varying affinities and multiple domains contributed to binding each substrate. The RRM and RG2 region appear to bind nucleic acids promiscuously while the ZnF displayed more selectivity for single-stranded structures. With these results, the structural underpinnings of EWS recognition and binding of R-loops and other nucleic acid structures is better understood.

Keywords: EWS; G-quadruplex; NMR; R-loop; intrinsically disordered protein.

MeSH terms

  • Cell Line, Tumor
  • Child
  • DNA
  • DNA-Binding Proteins
  • Humans
  • Nucleic Acids*
  • RNA
  • RNA-Binding Protein EWS / chemistry
  • RNA-Binding Protein EWS / genetics
  • RNA-Binding Protein EWS / metabolism
  • RNA-Binding Proteins*

Substances

  • RNA-Binding Protein EWS
  • RNA-Binding Proteins
  • DNA-Binding Proteins
  • DNA
  • RNA
  • Nucleic Acids