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

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.

Ewing Sarcoma Related protein 1 recognizes R-loops by binding DNA forks

“…Our study finds similar high affinity binding of EWSR1 to DNA and R-loops as in previous reports 16, 17 . These are also comparable to the highest affinities observed among FET protein and a nucleic acid target 12, 25 .…”

“…Protein-DNA interaction in hnRNP A1 and HNRPDL are both important for transcription regulation 33, 35 . Interaction between RGG and DNA G-quadraplexes has been observed in EWSR1 17, 36, 37 . This raises the possibility that when G-quadruplex structures are present in the single stranded DNA of the R-loop, EWSR1 recruitment can be improved 1, 18 .…”

“…However, unscheduled resolution of R-loops can result in head on collision with replication fork and transcription stalling which eventually leads to overall genomic stability found in many diseases 2, 4, 5 . In Ewing sarcoma, wildtype EWSR1 is essentially inactive, and the number of R-loops is up to 4x higher compared to normal healthy cells 3, 16, 17 . Our data provides a mechanism to involve EWSR1 in R-loop resolution through a general approach to recognize R-loops.…”

“…Recent studies have found EWSR1 can bind R-loops and has a role in their resolution, though much of the mechanism remains unknown 3, 15–17 . In Ewing Sarcoma, the oncogenic fusion protein EWS-FLI1 has a dominant negative effect on the wildtype EWSR1 protein activity 10 .…”

Intrinsic Disorder and Salt-dependent Conformational Changes of the N-terminal Region of TFIP11 Splicing Factor

“…9 LCDs are composed of a limited variety of amino acids and have distinct physico-chemical properties depending on the type of amino acid(s) each LCD is enriched with. 17 LCDs are mainly found in RNA- and DNA-binding proteins, such as the EWS 18 and TAR 19 proteins, where they enforce gene regulation and functioning through the formation of dynamic complexes in MLOs such as CBs. 20 Indeed, the LCDs can drive the formation of extended biomolecular condensates and MLOs by promoting LLPS.…”