HNRNPH1-dependent splicing of a fusion oncogene reveals a targetable RNA G-quadruplex interaction

Abstract

The primary oncogenic event in ∼85% of Ewing sarcomas is a chromosomal translocation that generates a fusion oncogene encoding an aberrant transcription factor. The exact genomic breakpoints within the translocated genes, EWSR1 and FLI1, vary; however, in EWSR1, breakpoints typically occur within introns 7 or 8. We previously found that in Ewing sarcoma cells harboring EWSR1 intron 8 breakpoints, the RNA-binding protein HNRNPH1 facilitates a splicing event that excludes EWSR1 exon 8 from the EWS-FLI1 pre-mRNA to generate an in-frame mRNA. Here, we show that the processing of distinct EWS-FLI1 pre-mRNAs by HNRNPH1, but not other homologous family members, resembles alternative splicing of transcript variants of EWSR1. We demonstrate that HNRNPH1 recruitment is driven by guanine-rich sequences within EWSR1 exon 8 that have the potential to fold into RNA G-quadruplex structures. Critically, we demonstrate that an RNA mimetic of one of these G-quadruplexes modulates HNRNPH1 binding and induces a decrease in the growth of an EWSR1 exon 8 fusion-positive Ewing sarcoma cell line. Finally, we show that EWSR1 exon 8 fusion-positive cell lines are more sensitive to treatment with the pan-quadruplex binding molecule, pyridostatin (PDS), than EWSR1 exon 8 fusion-negative lines. Also, the treatment of EWSR1 exon 8 fusion-positive cells with PDS decreases EWS-FLI1 transcriptional activity, reversing the transcriptional deregulation driven by EWS-FLI1. Our findings illustrate that modulation of the alternative splicing of EWS-FLI1 pre-mRNA is a novel strategy for future therapeutics against the EWSR1 exon 8 containing fusion oncogenes present in a third of Ewing sarcoma.

Anatomy of noncovalent interactions between the nucleobases or ribose and π-containing amino acids in RNA–protein complexes

“…Computer modeling has hinted that G:H π–π interactions are vital for aligning and binding RNA transcripts to the b-subunit of RNA polymerases ( 29 ), while CD72 uses several Y residues to form π–π interactions with RNA nucleobases in order to act as a regulatory protein for B lymphocytes ( 30 ). In the absence of structural data, π–π stacking between the RNA-binding protein heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) and an RNA G-quadraplex has been proposed to be related to alterative splicing and the onset of cancer ( 31 ). Although the RNA and protein components are anticipated to be relatively co-planar in the previous examples, nonparallel nucleobase–amino acid binding modes also occur.…”

Cancer biology functional genomics: From small RNAs to big dreams

“…One aspect of this and subsequent studies highlighted the dependency of about one‐third of Ewing sarcomas that harbor chromosome 22 translocations in intron 8 of the 5ʹ partner gene, EWSR1 . Specifically, our work demonstrated that EWS cells harboring EWSR1 ‐intron 8 breakpoints depend on the RNA binding protein HNRNPH1 to generate the in‐frame protein‐coding fusion mRNA in these tumor cells 107,108 …”

An Updated Focus on Quadruplex Structures as Potential Therapeutic Targets in Cancer

“…The last G4 structure included in this group is found in the EWS RNA binding protein 1, EWSR1 , one of the genes most commonly involved in sarcoma translocations, rendering novel transcription factors with tumoral effects [ 145 ]. EWSR1 mRNA contains a parallel-tetramolecular G4 structure within exon 8, which enables the recruitment of HNRNPH1, a component of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex required for the processing of distinct EWSR1 transcript variants [ 57 ].…”

“…For instance, the well-known cationic porphyrin TMPyP4 acts as a general stabilizer of quadruplexes including DNA [ 167 ] and RNA G4s [ 168 ], but also i-Motifs [ 169 ]. The same occurs with the pan-quadruplex binding molecule, Pyridostatin, which binds to all quadruplex structures [ 57 , 170 , 171 ]. In the case of BRACO-19, it shows binding affinity for both DNA G4s [ 172 ] and i-Motifs [ 170 ].…”