In the presence of physiological monovalent cations, thousands of RNA G-rich sequences can form parallel G-quadruplexes (G4s) unless RNA-binding proteins inhibit, destabilize, or resolve the formation of such secondary RNA structures. Here, we have used a disease-relevant model system to investigate the biophysical properties of the RNA-binding protein HNRNPH1’s interaction with G-rich sequences. We demonstrate the importance of two EWSR1-exon 8 G-rich regions in mediating the exclusion of this exon from the oncogenic EWS-FLI1 transcripts expressed in a subset of Ewing sarcomas, using complementary analysis of tumor data, long-read sequencing, and minigene studies. We determined that HNRNPH1 binds the EWSR1-exon 8 G-rich sequences with low nM affinities irrespective of whether in a non-G4 or G4 state but exhibits different kinetics depending on RNA structure. Specifically, HNRNPH1 associates and dissociates from G4-folded RNA faster than the identical sequences in a non-G4 state. Importantly, we demonstrate using gel shift and spectroscopic assays that HNRNPH1, particularly the qRRM1-qRRM2 domains, destabilizes the G4s formed by the EWSR1-exon 8 G-rich sequences in a non-catalytic fashion. Our results indicate that HNRNPH1’s binding of G-rich sequences favors the accumulation of RNA in a non-G4 state and that this contributes to its regulation of RNA processing.

The year 2021 marks the 20th anniversary of the first publications reporting the discovery of the gene silencing mechanism, RNA interference (RNAi) in mammalian cells. Along with the many studies that delineated the proteins and substrates that form the RNAi pathway, this finding changed our understanding of the posttranscriptional regulation of mammalian gene expression. Furthermore, the development of methods that exploited the RNAi pathway began the technological revolution that eventually enabled the interrogation of mammalian gene function-from a single gene to the whole genome-in only a few days. The needs of the cancer research community have driven much of this progress. In this perspective, we highlight milestones in the development and application of RNAi-based methods to study carcinogenesis. We discuss how RNAi-based functional genetic analysis of exemplar tumor suppressors and oncogenes furthered our understanding of cancer initiation and progression and explore how such studies formed the basis of genome-wide scale efforts to identify cancer or cancer-type specific vulnerabilities, including studies conducted in vivo. Furthermore, we examine how RNAi technologies have revealed new cancer-relevant molecular targets and the implications for cancer of the first RNAi-based drugs. Finally, we discuss the future of functional genetic analysis, highlighting the increasing availability of complementary approaches to analyze cancer gene function.