HNRNPH1 destabilizes the G-quadruplex structures formed by G-rich RNA sequences that regulate the alternative splicing of an oncogenic fusion transcript

Abstract

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.

HNRNPH1 regulates the neuroprotective cold-shock protein RBM3 expression through poison exon exclusion

“…It is worth noting that G-rich sequences have a tendency to form RNA G-quadruplex (rG4) secondary structures (Kharel et al, 2020), which are highly enriched at weak splice sites and promote cassette exon inclusion (Georgakopoulos-Soares et al, 2022; Huang et al, 2017). Intriguingly, HNRNPH1 has been recently reported as an interactor and destabilizer of rG4s (Vo et al, 2022). Therefore, destabilising rG4s by HNRNPH1 is likely to induce exon skipping, as reported for HNRNPH1-mediated exon exclusion in RNA-binding protein EWS (EWSR1) transcripts (Vo et al, 2022).…”

“…Intriguingly, HNRNPH1 has been recently reported as an interactor and destabilizer of rG4s (Vo et al, 2022). Therefore, destabilising rG4s by HNRNPH1 is likely to induce exon skipping, as reported for HNRNPH1-mediated exon exclusion in RNA-binding protein EWS (EWSR1) transcripts (Vo et al, 2022). Consistent with this hypothesis, sequence-based rG4 prediction suggests the presence of rG4s in the region between RBM3 Exon 3 and 4 that overlaps with the primary HNRNPH1 binding site (Fig.…”

“…Intriguingly, HNRNPH1 has been recently reported as an interactor and destabilizer of rG4s (Vo et al, 2022). Therefore, destabilising rG4s by HNRNPH1 is likely to induce exon skipping, as reported for HNRNPH1-mediated exon exclusion in RNA-binding protein EWS (EWSR1) transcripts (Vo et al, 2022).…”

Photosensitized Generation of Singlet Oxygen (1O2) from Pyropheophorbide a Specifically Bound to All Parallel-Stranded G-Quadruplex DNAs

“…are known to form G-quadruplexes . Structural stability and topology of a G-quadruplex have been shown to be largely affected by the presence of a 2′-OH groups in the ribose. − Intramolecular interactions associated with the 2′-OH groups contribute to enthalpic stabilization of G-quadruplex RNAs, and hence, G-quadruplex RNAs are generally more stable than their DNA counterparts. ,, In addition, the anti -conformation of the base with respect to the sugar moiety is stabilized relative to the syn -conformation due to the C3′-endo sugar pucker which is favored by the steric constraints imposed by the 2′-OH groups, and hence, G-quadruplex RNAs prefer an all-parallel topology where all four strands are oriented in the same direction, whereas G-quadruplex DNAs adopt a polymorphic topology. ,, In fact, G-rich RNA sequences that regulate the alternative splicing of an oncogenic fusion transcript have been recently shown to form an all parallel-stranded G-quadruplex RNA . Hence, knowledge about the interaction of the photosensitizers with all parallel-stranded G-quadruplex nucleic acids is critical for molecular design of photosensitizers to enhance their PDT efficacy.…”

The RNA binding proteins hnRNP H and F regulate splicing of a MYC dependent HRAS exon in Prostate Cancer Cells

“…Oncogenic splicing switches driven by hnRNP H include targets such as IG20/MADD in glioma (LeFave et al, 2011), TCF3 in lymphoma (Yamazaki et al, 2018), Mcl-1 and HER2 in breast cancer (Gautrey et al, 2015; Tyson-Capper and Gautrey, 2018), KHK in hepatocellular carcinoma (Li et al, 2016), and A-Raf in colon and head and neck cancers (Rauch et al, 2010). hnRNP H also regulates alternative splicing of the oncogenic fusion transcript EWS-FLI1 (Vo et al, 2022), and the RON protooncogene (Braun et al, 2018), and may alter translation in glioblastoma (Herviou et al, 2020). HnRNP F is less studied in the context of cancer cells but has been shown to be needed for the productive splicing of Sam68 in prostate cancer (Caggiano et al, 2019).…”