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

Abstract

Enhanced expression of the cold-shock protein RNA binding motif 3 (RBM3) is highly neuroprotective both in vitro and in vivo. Whilst upstream signalling pathways leading to RBM3 expression have been described, the precise molecular mechanism of RBM3 induction during cooling remains elusive. To identify temperature-dependent modulators of RBM3, we performed a genome-wide CRISPR-Cas9 knockout screen using RBM3-reporter human iPSC-derived neurons. We found that RBM3 mRNA and protein levels are robustly regulated by several splicing factors, with heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) being the strongest positive regulator. Splicing analysis revealed that moderate hypothermia significantly represses the inclusion of a poison exon, which, when retained, targets the mRNA for nonsense-mediated decay. Importantly, we show that HNRNPH1 mediates this cold-dependent exon skipping via its interaction with a G-rich motif within the poison exon. Our study provides novel mechanistic insights into the regulation of RBM3 and provides further targets for neuroprotective therapeutic strategies.

ASO targeting RBM3 temperature‐controlled poison exon splicing prevents neurodegeneration in vivo

“…These data suggest that temperature-controlled alternative splicing coupled to NMD is the main mechanism that controls RBM3 expression levels in the physiologically relevant temperature range. Consistent with this, recent work has implicated several splicing factors, among others HNRNPH1, in regulating RBM3 expression through poison exon splicing (Lin et al, 2022). Temperature-dependent phosphorylation of SR proteins likely contributes to this regulation (Preussner et al, 2017;Haltenhof et al, 2020), as the effect of temperature on RBM3 expression is strongly reduced in conditions with inhibited CDC-like kinases (CLKs;Fig EV2C).…”

ASO targeting temperature-controlledRBM3poison exon splicing prevents neurodegeneration in vivo

“…These data suggest that temperature-controlled alternative splicing coupled to NMD is the main mechanism that controls RBM3 expression levels in the physiologically relevant temperature range. Consistent with this, recent work has implicated several splicing factors, amongst others HNRNPH1, in regulating RBM3 expression through poison exon splicing (30). Temperature-dependent phosphorylation of SR proteins likely contributes to this regulation (8,31), as the effect of temperature on RBM3 expression is strongly reduced in conditions with inhibited CDC-like kinases (CLKs; Figure S2C).…”

“…These data suggest that temperature-controlled alternative splicing coupled to NMD is the main mechanism that controls RBM3 expression levels in the physiologically relevant temperature range. Consistent with this, recent work has implicated several splicing factors, amongst others HNRNPH1, in regulating RBM3 expression through poison exon splicing (Lin et al, 2022).…”

Manipulating and studying gene function in human pluripotent stem cell models

“…A strategy to overcome this limitation is to combine a GoF approach for efficient forward programming of hPSCs into highly homogeneous cortical neurons (NGN2 OPTi-OX) and a constitutively expressed dCas9 genome edited into the GAPDH locus to ensure its stable expression following differentiation [221]. After Dox-induced forward programming, neurons can be transduced with genome-wide sgRNA libraries, and subjected to a combination of selective pressures (i.e., tunicamycin [222] or cold shock [221]) and FACS enrichment, and analyzed by bulk NGS. Another possibility to achieve consistent Cas9 expression during pooled CRISPRn screen is via genome editing into the AAVS1 locus of hPSCs [25].…”

“…Genome‐wide screenings require a relatively homogeneous and sufficiently large starting cell population to ensure coverage of all perturbations (~100–1000 cells per sgRNA); this can be challenging in hPSC‐derived lineages due to inefficient, poorly scalable, and highly variable differentiation protocols. A strategy to overcome this limitation is to combine a GoF approach for efficient forward programming of hPSCs into highly homogeneous cortical neurons (NGN2 OPTi‐OX) and a constitutively expressed dCas9 genome edited into the GAPDH locus to ensure its stable expression following differentiation [221]. After Dox‐induced forward programming, neurons can be transduced with genome‐wide sgRNA libraries, and subjected to a combination of selective pressures (i.e., tunicamycin [222] or cold shock [221]) and FACS enrichment, and analyzed by bulk NGS.…”