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 cold induction 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 thermosensitive 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.

Cold-shock proteome of myoblasts reveals role of RBM3 in promotion of mitochondrial metabolism and myoblast differentiation

“…2c, d ). This underlines the importance of post-transcriptional RNA regulation in cold response in agreement with previously reported studies where RBM3 has been shown to regulate or bind to proteins involved in splicing 31 and translation 47 , 56 . The cold-responsive RNA binding protein, RBM3 16 , was also shown to be upregulated at 6 h (Fig.…”

“…At lower temperatures, a poison exon (E3a) positioned at exon 3 of RBM3 mRNA gets spliced, thus there is no degradation of RBM3 mRNA by NMD 30 . Another study in human iPSC‐derived neurons suggests that an alternative splicing factor, heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) enhances the splicing of the poison exon by strongly binding to G-rich motifs in RBM3 mRNA 31 . A few downstream effector proteins of RBM3 have been identified, for example, RTN3 in HEK293 and a mice model of prion disease 32 .…”

Stabilizing mammalian RNA thermometer confers neuroprotection in subarachnoid hemorrhage

“…We next aimed to further substantiate the connection between G-rich elements and temperature-sensitive alternative splicing. We focused on the cold-inducible and neuroprotective protein RBM3 57,58 , which is controlled by temperature-regulated alternative splicing of a poison exon (exon 3a) leading to heat-induced NMD 46,47 . In RNA-Seq data, RBM3’s exon 3a inclusion level remained consistently below 0.1 at or below 37°C.…”

“…Since RBM3 transcripts containing exon 3a are extremely efficiently degraded via the NMD pathway 46,47 , we cloned human RBM3 (hRBM3) and mouse RBM3 (mRBM3) minigenes consisting of exon 3, exon 3a, exon 4, and the introns flanking exon 3a. The RNA produced by the two minigenes is not translated, and therefore is not degraded by NMD ( Figure 3B ).…”

“…Using the neuroprotective and cold-induced RBM3 as an example, we characterize a specific rG4 in detail. We find that stabilizing rG4s located proximal to the splice sites of RBM3’s cold-repressed poison exon 46,47 , either through low temperature or high potassium concentration, promotes exon skipping, thereby increasing RBM3 expression by evading nonsense-mediated decay (NMD) 46,47 . Importantly, increasing intracellular potassium level via a potassium channel blocker (4-aminopyridine, 4-AP) stabilizes rG4s and confers RBM3-dependent neuroprotection in a subarachnoid hemorrhage mouse model.…”