hnRNP A1 and hnRNP F Modulate the Alternative Splicing of Exon 11 of the Insulin Receptor Gene

Abstract

Exon 11 of the insulin receptor gene (INSR) is alternatively spliced in a developmentally and tissue-specific manner. Linker scanning mutations in a 5′ GA-rich enhancer in intron 10 identified AGGGA sequences that are important for enhancer function. Using RNA-affinity purification and mass spectrometry, we identified hnRNP F and hnRNP A1 binding to these AGGGA sites and also to similar motifs at the 3′ end of the intron. The hnRNPs have opposite functional effects with hnRNP F promoting and hnRNP A1 inhibiting exon 11 inclusion, and deletion of the GA-rich elements eliminates both effects. We also observed specific binding of hnRNP A1 to the 5′ splice site of intron 11. The SR protein SRSF1 (SF2/ASF) co-purified on the GA-rich enhancer and, interestingly, also competes with hnRNP A1 for binding to the splice site. A point mutation -3U→C decreases hnRNP A1 binding, increases SRSF1 binding and renders the exon constitutive. Lastly, our data point to a functional interaction between hnRNP F and SRSF1 as a mutant that eliminates SRSF1 binding to exon 11, or a SRSF1 knockdown, which prevents the stimulatory effect of hnRNP F over expression.

Adipose tissue INSR splicing in humans associates with fasting insulin level and is regulated by weight loss

“…We found a correlation between INSR splicing and expression of HNRNPA1 , SF3A1 and SFRS7 in the KOBS study (ESM Table 4). The strongest negative correlation was with HNRNPA1 , previously reported to inhibit exon 11 inclusion in HepG2 and HEK293 cells [6]. We acknowledge that other regulators of INSR splicing exist [6], and they may also be modified by weight loss.…”

“…The strongest negative correlation was with HNRNPA1 , previously reported to inhibit exon 11 inclusion in HepG2 and HEK293 cells [6]. We acknowledge that other regulators of INSR splicing exist [6], and they may also be modified by weight loss. One limitation of this study is that INSR protein isoforms created by alternative splicing could not be detected because the difference between the protein isoforms is only 1 kDa (data not shown).…”

“…1e) in both subcutaneous and visceral fat (ESM Table 1), suggesting common regulatory mechanisms related to insulin action. Finally, we observed a correlation between alternatively spliced INSR variants and expression of HNRNPA1, a known regulator of INSR splicing [6]. Weight loss resulted in higher expression of INSR-B, the more active isoform in insulin signalling [7].…”

“…Therefore, we analysed the effect of weight loss on the adipose tissue expression of these genes and the association with INSR splicing. The expression of HNRNPA1, a known regulator of INSR splicing [6], was increased in response to surgery-induced weight loss (p =0.001, data not shown) and its expression correlated negatively with INSR-B expression in the KOBS (r = −0.427, p = 1×10 −5 ), but not significantly in the VLCD study (r = −0.222, p = 0.238). However, after pooling adipose tissue samples from all time points of the KOBS and VLCD studies and after controlling for insulin levels the correlation between INSR-B and HNRPNA1 was significant in both KOBS and VLCD studies (r =−0.226 , p = Table 4).…”

A natural tapasin isoform lacking exon 3 modifies peptide loading complex function

“…Interestingly, the splice regulators hnRNPF/H predicted to bind to the G-runs by the ESRsearch tool (http://esrsearch.tau. ac.il/) have been described to control alternative splicing in several cases [32][33][34]. Specifically, it has been shown that these proteins are able to enhance the usage of upstream splice donor sites [35] as observed for the G-runs in intron 3 of tpn.…”