Abstract
In the nematode C. elegans, insulin signaling regulates development and aging in response to the secretion of numerous insulin peptides. Here, we describe a novel, non-signaling isoform of the nematode insulin receptor (IR), DAF-2B, that modulates insulin signaling by sequestration of insulin peptides. DAF-2B arises via alternative splicing and retains the extracellular ligand binding domain but lacks the intracellular signaling domain. A daf-2b splicing reporter revealed active regulation of this transcript through development, particularly in the dauer larva, a diapause stage associated with longevity. CRISPR knock-in of mScarlet into the daf-2b genomic locus confirmed that DAF-2B is expressed in vivo and is likely secreted. Genetic studies indicate that DAF-2B influences dauer entry, dauer recovery and adult lifespan by altering insulin sensitivity according to the prevailing insulin milieu. Thus, in C. elegans alternative splicing at the daf-2 locus generates a truncated IR that fine-tunes insulin signaling in response to the environment.
Highlights
Alternative splicing of messenger RNA provides a mechanism for generating protein diversity, and it has been suggested that up to 95% of the human genome undergoes some form of alternative splicing (Chen and Manley, 2009)
It is well established that alternative splicing at the insulin receptor (IR) locus in mammals leads to the expression of two isoforms of the IR that differ in their affinity for ligands (Belfiore et al, 2009)
Failure to utilize this splice site would result in the addition of 46 bp of intronic sequence before reaching an in-frame stop codon (Figure 1B, Figure 1—figure supplement 1). Downstream of this stop codon we observed multiple possible polyA sites, including a variant (AAUGAA) 84 bp away and matching sequence from the expressed sequence tag (EST) (Figure 1—figure supplement 1). These EST sequences previously formed the basis for the existence of the daf-2b isoform, which was predicted to share the first 11 exons with the daf-2a full-length receptor but lack the transmembrane and intracellular tyrosine kinase domains encoded by exons 12–17 (Figure 1A and B)
Summary
Alternative splicing of messenger RNA provides a mechanism for generating protein diversity, and it has been suggested that up to 95% of the human genome undergoes some form of alternative splicing (Chen and Manley, 2009). In Drosophila, an insulin binding protein (SDR) with significant homology to the extracellular domain of the fly insulin-like receptor (InR) has been shown to antagonize insulin signaling by binding insulin-like peptides (Okamoto et al, 2013) This variant is encoded by a distinct gene rather than arising via alternative splicing of the InR locus. Analysis of dauer formation and recovery, established paradigms for insulin-like peptide activity (Cornils et al, 2011), indicated that DAF-2B modifies insulin signaling both positively and negatively, in a manner consistent with the sequestration of insulin peptides This mechanism was confirmed by co-expression of DAF-2B with both agonist and antagonist insulin-like peptides. Our results indicate that a truncated IR isoform arising via alternative splicing represents a new fundamental principle for how the insulin signaling axis is regulated
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