We have previously characterized a truncated isoform of the worm insulin receptor, DAF-2B which alters insulin signaling phenotypes via sequestration of insulin-like peptides. The DAF-2B isoform is generated by alternative splicing, whereby inclusion of exon 11.5 is followed by a failure to splice to exon 12, leading to the retention of a short in-frame intronic sequence. However, the factors which control these splicing events are not known. In C. elegans, rsp genes encode members of the SR protein family which control both constitutive and alternative splicing. To determine if SR-proteins play a role in regulation of the DAF-2B isoform, we examined the effect of RNAi against rsp genes in a fluorescent
daf-2b splicing reporter strain. We found that knockdown of the SRp40 homologue,
rsp-2 significantly increased fluorescence expression in all larval stages, most prominently in early larval stages. Increased reporter expression, as well as increased expression of the endogenous
daf-2b transcript, was confirmed using a deletion mutation,
rsp-2(delta). These observations suggest that the normal function of RSP-2 is to inhibit expression of the
daf-2b transcript. Since
rsp-2(delta) is associated with higher
daf-2b expression, we hypothesized that
rsp-2(delta) would mimic phenotypes associated with high expression of DAF-2B, such as increased dauer entry and reduced dauer exit. Consistent with this, we found that
rsp-2(delta);
pdk-1 double mutant animals had higher rates of dauer formation and reduced dauer recovery. Epistasis experiments between
daf-2b and
rsp-2 in the
pdk-1 background show that
daf-2b is downstream of
rsp-2 but only partially required, suggesting that RSP-2 may more broadly be a target of the insulin signaling pathway. To investigate this link, we used CRISPR/Cas9 to generate mutations that target a predicted Akt phosphorylation site in
rsp-2. Animals bearing a phosphorylation deficient mutation showed enhanced dauer entry and higher
daf-2b expression whereas a phosphorylation mimetic mutation showed reduced dauer entry and no change in
daf-2b. Therefore, RSP-2 regulation of DAF-2B appears to be linked to the phosphorylation cascade of the insulin-signaling pathway.