Endocrine Abstracts

Background: The insulin receptor (INSR) is alternatively spliced in a developmental and tissue specific manner into two isoforms, IR-A and IR-B. IR-A excludes exon 11 and is widely expressed whereas IR-B includes exon 11 and is expressed in insulin sensitive tissues. The severe short stature disorder 3-M syndrome is caused by mutations in CUL7, OBSL1 and CCDC8 and we have recently associated these proteins with the major mRNA splicing pathways including the heterogeneous nuclear ribonucleoprotein (HNRNP) complex.

Objective: To determine if the 3-M proteins regulate the alternative splicing of INSR.

Methods: We co-transfected expression vectors for the 3-M genes (CUL7, OBSL1 and CCDC8) and an INSR minigene into HEK293 cells, the INSR minigene was also transfected into fibroblast cells from healthy controls and 3-M syndrome patients. Analysis of exon 11 splicing was determined by RT-PCR and calculating alteration of the ratio IR-B/IR-A ratio by gel densitometry.

Results: Over-expression of the 3-M genes in HEK293 cells causes a decrease in exon 11 inclusion resulting in a reduction in the ratio of IR-B/IR-A expression (P<0.001). While reduced expression of the 3-M genes in patient cells has the opposite effect with an increase in exon 11 inclusion and an increase in the ratio of IR-B/IR-A expression (P<0.001).

Conclusion: Alternative INSR splicing has been previously associated with different HNRNP proteins and we now demonstrate that the interaction of the 3-M proteins within this complex can also modulate exon 11 splicing. This may in part explain the reduced growth seen in short stature patients as IR-A exhibits the mitogenic effects of insulin while IR-B exhibits its metabolic effects. Conversely elevated expression of CUL7 and IR-A have been associated with increased risk of tumours and cancer progression suggesting a more widespread role of the 3-M genes as growth regulators beyond stature.