Identifying biological pathways that underlie primordial short stature using network analysis

  1. Peter E Clayton1,2
  1. 1Institute of Human Development, Faculty of Medical and Human Sciences, The University of Manchester, Oxford Road, Manchester M13 9WL, UK
    2Manchester Academic Health Sciences Centre (MAHSC), Central Manchester University Hospitals NHS Foundation Trust, Manchester M13 9WL, UK
  1. Correspondence should be addressed to D Hanson; Email: Daniel.hanson{at}
  1. Figure 1

    The 3-M interactome. (A) Cytoscape grid layout of the 131 proteins with 721 connections between them that form the 3-M interactome. Network was generated through identifying proteins present in both the IP/MS network and the transcriptomic network. Physical interactions are shown by orange connections and interactions which are both physical and genetic shown by blue connections. Nodes are assigned and coloured according to the central node where they most belong. (B) ModuLand network representing the key nodes within the overall network designated by degree of interactions and network centrality. (C) Immunoprecipitation of V5-OBSL1-overexpressing HEK293 cells (left panel, OBSL1-V5 IP), CUL7-overexpressing HEK293 cells (middle panel, CUL7 IP) and CCDC8 overexpressing HEK293 cells (right panel, CCDC8 IP) with western blotting to identify co-immunoprecipitated proteins to confirm the putative interactions identified by IP/MS. Protein inputs (Input) and control IPs with no antibody (no Ab IP) are shown for each panel.

  2. Figure 2

    INSR minigene assay. (A) Quantification of alternative splicing of INSR minigene in fibroblast cells. Control cells (n=3) and fibroblasts from 3-M syndrome patients, CUL7−/−, OBSL1−/− and CCDC8−/−, were transfected with an INSR minigene construct and relative levels of INSR were measured by RT-PCR analysis. Graph indicates the relative expression of IR-B/IR-A as a mean for n=10 transfection experiments for each cell type, a representative gel is shown below the graph. Error bars represent s.e.m. (B) Quantification of alternative splicing of INSR minigene in HEK293 cells. HEK293 cells were transfected with INSR minigene construct only (labelled HEKs, n=8 transfection experiments) or with minigene and a CUL7 expression vector (HEKs+CUL7, n=5 transfection experiments), with minigene and a OBSL1 expression vector (HEKs+OBSL1, n=5 transfection experiments) and with minigene and a CCDC8 expression vector (HEKs+CCDC8, n=5 transfection experiments). Graph indicates the mean relative expression of IR-B/IR-A for each combination of transfections as indicated, a representative gel is shown below the graph. Error bars represent s.e.m.

  3. Figure 3

    The CUL7-OBSL1-CCDC8 pathway and its predicted role in cell growth. OBSL1 interacts with both CUL7 and CCDC8 (solid connections shows protein–protein interactions) all three associate with the mRNA splicing machinery with particularly high abundance of HNRNPs in the 3-M interactome. Alternative splicing of the Insulin receptor (INSR) is modulated by CUL7, OBSL1 and CCDC8, IRS-1 is also a target of the CUL7 E3 ubiqutin ligase and this impacts on downstream signalling upon growth factor stimulation leading to dysfunction in MAPK and AKT activation. This subsequently results in a reduction of cell proliferation in cells derived from 3-M syndrome patients.

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