Alternatively Splicing Interactomes Identify Novel Isoform-Specific Partners for NSD2

Abstract

Nuclear receptor SET domain protein (NSD2) plays a fundamental role in the pathogenesis of Wolf-Hirschhorn Syndrome (WHS) and is overexpressed in multiple human myelomas, but its protein-protein interaction (PPI) patterns, particularly at the isoform/exon levels, are poorly understood. We explored the subcellular localizations of four representative NSD2 transcripts with immunofluorescence microscopy. Next, we used label-free quantification to perform immunoprecipitation mass spectrometry (IP-MS) analyses of the transcripts. Using the interaction partners for each transcript detected in the IP-MS results, we identified 890 isoform-specific PPI partners (83% are novel). These PPI networks were further divided into four categories of the exon-specific interactome. In these exon-specific PPI partners, two genes, RPL10 and HSPA8, were successfully confirmed by co-immunoprecipitation and Western blotting. RPL10 primarily interacted with Isoforms 1, 3, and 5, and HSPA8 interacted with all four isoforms, respectively. Using our extended NSD2 protein interactions, we constructed an isoform-level PPI landscape for NSD2 to serve as reference interactome data for NSD2 spliceosome-level studies. Furthermore, the RNA splicing processes supported by these isoform partners shed light on the diverse roles NSD2 plays in WHS and myeloma development. We also validated the interactions using Western blotting, RPL10, and the three NSD2 (Isoform 1, 3, and 5). Our results expand gene-level NSD2 PPI networks and provide a basis for the treatment of NSD2-related developmental diseases.

Keywords: NSD2; RPL10; alternatively splicing; isoform; protein–protein interaction.

FIGURE 1

NSD2 transcripts and protein isoforms have different expression patterns. (A) The gene model and exon distributions in transcripts of NSD2. (B) Four transcripts of NSD2 were expressed in human tissues by using the GTEx database. (C) The subcellular locations of four protein isoforms of NSD2.

FIGURE 2

NSD2 isoform-level interactors exhibit importance in the interactome network. (A) The pipeline of IP-MS to identify interactions. (B) Silver stainings of NSD2 isoforms. (C) The diagram of overlapped partners between isoforms. (D) Distribution of paired NSD2 isoforms in the interaction profile differences between all possible pairs of alternative isoforms from Yang et al. (2016). The Jaccard distance of 0 means that both isoforms share all interaction partners, whereas a distance of 1 means the isoforms have no shared partners. The above heatmap shows the value of Jaccard distance in paired isoforms of NSD2. (E) The histogram represents the number of the isoform-level interactors according to the isoform type, reference (light green), or non-reference (light blue). The Venn diagram below represents the number of the gene-level PPIs (a total of 442), reference (light green), or non-reference (light blue). Some genes have PPIs shared by the reference and the non-reference isoforms.

FIGURE 3

NSD2 isoform-specific function enrichments. (A) The network of the gene and isoform level partners of NSD2. The heatmap shows the degree of each node (Isoform 1, 3, 5, 7, and NSD2). (B) The Venn diagram shows the overlapped interactors between NSD2 isoforms and reference interactors of NSD2 from BioGrid. (C) The enrichment results of BioGrid and our isoform interactors. The histogram in light green represents genes from BioGrid, and the light blue represents genes from our NSD2 isoform interactions. (D) The Venn diagram shows the overlapped PPIs between Isoform 1 and the other three isoforms. The histogram represents the enrichment categories of both interactors.

FIGURE 4

NSD2 exon junction specific clusters show different function. (A) The junctional patterns of exons into clusters. (B) The diagram shows the shared genes between exon specific isoforms, and the histogram in the same color represents enrichment results corresponding to the diagram genes. (C) Western blots of all four NSD2 isoforms and RPL10. (D) Western blots of all four NSD2 isoforms and HSPA8.