Widespread Expansion of Protein Interaction Capabilities by Alternative Splicing

Abstract

While alternative splicing is known to diversify the functional characteristics of some genes, the extent to which protein isoforms globally contribute to functional complexity on a proteomic scale remains unknown. To address this systematically, we cloned full-length open reading frames of alternatively spliced transcripts for a large number of human genes and used protein-protein interaction profiling to functionally compare hundreds of protein isoform pairs. The majority of isoform pairs share less than 50% of their interactions. In the global context of interactome network maps, alternative isoforms tend to behave like distinct proteins rather than minor variants of each other. Interaction partners specific to alternative isoforms tend to be expressed in a highly tissue-specific manner and belong to distinct functional modules. Our strategy, applicable to other functional characteristics, reveals a widespread expansion of protein interaction capabilities through alternative splicing and suggests that many alternative "isoforms" are functionally divergent (i.e., "functional alloforms").

Figure 1. Cloning of Novel Alternatively Spliced Isoforms Using ORF-Seq

(A) Comparative functional profiling of alternative isoforms. (B) Pipeline for systematic cloning of alternatively spliced ORFs, or “altORFs”. (C) Fraction of novel exon-exon junctions vs. novel full-length isoforms among cloned altORFs. (D) Distribution of endogenous transcript abundance for reference and alternatively spliced isoform clones. (E) Heatmap distinguishing cases where the reference isoform (yellow) or an alternatively spliced isoform (blue and light blue) was the major isoform detected. See also Figure S1 and Table S1.

Figure 2. Comprehensive Binary PPI Mapping for Protein Isoforms

(A) Comparative binary PPI profiling pipeline. (B) Western blot analysis showing comparable expression of four DB-BTRC alternative protein isoforms using an anti-Gal4-DB antibody (see red arrows). Interaction profiles are shown at bottom right with black and white boxes representing positive and negative interactions, respectively. (C) Validation of protein isoform interaction dataset. Shown is the fraction of pairs recovered by an orthogonal protein complementation assay (PCA) relative to increasing assay stringency. Shading indicates the standard error of the fraction. (D) Protein isoform interactome subnetworks. Each subnetwork displays relationships between genes, isoforms, and interaction partners with interactions mediated by reference protein isoforms shown in blue, and those mediated by novel alternatively spliced protein isoforms shown in red. See also Figure S2 and Table S2.

Figure 3. Contiguous Sequence Regions Associated with Isoform-Specific PPIs

(A) Four categories of isoform specific regions (ISRs) according to their effects on interactions (promoting, inhibiting, promoting or inhibiting, or complex). (B) Fraction of interaction partners classified in each of the four categories for all genes encoding at least two (left) or three (right) isoforms. (C) Box plot showing the average number of linear motifs per residue in promoting and inhibiting ISRs. P values from two-sided Wilcoxon rank test. (D) Schematic diagram illustrating interaction modulation potentially explained by differential splicing of linear motifs within exons. (E) Histogram showing the fraction of interaction partners that contain linear motif binding domains (LMBDs) and exhibit isoform-specific interactions associated with promoting regions or not. P values from two-sided Fisher's exact test; error bars represent the standard error of the fraction, estimated using bootstrapping with 100 resamplings. (F) Histogram showing the fraction of isoforms with interaction loss where a predicted interaction domain was disrupted by alternative splicing. P values from two-sided Fisher's exact test; error bars represent the standard error of the fraction, estimated using bootstrapping with 100 resamplings. (G) Three dimensional structure of BCL2-xL (grey; PDB code 1g5j) in complex with BAD (blue). The interaction interface is disrupted in the BCL2-xS isoform with the 3′-end of the first exon spliced out (pink). See Figure S3 for more structure examples. (H) Schematic diagram illustrating the interaction modulation of alternatively spliced isoforms of BCL2L1 potentially explained by differential splicing of BCL2 domain. See also Figure S3 and Table S3.

Figure 4. Comparison of Interaction Profiles for Alternative Isoforms

(A) Representative examples of alternative isoforms displaying identical, intermediate, or distinct interaction profiles. (B) Distribution of interaction profile differences between all possible pairs of alternative isoforms as measured by Jaccard distance. A Jaccard distance of 0 means that both isoforms share all interaction partners, whereas a distance of 1 means the isoforms have no shared partners. Isoforms for which no interactions were detected were omitted from the graph. See also Figure S4 and Table S4.

Figure 5. Functional Differences between Isoforms Revealed by Properties of Isoform Interaction Partners

(A) Schematic showing two different partners (blue nodes) interacting with either a single protein (left panel), alternative isoforms encoded by a common gene (middle panel), or the protein products of different genes (right panel). (B) Average network distance of pairs of partners interacting with a single protein, alternative isoforms, or the protein products of different genes. Error bars represent standard errors of the mean. (C) Fraction of pairs of partners interacting with a single protein, alternative isoforms, or the protein products of different genes, and showing positively correlated mRNA levels across 16 human tissues (Illumina Human Body Map 2.0). Error bars represent standard errors of the mean. (D) Mean Jaccard index of disease subnetwork co-occurrence of pairs of partners interacting with a single protein, alternative isoforms, or the protein products of different genes. Error bars represent standard errors of the mean. (E) Example of alternative isoforms interacting exclusively with proteins from different disease subnetworks. Pink nodes represent two protein isoforms encoded by the CD99L2 gene. Blue nodes represent the respective isoform interaction partners. Red nodes represent two different proteins encoded by genes associated with distinct diseases. See also Figure S5.

Figure 6. Protein Isoforms with Change-Over Interaction Profiles Exhibit Different Tissue-Specificities

(A) Distribution of four types of PPI differences exhibited by protein isoform pairs: change-over, each protein isoform has at least one exclusive interaction partner; on/off, one protein isoform lacks all interactions relative to another protein isoform from the same gene; subset on/off, one protein isoform lacks a subset of interactions; no difference, no differences observed in interaction partners for protein isoform pairs. (B) Comparison of the fraction of tissue-specific interaction partners, as estimated from the range of normalized log₂ RNA-Seq read counts from 16 human tissues (Illumina Human Body Map 2.0) for change-over interaction partners and other partners. P value from Fisher's exact test; error bars represent the standard error of the fraction, estimated using bootstrapping with 100 resamplings. (C) Example of a change-over isoform pair from the ZNF688 gene where each isoform interacts with a different protein whose mRNA is detected in very distinct sets of tissues. (D, E) Yeast complementation assays. Pictures on top show the growth status of yeast thermosensitive mutants transformed with different isoforms of the DOLK (panel D) or YARS (panel E) genes. GFP is used as negative control. Diagrams at the bottom show interactions and complementation mediated by the two isoforms. See also Figure S6 and Table S4.