Integrative Proteogenomics for Differential Expression and Splicing Variation in a DM1 Mouse Model

Abstract

Dysregulated mRNA splicing is involved in the pathogenesis of many diseases including cancer, neurodegenerative diseases, and muscular dystrophies such as myotonic dystrophy type 1 (DM1). Comprehensive assessment of dysregulated splicing on the transcriptome and proteome level has been methodologically challenging, and thus investigations have often been targeting only few genes. Here, we performed a large-scale coordinated transcriptomic and proteomic analysis to characterize a DM1 mouse model (HSA^LR) in comparison to wild type. Our integrative proteogenomics approach comprised gene- and splicing-level assessments for mRNAs and proteins. It recapitulated many known instances of aberrant mRNA splicing in DM1 and identified new ones. It enabled the design and targeting of splicing-specific peptides and confirmed the translation of known instances of aberrantly spliced disease-related genes (e.g., Atp2a1, Bin1, Ryr1), complemented by novel findings (Flnc and Ywhae). Comparative analysis of large-scale mRNA and protein expression data showed quantitative agreement of differentially expressed genes and splicing patterns between disease and wild type. We hence propose this work as a suitable blueprint for a robust and scalable integrative proteogenomic strategy geared toward advancing our understanding of splicing-based disorders. With such a strategy, splicing-based biomarker candidates emerge as an attractive and accessible option, as they can be efficiently asserted on the mRNA and protein level in coordinated fashion.

Keywords: alternative splicing; myotonic dystrophy type 1 (DM1); proteogenomics.

Graphical abstract

Fig. 1

Gene sets showing differential gene expression and differential alternative splicing and overlaps.A, adjusted p-value cut-off p < 0.01 for both DGE and DAS. B, adjusted p-value cut-off p < 0.01 for both DGE and DAS and an additionally for DGE, an absolute abundance fold change of >2. DAS, differential alternative splicing; DGE, differential gene expression.

Fig. 2

Differential analysis of transcripts and proteins, Differential analysis of (A) transcripts and (B) proteins identified in WT compared to DM1 mice (volcano plots). The coordinates are log-transformed and correspond to adjusted p-values and abundance fold change calculated by limma test with Benjamini–Hochberg correction. The red dots represent differentially expressed transcripts and proteins (p-value <0.01 and absolute abundance fold change >2). C, the correlation between observed transcript and protein abundance fold changes in log-transformed coordinates. D, the intersection between gene sets with significant differential gene expression (DGE) on transcript and protein levels (note that 65 differential expression proteins correspond to 64 genes) and (E) their intersection with genes showing differential alternative splicing (DAS). DM1, myotonic dystrophy, type 1.

Fig. 3

Correlation of fold changes on transcript and protein levels.A, the Pearson correlation of abundance fold changes and (B) the number of common differential expression genes at transcript and protein levels. Each heatmap cell corresponds to different significance thresholds, the colour represents (A) the value of Pearson correlation and (B) the number of common genes.

Fig. 4

Comparison of instances of differential alternative splicing at transcriptional and protein level for Atp2a1 and Bin1 genes. Panels on the left show LeafCutter AS intron clusters with relative exon usage in the DM1 and WT control groups. Panels in the middle show qRT-PCR results with the splicing-specific probes (see Experimental Procedures) covering the corresponding exon inclusion (gray) and exon exclusion (blue). Panels on the right show the corresponding relative abundances of peptides specific to exon inclusion or exclusion in the same sample groups. See also supplemental Fig. S4 for a UCSC genome browser-based display of these LeafCutter AS intron clusters. AS, alternative splicing; DM1, myotonic dystrophy, type 1.

Fig. 5

The correlation between log-transformed splice event ratio observed in targeted Px (Equation 2andBox 2) and Tx (Equation 3andBox 2) for 14 genes, corresponding to 16 splice events and 19 peptide pairs detected in DM1 and WT. The superscript numbers correspond to different clusters/splice events in the same gene (e.g., Neb¹ is an inclusion event in cluster 5321, Neb^2&3 correspond to different peptides covering the same inclusion event in cluster 5320, and Neb⁴ is an exclusion event in that same cluster 5320. For all other events, see supplemental Table S3, “AS event ratio” list). The Pearson correlation for all examined events is 0.95 (0.98 without two outliers, Atp2a1 and Pdlim7). AS, alternative splicing; DM1, myotonic dystrophy, type 1; Px, proteomics; Tx, transcriptomics.