Abstract
Increasing evidence suggests that defective RNA processing contributes to the development of amyotrophic lateral sclerosis (ALS). This may be especially true for ALS caused by a repeat expansion in C9orf72 (c9ALS), in which the accumulation of RNA foci and dipeptide-repeat proteins are expected to modify RNA metabolism. We report extensive alternative splicing (AS) and alternative polyadenylation (APA) defects in the cerebellum of c9ALS subjects (8,224 AS and 1,437 APA), including changes in ALS-associated genes (for example, ATXN2 and FUS), and in subjects with sporadic ALS (sALS; 2,229 AS and 716 APA). Furthermore, heterogeneous nuclear ribonucleoprotein H (hnRNPH) and other RNA-binding proteins are predicted to be potential regulators of cassette exon AS events in both c9ALS and sALS. Co-expression and gene-association network analyses of gene expression and AS data revealed divergent pathways associated with c9ALS and sALS.
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Acknowledgements
We are extremely grateful to all individuals who agreed to donate their brains to research. This work was supported by the National Institutes of Health/National Institute on Aging (R01AG026251 and P50AG016574 to L.P.), the National Institutes of Health/National Institute of Neurological Disorders and Stroke (R21NS089979 to T.F.G. and K.B.B.; R21NS084528, R01NS088689 and R01NS077402 to L.P.; R01NS063964 to L.P. and C.D.L.; P01NS084974 to L.P., D.W.D., R.R. and K.B.B.), the National Institute of Environmental Health Sciences (R01ES20395 to L.P.), the Department of Defense (ALSRP AL130125 to L.P.), the Mayo Clinic Foundation (L.P.), the Mayo Clinic Center for Individualized Medicine (L.P. and K.B.B.), the ALS Association (K.B.B., L.P., M.P. and T.F.G.), the Robert Packard Center for ALS Research at Johns Hopkins (L.P.), Target ALS (L.P.), the ALS Association (Milton Safenowitz postdoctoral fellowships to V.V.B. and M.P.), the Canadian Institutes of Health Research (postdoctoral fellowship to V.V.B.), the Siragusa Foundation (Career Development Award for Young Investigators to V.V.B.), and the Robert and Clarice Smith & Abigail Van Buren Alzheimer's Disease Research Foundation (postdoctoral fellowship to V.V.B.). H.L. and M.E.M. are supported by the Mayo Clinic Center for Individualized Medicine and the Donors Cure Foundation.
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M.P., V.V.B., R.B. and C.A.R. contributed equally to this work. M.P., V.V.B. and L.P. contributed to the conception and design of the study. M.P., V.V.B., L.J.P., M.E.M., K.K.O., A.E.P.-J., P.D., M.D., M.D.D., M.C.B., R.B.P., K.B.B. and D.W.D. contributed to tissue selection and collection. M.P., L.J.P. and M.D.D. performed RNA extractions. M.P., V.V.B. and M.D.D. made cDNA. M.P. ran qRT-PCRs for expression and AS validation. R.B., C.A.R. and H.L. performed expression and WGCNA bioinformatics analyses. R.B. conducted AS, APA and system network analyses. M.P. and R.B. carried out GO analyses. H.L. supervised the bioinformatics analyses. T.F.G. and K.B. performed histological analyses. M.P., V.V.B., R.B., C.A.R., T.F.G., C.D.L., H.L. and L.P. interpreted the data and prepared the manuscript. All authors contributed to critical revision of the manuscript for important intellectual content and approved the final version for publication.
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Integrated supplementary information
Supplementary Figure 1 Principal-component analyses reveal divergence between c9ALS and sALS.
Principal-component analysis was performed using Log2(n+1) transformed RPKM data of the top 5,000 differentially expressed c9ALS genes from (a) 26 cerebellar and (b) 27 frontal cortex tissues. Green circles represent controls, purple circles represent sALS cases, and black circles represent c9ALS cases. Numbers for each case were randomly assigned and correspond to the cases described in Supplementary Table 1.
Supplementary Figure 2 Hierarchical clustering of differentially expressed genes in c9ALS cerebellum with clinical features shown for each case.
Hierarchical clustering representation of differentially expressed c9ALS transcripts in cerebellum with clinical features shown for each case. Each row of the heat map corresponds to a control (N = 8), sALS case (N = 10) or c9ALS case (N = 8), as designated by the color-coded bar to the left of the heat maps. Clinical features of each case, also color-coded, are shown to the right of the heat maps. Note that clinical information on the second column represents family history of neurodegeneration. Complete clinical information can be found in Supplementary Table 1.
Supplementary Figure 3 Hierarchical clustering of differentially expressed genes in c9ALS frontal cortex with clinical features shown for each case.
Hierarchical clustering representation of differentially expressed c9ALS transcripts in frontal cortex with clinical features shown for each case. Each row of the heat map corresponds to a control (N = 9), sALS case (N = 10) or c9ALS case (N = 9), as designated by the color-coded bar to the left of the heat maps. Clinical features of each case, also color-coded, are shown to the right of the heat maps. Note that clinical information on the second column represents family history of neurodegeneration. Complete clinical information can be found in Supplementary Table 1.
Supplementary Figure 4 Validation of top differentially expressed genes in c9ALS cerebellum and frontal cortex.
Bar graphs (mean ± s.e.m.) showing qRT-PCR validations, and the corresponding P values when comparing c9ALS (N = 9) and sALS (N = 10) to controls (N = 9 cerebellum, N = 8 frontal cortex) below, using RNA from cerebellum (a,b) and frontal cortex (c,d) for top differentially expressed genes in c9ALS (P < 0.05, │log2FC│ ≥ 2). Relative mRNA levels are normalized to the endogenous control, RPLP0, and the control group (mean value set to 1). Statistical differences were calculated by one-way ANOVA with Bonferroni post-hoc test (*P < 0.05, **P < 0.01, ***P < 0.005). The full list of oligonucleotides used in this study can be found in Supplementary Table 9.
Supplementary Figure 5 Intron retention is a common AS event in c9ALS.
Venn diagrams and bar graphs depicting the number of unique and common introns that are more (red) or less (blue) retained in c9ALS and sALS cerebellum (a) or frontal cortex (b) (FDR < 0.05). The black section of bar graph (a) shows common intron retention events but going in opposite directions. c9ALS (N = 8), sALS (N = 10), controls (N = 8 cerebellum, N = 9 frontal cortex).
Supplementary Figure 6 Hierarchical clustering of AS cassette exon variants in c9ALS cerebellum and clinical features for all cases.
Hierarchical clustering of down- or up-regulated c9ALS AS cassette exon-inclusion events in cerebellum from c9ALS (N = 8), sALS (N = 10) and controls (N = 8) with corresponding clinical features. Each row of the heat map corresponds to either a control, sALS or c9ALS case, as designated by the color-coded bar to the left of the heat map. Clinical features for all cases, also color-coded, are shown to the right of the heat maps. Note that clinical information on the second column represents family history of neurodegeneration. Complete clinical information can be found in Supplementary Table 1.
Supplementary Figure 7 Hierarchical clustering of AS cassette exon variants in c9ALS frontal cortex and clinical features for all cases.
Hierarchical clustering of down- or up-regulated c9ALS AS cassette exon-inclusion events in frontal cortex tissues from c9ALS (N = 8), sALS (N = 10) and controls (N = 9) with corresponding clinical features. Each row of the heat map corresponds to either a control, sALS or c9ALS case, as designated by the color-coded bar to the left of the heat map. Clinical features for all cases, also color-coded, are shown to the right of the heat maps. Note that clinical information on the second column represents family history of neurodegeneration. Complete clinical information can be found in Supplementary Table 1.
Supplementary Figure 8 Additional validation of AS cassette exon events in c9ALS cerebellum.
(a) RNA-Seq wiggle plots and bar graphs (mean ± s.e.m.) showing additional qRT-PCR validations in the cerebellum (see Fig. 5a in the main text for other validations). Shown is an example of the three technical qRT-PCR replicates performed for each AS event. Relative mRNA levels were normalized to the endogenous control, RPLP0, and controls (mean value set to 1). Note that inclusion of the cassette exon (of a mutually exclusive CE event) in U2AF1 is indicated on the wiggle plot by the arrow. The full list of primers used in this study can be found in Supplementary Table 9. (b,c) P values when comparing the levels of cassette exon inclusion, from qRT-PCR validations, in c9ALS (N = 9) and sALS (N = 10) to either non-neurological disease controls (controls, N = 9), (b) or to other neurological disease (PSP, N = 13) group (c). Statistical differences were calculated by one-way ANOVA with Bonferroni post-hoc test (*P < 0.05, **P < 0.01, #P < 0.0001). ND, not determined.
Supplementary Figure 9 Validation of AS cassette exon events in c9ALS frontal cortex.
(a) RNA-Seq wiggle plots and bar graphs (mean ± s.e.m.) showing qRT-PCR validations in the frontal cortex. Relative mRNA levels were normalized to the endogenous control, RPLP0, and controls (mean value set to 1). Note that inclusion of the cassette exon (of a mutually exclusive CE event) in U2AF1 is indicated on the wiggle plot by the arrow. The full list of primers used in this study can be found in Supplementary Table 9. (b) P values when comparing the levels of cassette exon inclusion, from qRT-PCR validations, in c9ALS (N = 9) and sALS (N = 10) to non-neurological disease controls (controls, N = 8). Statistical differences were calculated by one-way ANOVA with Bonferroni post-hoc test (*P < 0.05, #P < 0.0001).
Supplementary Figure 10 Validation of AS cassette exon events in c9ALS motor cortex.
(a) Bar graphs (mean ± s.e.m.) showing qRT-PCR validations of significantly misregulated cassette exons in the motor cortex. The full list of primers used in this study can be found in Supplementary Table 9. (b) P values when comparing the levels of cassette exon inclusion, from qRT-PCR validations, in c9ALS (N = 10) and sALS (N = 9) to non-neurological disease controls (controls, N = 10). Relative mRNA levels were normalized to the endogenous control, RPLP0, and controls (mean value set to 1). Statistical differences were calculated by one-way ANOVA with Bonferroni post-hoc test (*P < 0.05, ***P < 0.005).
Supplementary Figure 11 RNA-binding protein motif enrichment analysis of AS cassette exons highlights hnRNPH binding motifs.
RNA-binding protein (RBP) motif enrichment analysis was performed using AS cassette exon sequences and their flanking intronic regions of misspliced cassette exon events occurring in cerebellum and frontal cortex of c9ALS or sALS cases (c9ALS, N = 8; sALS, N = 10; controls, N = 8 cerebellum and N = 9 frontal cortex. RBP motif enrichment P values, shown in parentheses, were calculated using MEME-ChIP software. The resulting enriched motifs were cross-referenced with known RBP motifs from Ray and colleagues (2013)1 and an RBP database (http://rbpdb.ccbr.utoronto.ca).
1Ray, D. et al. A compendium of RNA-binding motifs for decoding gene regulation. Nature 499, 172–177 (2013).
Supplementary Figure 12 Gene-association network of top 1,000 genes of mis-spliced cassette exons in c9ALS cerebellum.
Genes with the top most significant AS cassette exon events in the c9ALS cerebellum (FDR < 0.05, │dl│ ≥ 0.1) were selected to construct a gene-association network using String v9.11 and Cytoscape 3.1.12 software. dl: differential index value. c9ALS (N = 8), sALS (N = 10), controls (N = 8). Note that this is a more complete version of Figure 5c shown in the main text. Genes are represented by nodes of different colors, which vary according to degree. The size of the node denotes neighborhood connectivity: nodes are bigger if they are connected to other nodes with higher connectivity. Edges are colored according to edge betweenness to indicate the proximity to other nodes, with low betweeness (closer proximity) meaning larger influence to other nodes. 1Franceschini, A. et al. STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 41, D808–D815 (2013). 2Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).
Supplementary Figure 13 Gene-association network for all mis-spliced cassette exons in sALS cerebellum.
All genes presenting AS cassette exons in sALS cerebellum (FDR < 0.05, │dl│ ≥ 0.1) were used to construct a gene-association network using String v9.11 and Cytoscape 3.1.12 software. dl: differential index value. c9ALS (N = 8), sALS (N = 10), controls (N = 9). Genes are represented by nodes of different colors, which vary according to degree. The size of the node denotes neighborhood connectivity: nodes are bigger if they are connected to other nodes with higher connectivity. Edges are colored according to edge between-ness to indicate the proximity to other nodes, with low between-ness (closer proximity) meaning larger influence to other nodes. Gene ontology terms of different interconnected cellular pathways are indicated.
1Franceschini, A. et al. STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 41, D808–D815 (2013).
2Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).
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Prudencio, M., Belzil, V., Batra, R. et al. Distinct brain transcriptome profiles in C9orf72-associated and sporadic ALS. Nat Neurosci 18, 1175–1182 (2015). https://doi.org/10.1038/nn.4065
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DOI: https://doi.org/10.1038/nn.4065
