Aberrant splicing exonizes C9ORF72 repeat expansion in ALS/FTD

Abstract

A nucleotide repeat expansion (NRE) in the first annotated intron of the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). While C9 NRE-containing RNAs can be translated into several toxic dipeptide repeat proteins, how an intronic NRE can assess the translation machinery in the cytoplasm remains unclear. By capturing and sequencing NRE-containing RNAs from patient-derived cells, we found that C9 NRE was exonized by the usage of downstream 5' splice sites and exported from the nucleus in a variety of spliced mRNA isoforms. C9ORF72 aberrant splicing was substantially elevated in both C9 NRE⁺ motor neurons and human brain tissues. Furthermore, NREs above the pathological threshold were sufficient to activate cryptic splice sites in reporter mRNAs. In summary, our results revealed a crucial and potentially widespread role of repeat-induced aberrant splicing in the biogenesis, localization, and translation of NRE-containing RNAs.

Keywords: ALS/FTD; C9ORF72; cryptic splice site; mRNA splicing; nucleotide repeat expansion.

Figure 1. ASO-based capture of NRE-containing transcripts.

(a) Schematics of NRE-RAP-seq. (b) RT-qPCR quantification of the enrichment of C9 NRE-flanking region and GAPDH by NRE-RAP relative to input. (c) Comparison of normalized NRE-RAP-seq read counts for each gene between C9 NRE⁺ and NRE⁻ samples. C9ORF72 is indicated in red.

Figure 2. Exonization of C9 NRE by downstream 5ʹ splice site usage.

(a) Read coverage of FB504 cytoplasmic RNA input (top) and ASO-captured NRE-containing RNAs (bottom). Dotted line rectangle indicates the region shown in (b). (b) Splice junctions within and near intron 1 detected in NRE-RAP-seq results. The three most abundant splice junctions are shown in red. (c) Schematics illustrating C9 NRE-exonized transcript isoforms. (d) Quantification of polyGA and polyGP abundance in siRNA-treated C9 NRE⁺ fibroblasts. siNT, non-targeting siRNA. siEx2, a mix of two siRNAs targeting exon 2. *, p<0.05; **, p<0.01, two-tailed ratio t test.

Figure 3. Nucleocytoplasmic distribution of exonized C9 NRE.

(a) Comparison of normalized read count per gene between nuclear and cytoplasmic input RNAs, showing the most enriched transcripts. (b) Read coverage of FB504 nuclear RNA input (top, middle) and ASO-captured C9 NRE-containing RNAs (bottom). Dotted line rectangle indicates the region shown in the middle and bottom plots. The three most abundant splice junctions detected in NRE-RAP-seq results are shown in red. (c) RT-qPCR quantification of the distribution of individual C9ORF72 region between nucleus and cytoplasm. Ex2-Ex3 junction, a proxy for canonical C9ORF72 mRNA, was measured using the input RNA, whereas all other regions were measured using ASO-captured RNA.

Figure 4. C9ORF72 aberrant splicing in motor neurons and ALS/FTD brains.

(a) RT-qPCR quantification of the Ex2-Ex3 splice junction, NRE-flanking region, and Ex1c-Ex2 splice junction, comparing C9 NRE⁺ and NRE⁻ iPS-MNs. (b) RNA-seq quantification of the Ex2-Ex3 splice junction, intron 1, Ex1c-Ex2, and Ex1d-Ex2 splice junctions, comparing C9 NRE⁺ and NRE⁻ samples in each brain region. CB, cerebellum. FC, frontal cortex. MC, motor cortex. p values, Mann-Whitney-Wilcoxon tests.

Figure 5. Repeat length-dependent activation of cryptic splice sites by C9 NRE.

(a) RT-qPCR quantification of the splicing efficiency of reporter mRNAs with varying (GGGGCC)_n inserts within a chimeric intron. (b) Normalized activities of dual-luciferase with varying (GGGGCC)_n inserts between FLuc and RLuc coding sequences. ***, p<0.001, two-tailed t test. (c) Top, RNA-seq read coverage and splice junctions of the reporter containing (GGGGCC)₃₃. Splicing efficiencies of the J1 and J2 splice junctions are shown. Bottom, RT-qPCR quantification of the splicing efficiency of the J1 splice junction in reporters with varying (GGGGCC)_n inserts. *, p<0.05; **, p<0.01, two-tailed t test. (d) RNA-seq quantification of all splice junctions, comparing cytoplasmic RNAs between HEK293T cells expressing reporters with no NRE and (GGGGCC)₃₃. J1 and J2 splice junctions are shown in red.