We demonstrate that a mutation in one of the multicopy mouse U2 snRNA genes causes defects in pre-mRNA splicing leading to neurodegeneration. U2 snRNAs play an essential role in formation of the catalytically active spliceosome by base pairing with both the intron branch point and the U6 snRNA (Wahl et al., 2009
). Expression of the mutant U2 snRNA alters pre-mRNA splicing at selective splice sites that are often associated with alternative splicing demonstrating that U-snRNA dysfunction, like downregulation of core spliceosomal proteins, can influence splice site choice (Corioni et al., 2011
; Park et al., 2004
; Saltzman et al., 2011
; Shaw et al., 2007
One of the main pathological features of the NMF291−/−
cerebellum is the increased retention of small introns, which likely represent a unique spliceosomal substrate. Unlike splicing of large introns, which is thought to first occur by pairing of splice sites across the exons (“exon definition”), splicing of short introns likely occurs by pairing of 5' and 3' splice sites across the intron (“intron definition”) (Berget, 1995
). Furthermore, even under normal physiological conditions many of the highly retained introns in the mutant cerebellum are not fully spliced, consistent with the observation that weak splicing sites often flank small introns (Lim and Burge, 2001
; Sakabe and de Souza, 2007
). Given the disruption of short intron splicing and other alternative splicing events, it is plausible that mutant U2 snRNPs are fully functional on optimal, but not suboptimal, substrates ().
Interestingly, we found that mRNA-processing genes were significantly enriched among the alternative splicing events differentially expressed between the wild type and mutant cerebellum, suggesting that neurons may utilize alternative splicing to regulate the function of proteins involved in RNA splicing and processing in an effort to restore splicing homeostasis. Indeed, studies have shown that a number of splicing regulators autoregulate their expression and activity via transcriptional feedback loops and alternative splicing (Ni et al., 2007
; Saltzman et al., 2011
; Wollerton et al., 2004
). However, rather than reestablishing homeostasis, these splicing alterations could in fact act to amplify the amount of abnormal splicing and ultimately prove deleterious. It will be intriguing to see whether the expression level and/or alternative splicing status of these mRNA-processing genes is also affected in SMA patients or ALS/FTD patients with mutations in TDP-43 or FUS/TLS.
The ultimate cause of neuron death in the mutant cerebellum is unclear. Cell death could be caused by the generation of proteins with altered function or by the production of RNAs containing abnormal sequences, which could themselves be toxic as previously reported for trinucleotide repeat expansion diseases (Li et al., 2008
). Retained introns may also contribute to neuron death by sequestering splicing regulators and/or other RNA binding proteins. In addition, many of these introns likely harbor premature translation termination codons (PTCs) that would be predicted to trigger the nonsense-mediated mRNA-decay (NMD) pathway. In addition to its role in degradation of abnormal transcripts, NMD also regulates many natural PTC-containing transcripts, including those involved in synaptic physiology and cellular stress (Gardner, 2010
; Giorgi et al., 2007
). Enhanced and/or prolonged NMD activation could overwhelm the NMD pathway, causing dysregulation of natural NMD targets that might be essential for cell survival, or itself cause cellular stress.
Finally, our data demonstrate the potential for disease-causing mutations in multicopy genes. While a single U2 gene is present in yeast, multiple copies of these genes exist in higher organisms, each producing identical or nearly identical products. Indeed, the region on human Chromosome 17p21 homologous to the (Rnu2–6
-containing) region on mouse Chromosome 11 also contains a cluster of stably inherited U2 genes that vary in copy number from 5 to 25 (Van Arsdell and Weiner, 1984
; Westin et al., 1984
). In addition to our findings of temporal and spatial regulation of a mouse U2 gene, developmental regulation of other snRNA genes, including U2 snRNA genes, has been reported (Forbes et al., 1984
; Lund et al., 1985
; Sierra-Montes et al., 2005
; Stefanovic et al., 1991
). Furthermore, the developmental arrest associated with homozygosity for a mutation in one of twelve C. elegans U1 genes raises possibility of differences in expression and/or function between C. elegans U1 genes (Zahler et al., 2004
). Whether individual human U2s (or other multicopy genes) are differentially expressed during development, in different cell types, or even as a result of pathogenic processes is unknown. However, the potential for discrete regulation of individual members of multigene families, combined with their potential for copy number variation, increases the prospect of uncovering novel disease-causing mutations in repetitive genes.