Splicing is fundamental to gene expression in all eukaryotic organisms. Indeed, aberrant splicing is associated with many inherited diseases such as spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), myotonic dystrophy (DM), microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1) and most recently amyotrophic lateral sclerosis (ALS). The process of splicing is co-transcriptional and is executed by the spliceosome of which there are two types, i.e. the major and the minor spliceosome. As the name suggests, majority of the splicing in humans is executed by the major spliceosome, but there are <2% of the genes in humans that have at least one intron that it cannot splice. This minor subset of introns is spliced by the aptly named minor spliceosome. It must be noted that the efficiency of minor intron splicing is lower than its major introns and is attributed to low levels of these snRNAs. Moreover, these minor introns are found in ~450 human genes that execute various functions in the cell. Therefore, the function of the minor spliceosome including its inefficient splicing can have a profound impact on expression of these genes. Although many diseases are now being linked to aberrant minor spliceosome activity, the role of minor spliceosome in mammalian neuronal development, function and disease remain unexplored. Thus, our overall objective is to understand the role of minor introns and the minor spliceosome in neuronal development, function and disease. The focus of the current proposal is to test the model proposed for the mechanism of disease pathogenesis of ALS. Briefly, specific point mutation P525L in FUS is linked to autosomal dominant ALS. Despite having one wild-type allele, the mutant FUSP525L causes the disease. Loss of function of FUS in mice does not develop ALS-like symptoms, which suggests that the mutant FUS gains a toxic function. Normally, FUS shuttles between the nucleus and the cytoplasm, but the mutant FUSP525L cannot go into the nucleus and is found as aggregates in the cytoplasm. A recent (June 1st, 2016) report by Reber et al., in EMBO journal showed that U11 snRNA, a crucial component of the minor spliceosome was sequestered in these cytoplasmic aggregates of FUSP525L. The proposed model is that sequestration of U11 in the cytoplasmic FUSP525L aggregates results in minor spliceosome inactivation and mis-expression of minor intron-containing genes (MIGs) and disease. A direct test of this hypothesis is the ablation U11 to inactivate the minor spliceosome independent of the FUS mutation. The expectation would be that we would observe ALS-like phenotype. Serendipitously, we have already generated U11 conditional knockout (cKO) mouse to produce mouse model for MOPD1 and will now extend our studies to modeling ALS. The results of the proposed experiments will show whether inactivation of the minor spliceosome due to sequestration of U11 is the primary cause of ALS, or is the mutant FUS is required in conjunction with aberrant activity of the minor spliceosome. In all, findings of the current proposal will move the field of ALS forward in resolving the underlying molecular mechanism.
Autosomal dominant mutations in FUS result in cytoplasmic aggregates that are thought to cause ALS. Recently it was shown that U11 snRNA is sequestered to these cytoplasmic aggregates thereby resulting in the inactivation of the minor spliceosome. We will test this model through our U11 conditional knockout mouse.
