Mislocalised FUS mutants stall spliceosomal snRNPs in the cytoplasm

Background: Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are associated with defects in proteins involved in RNA metabolism (TDP43 and FUS, and SMN, respectively). SMN, the causative factor in SMA, is crucial for the biogenesis of the spliceosomal snRNPs. FUS forms cytoplasmic aggregates, as a consequence of disturbed nuclear import due to disease-causing mutations. It is extremely likely that the cytoplasmic aggregates are cytotoxic because they trap important factors; the nature of these factors, however, remains to be elucidated. Objectives: We investigated whether mutant FUS and reduced SMN might disturb the same pathway, namely the biogenesis of snRNPs, looking for mutual interactions and disturbed expression of these factors. Methods: Mouse motoneuronal NSC34 cells transfected with wild-type or mutant FUS were used as cellular models of ALS. qPCR coupled to immunoprecipitation, as well as to immunofl uorescence analysis and fl uorescence in situ hybridisation (FISH), was used to assess expression, FUS binding, and subcellular distribution of the snRNPs. A splice reporter plasmid including the exon 7 of human SMN2 was used to monitor alternative splicing variations by FUS. Results: To test the hypothesis that FUS might be involved in snRNP biosynthesis, we checked for a physical association with SMN, the catalyst of snRNP assembly, and with the snRNAs themselves. We found that FUS and SMN associated with each other, and FUS bound to Sm-snRNPs. Mutations in FUS did not affect association with the snRNPs, but caused their retention in the cytoplasm. Since the total snRNP concentration did not change, this reduced the availability of functional snRNPs in the nucleus. As a result, alterations in the alternative splicing of a reporter plasmid were observed. Discussion and conclusion: Our results suggest that aggregated FUS may indeed be toxic because they sequester spliceosomal snRNPs in the cytoplasm, lowering their availability in the nucleus and thus leading to changes in alternative splicing patterns. In this sense, the FUS mutations and genetic depletion of SMN interfere with the same pathway, which might represent a unifying theme in the FUS-related ALS and SMA. Acknowledgments: This work was supported by ARiSLA (MTC, MC) SMA Europe (TA), and CARIPLO foundation (MTC and TA).