Pathogenic FUS mutations retain spliceosomal snRNPs in the cytoplasm

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, a nuclear protein which has been implicated in alternative splicing or mRNA localisation, 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. Based on our previous findings, showing that FUS and SMN associated with each other, and that FUS binds to Sm-snRNPs, we investigated whether aggregation-prone FUS mutants might interfere with the localisation and biogenesis of snRNPs. Indeed, in transfected mouse motoneuronal-like NSC34 cells, mutations in FUS do not affect association to the snRNPs. However, mutant FUS and SMN co-localise in multiple cytoplasmic aggregates, and snRNAs are retained into the cytoplasm. As a result, alterations in the alternative splicing of a reporter plasmid are observed. Alternative splicing defects as well as alterations in SMN sub-cellular distribution are also observed in cells depleted of FUS by RNA interference, suggesting that aggregated mutant FUS might disturb the nuclear import of SMN, thus reducing the availability of functional snRNPs in the nucleus. Experiments are undergoing to further characterise this issue. Overall, our observations indicate that FUS mutations and genetic depletion of SMN converge onto the same pathway, i.e. alternative splicing changes, that might represent a unifying theme in the FUS-related ALS and SMA.