Investigation of the role of the survival of motor neurons protein in the biogenesis of small nucleolar ribonucleoprotein complexes
Finch, Sarah Emily Whitehead
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Deletion or mutation of the SMN1 (survival of motor neurons) gene causes the fatal neuromuscular disease spinal muscular atrophy, SMA. The SMN protein is important in small nuclear ribonucleoprotein (snRNP) assembly. We discovered the interaction of SMN with core protein components of the two major families of small nucleolar RNPs, fibrillarin and GAR1,suggesting that SMN may also function in the assembly of small nucleolar RNPs. Here we present results that indicate that the interaction of SMN with GAR1 is mediated by the Tudordomain of SMN. Single point mutations within the Tudor domain, including a spinal muscular atrophy patient mutation, impair the interaction of SMN with GAR1. Furthermore, we find that either of the two arginine/glycine-rich domains of GAR1 can provide for interaction with SMN,but removal of both results in loss of the interaction. Finally, we have found that unlike the interaction of SMN with the Sm snRNP proteins, interaction with GAR1 and fibrillarin is not enhanced by arginine dimethylation. Our results argue against post-translational arginine dimethylation as a general requirement for SMN recognition of proteins bearing arginine/glycine-rich domains.To address the hypothesis that SMN assists in the assembly of snoRNPs, we utilized RNA interference (RNAi) assays to reduce in vivo levels of the SMN protein and subsequently performed immunoprecipitations with a trimethylguanosine (TMG) cap antibody, specific for the cap modification found on most snRNAs and a few snoRNAs. We found that all newly-made major U snRNAs demonstrated decreased accumulation when SMN was substantially reduced.Additionally, all three newly-made TMG-capped snoRNAs, U3, U8, and U13 revealed a similar reduction under conditions of SMN knockdown. Decreased accumulation of newly-made TMG-capped snoRNAs may indicate a RNP assembly defect, as RNA molecules unprotected by proteins are more susceptible to rapid degradation by nucleases. Therefore, the reduced accumulation of newly-made TMG-capped snoRNAs when SMN is present at very low levels provides evidence that SMN chaperones the assembly of snoRNPs, as well as snRNPs.Elucidating novel roles for the SMN protein in vivo is vital to ultimately discovering and implementing a cure for the deadly disease, SMA.