Mutant ataxin‐3 alters the functional organization of the nucleus

The effect of nuclear inclusions formed by disease proteins on the functional organization of the nucleus is largely unexplored. One such protein is ataxin‐3, which is mutated in the neurological disorder Machado‐Joseph disease. By live‐cell analysis, we have demonstrated that two nuclear suborganelles known as Cajal bodies (CBs) and PML(Promyeolocytic leukemia) bodies can co‐associate for at least ten minutes with little movement. In contract, mutant ataxin‐3 with an expanded polyglutamine tract that forms nuclear inclusions associates with CBs for up to two hours. Cell biological and binding studies indicate that interactions between coilin (a CB protein) and PIASy (a PML body protein) partially link CBs to PML bodies and inclusions. Since the CB is implicated in the maturation of small nuclear ribonucleoproteins (snRNPs), we hypothesize that mutant ataxin‐3 affects nuclear functions by reducing the efficiency of pre‐mRNA splicing. To test our hypothesis, an artificial splicing substrate was constructed within plasmids expressing GFP‐Ataxin‐3 containing normal or expanded CAG repeats. The constructs were transiently transected into HeLa cells. RT‐PCR assays were employed to evaluate the splicing efficiencies within the cells expressing normal or mutant ataxin‐3. We found that the cells expressing mutant ataxin‐3 have more unspliced products than the ones that express normal ataxin‐3, consistent with our hypothesis. Further work is being done using RNase protection assay to confirm the RT‐PCR results.