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.

Friedreich’s ataxia (FRDA) is an autosomal recessive trinucleotide repeat disease with no effective therapy. Expanded GAA repeats in the first intron of the FRDA gene are thought to form unusual non-B DNA conformations that decrease transcription and subsequently reduce levels of the encoded protein, frataxin. Frataxin plays a crucial role in iron metabolism and detoxification. To discover small molecules that increase transcription through the GAA repeat region in FRDA, we have made stable cell lines containing a portion of expanded intron 1 fused to a GFP reporter. Small molecules identified using the competition dialysis method were found to increase FRDA-intron 1-reporter gene expression. One of these compounds, pentamidine, increases frataxin levels in patient cells. Thus our approach can be used to detect small molecules of potential therapeutic value in FRDA.

The eukaryotic nucleus contains a variety of dynamic structures, yet studies into the functional relationship of one type of subnuclear domain to another have been limited. For example, PML bodies and Cajal bodies associate, but the functional consequence of this association and the mediating factors are unknown. Here we report that an associated PML body/Cajal body can co-localize to an snRNA gene locus, with the Cajal body invariably situated between the PML body and the snRNA locus. Binding studies demonstrate that coilin (a Cajal body protein) directly interacts with PIASy (a PML body protein). Cell biological experiments using coilin and PIASy knockout cell lines demonstrate that interactions between coilin and PIASy account in part for the observed association of Cajal bodies with PML bodies. When the PIASy interaction region on coilin is deleted, the frequency of the association between Cajal bodies and PML bodies is reduced. These studies provide another example of coilin's role in the functional organization of the nucleus.