RNF220: a novel ubiquitin E3 ligase associated to autosomal recessive leukodystrophy with ataxia and deafness AR-LAD: is involved in nuclear lamina integrity control

Autosomal recessive leukodystrophy with ataxia and deafness (AR-LAD) is a rare white matter disease that slowly develops cerebellar ataxia and deafness. As disease progresses, the patients develop a fatal cardiomyophathy that strongly recalls that observed in patients affected by laminopathies, a group of disorders caused by mutations in A-type lamins (Lamin A/C), the major components of nuclear lamina. Linkage analysis and positional cloning experiments allowed us to identify the causative gene as RNF220 and two different homozygous missense mutations in highly conserved residues in exon 8 (c.1088G>A and c.1094G>A) (Dr. Bertini, unpublished data). RNF220 gene encodes for a RING finger ubiquitin ligase with a predicted molecular weight of 62.7 kDa highly expressed in liver, kidney and brain. Ubiquitin ligases catalyze the covalent attachment of ubiquitin to the substrate and play a key role in the ubiquitin-mediated proteolysis, however to date the role of RNF220 and the effects of pathological mutations associated with AR-LAD still need to be demonstrated. Our study has been focused on the functional characterization of RNF220, particularly we proposed to: 1) study the expression and subcellular distribution of RNF220, 2) verify if RNF220 may be involved in protein degradation via UPS and eventually determinate the spatial relationship between RNF220 and the different compartments associated with the proteasome, 3) investigate the involvement of RNF220 in the maintenance of nuclear lamina integrity, 4) investigate the possible interaction of RNF220 with molecules involved in the autophagic pathway. The results of this study have shown that: 1) RNF220 is an ubiquitin ligase with a nuclear distribution, 2) RNF220 co-localized with the 20S subunit, the catalytic core of the 26S proteasome, in nuclear speckles where is likely involved in protein degradation via the ubiquitin system, 3) RNF220 is a key player in the maintenance of nuclear integrity as shown by alteration/delocalization of Lamin A/C in AR-LAD fibroblast and in COS-1 cells ectopically expressing RNF220, 3) RNF220 forms a complex with Ambra1, a novel autophagic molecule, and co-localized with it in the nucleus, suggesting a RNF220 implication in autophagic pathway. Taken together, our results suggest that RNF220, the defective protein of AR-LAD is a key player of a novel degradation complex at nuclear level, connecting autophagic and proteosomal pathways and playing a crucial role in maintaining nuclear integrity.