Ullrich congenital muscular dystrophy is a severe genetically and clinically heterogeneous muscle disorder linked to collagen VI deficiency. The pathogenesis of the disease is unknown. To assess the potential role of mitochondrial dysfunction in the onset of muscle fiber death in this form of dystrophy, we studied biopsies and myoblast cultures obtained from patients with different genetic defects of collagen VI and variable clinical presentations of the disease. We identified a latent mitochondrial dysfunction in myoblasts from patients with Ullrich congenital muscular dystrophy that matched an increased occurrence of spontaneous apoptosis. Unlike those in myoblasts from healthy donors, mitochondria in cells from patients depolarized upon addition of oligomycin and displayed ultrastructural alterations that were worsened by treatment with oligomycin. The increased apoptosis, the ultrastructural defects, and the anomalous response to oligomycin could be normalized by Ca 2؉ chelators, by plating cells on collagen VI, and by treatment with cyclosporin A or with the specific cyclophilin inhibitor methylAla 3 ethylVal 4 -cyclosporin, which does not affect calcineurin activity. Here we demonstrate that mitochondrial dysfunction plays an important role in muscle cell wasting in Ullrich congenital muscular dystrophy. This study represents an essential step toward a pharmacological therapy of Ullrich congenital muscular dystrophy with cyclosporin A and methylAla 3 ethylVal 4 cyclosporin.collagen VI ͉ mitochondria ͉ permeability transition

Kinetochores attach the replicated chromosomes to the mitotic spindle and orchestrate their transmission to the daughter cells. Kinetochore–spindle binding and chromosome segregation are mediated by the multi-copy KNL1Spc105, MIS12Mtw1 and NDC80Ndc80 complexes that form the so-called KMN network. KMN–spindle attachment is regulated by the Aurora BIpl1 and MPS1Mps1 kinases. It is unclear whether other mechanisms exist that support KMN activity during the cell cycle. Using budding yeast, we show that kinetochore protein Cnn1 localizes to the base of the Ndc80 complex and promotes a functionally competent configuration of the KMN network. Cnn1 regulates KMN activity in a spatiotemporal manner by inhibiting the interaction between its complexes. Cnn1 activity peaks in anaphase and is driven by the Cdc28, Mps1 and Ipl1 kinases.

The conserved protein kinase Rio1 localizes to the cytoplasm and nucleus of eukaryotic cells. While the roles of Rio1 in the cytoplasm are well characterized, its nuclear function remains unknown. Here we show that nuclear Rio1 promotes rDNA array stability and segregation in Saccharomyces cerevisiae. During rDNA replication in S phase, Rio1 downregulates RNA polymerase I (PolI) and recruits the histone deacetylase Sir2. Both interventions ensure rDNA copy-number homeostasis and prevent the formation of extrachromosomal rDNA circles, which are linked to accelerated ageing in yeast. During anaphase, Rio1 downregulates PolI by targeting its subunit Rpa43, causing PolI to dissociate from the rDNA. By stimulating the processing of PolI-generated transcripts at the rDNA, Rio1 allows for rDNA condensation and segregation in late anaphase. These events finalize the genome transmission process. We identify Rio1 as an essential nucleolar housekeeper that integrates rDNA replication and segregation with ribosome biogenesis. A t anaphase onset, the replicated chromosomes separate and then segregate along the mitotic spindle into the daughter cells. In the budding yeast Saccharomyces cerevisiae, the locus containing the genes that encode the ribosomal RNAs (rDNA) segregates after the rest of the genome, in late anaphase [1][2][3][4] . The rDNA locus exists as a tandem-repeat array comprising B150 rDNA units containing the 35S and 5S genes, which are transcribed by RNA polymerase I (PolI) and PolIII, respectively. Processing of the 35S pre-rRNA generates 5.8S, 18S and 25S rRNA that, together with the 5S rRNA, become the catalytic backbones of each ribosome 5,6 . Only in anaphase does yeast repress rDNA transcription 4 , which allows the sister rDNA loci to condensate and segregate. PolI downregulation in anaphase is mediated by the Cdc14 phosphatase acting on PolI subunit Rpa43 (ref. 4), resulting in PolI dissociating from the 35S rDNA. The removal of PolI and the local resolution of its transcripts allow the condensin complex to bind. The latter compacts the rDNA array and recruits the DNA decatenating enzyme topoisomerase II (refs 1,3,4,7) resulting in the physical separation and subsequent segregation of the sister rDNA loci.S. cerevisiae Rio1 belongs to the atypical RIO protein kinase family whose members lack the activation loop and substrate recognition domain present in canonical eukaryotic protein kinases [8][9][10][11] . Noteworthy, the RIO kinases may act especially as ATPases as they exhibit o0.1% kinase activity in vitro [12][13][14] . Cytoplasmic Rio1 contributes to pre-40S ribosome biogenesis by promoting 20S pre-rRNA maturation and by stimulating the recycling of trans-acting factors at the pre-40S subunit, both in yeast 12,[15][16][17][18] and human cells 19,20 . Roles in the nucleus are unknown for any RIO member, either in yeast or eukaryotes beyond. Using S. cerevisiae, we now describe the first activities of Rio1 in the nucleus. Foremost, Rio1 downregulates PolI transcription through the cell cycle. In G...

The Saccharomyces cerevisiae kinase/adenosine triphosphatase Rio1 regulates rDNA transcription and segregation, pre-rRNA processing and small ribosomal subunit maturation. Other roles are unknown. When overexpressed, human ortholog RIOK1 drives tumor growth and metastasis. Likewise, RIOK1 promotes 40S ribosomal subunit biogenesis and has not been characterized globally. We show that Rio1 manages directly and via a series of regulators, an essential signaling network at the protein, chromatin and RNA levels. Rio1 orchestrates growth and division depending on resource availability, in parallel to the nutrient-activated Tor1 kinase. To define the Rio1 network, we identified its physical interactors, profiled its target genes/transcripts, mapped its chromatin-binding sites and integrated our data with yeast’s protein–protein and protein–DNA interaction catalogs using network computation. We experimentally confirmed network components and localized Rio1 also to mitochondria and vacuoles. Via its network, Rio1 commands protein synthesis (ribosomal gene expression, assembly and activity) and turnover (26S proteasome expression), and impinges on metabolic, energy-production and cell-cycle programs. We find that Rio1 activity is conserved to humans and propose that pathological RIOK1 may fuel promiscuous transcription, ribosome production, chromosomal instability, unrestrained metabolism and proliferation; established contributors to cancer. Our study will advance the understanding of numerous processes, here revealed to depend on Rio1 activity.

Reference 48 was omitted in the Methods section. This reference should be cited as follows: "GST-MPS1 (ref. 48) was expressed in E. coli BL21 (DE3)plysS cells", under the heading 'Recombinant production, in vitro phosphorylation and phospho-mapping of Cnn1' . The original reference 48 has been re-numbered as reference 49, and the new reference 48 is as follows: