The ribosomal DNA metaphase loop of Saccharomyces cerevisiae gets condensed upon heat stress in a Cdc14-independent TORC1-dependent manner

Chromosome morphology in Saccharomyces cerevisiae is only visible at the microscopic level in the ribosomal DNA array (rDNA). The rDNA has been thus used as a model to characterize condensation and segregation of sister chromatids in mitosis. It has been established that the metaphase structure (“loop”) depends, among others, on the condensin complex; whereas its segregation also depends on that complex, the Polo-like kinase Cdc5 and the cell cycle master phosphatase Cdc14. In addition, Cdc14 also drives rDNA hypercondensation in telophase. Remarkably, since all these components are essential for cell survival, their role on rDNA condensation and segregation was established by temperature-sensitive (ts) alleles. Here, we show that the heat stress (HS) used to inactivate ts alleles (25 ºC to 37 ºC shift) causes rDNA loop condensation in metaphase-arrested wild type cells, a result that can also be mimicked by other stresses that inhibit the TORC1 pathway. Because this condensation might challenge previous findings with ts alleles, we have repeated classical experiments of rDNA condensation and segregation, yet using instead auxin-driven degradation alleles (aid alleles). We have undertaken the protein degradation at lower temperatures (25 ºC) and concluded that the classical roles for condensin, Cdc5, Cdc14 and Cdc15 still prevailed. Thus, condensin degradation disrupts rDNA higher organization, Cdc14 and Cdc5 degradation precludes rDNA segregation and Cdc15 degradation still allows rDNA hypercompaction in telophase. Finally, we provide direct genetic evidence that this HS-mediated rDNA condensation is dependent on TORC1 but, unlike the one observed in anaphase, is independent of Cdc14.     

Cdc14 and condensin control the dissolution of cohesin-independent chromosome linkages at repeated DNA

Chromosome segregation is triggered by the cleavage of cohesins by separase. Here we show that in budding yeast separation of the ribosomal DNA (rDNA) and telomeres also requires Cdc14, a protein phosphatase known for its role in mitotic exit. Cdc14 shares this role with the FEAR network, which activates Cdc14 during early anaphase, but not the mitotic exit network, which promotes Cdc14 activity during late anaphase. We further show that CDC14 is necessary and sufficient to promote condensin enrichment at the rDNA locus and to trigger rDNA segregation in a condensin-dependent manner. We propose that Cdc14 released by the FEAR network mediates the partitioning of rDNA by facilitating the localization of condensin thereto. This dual role of the FEAR network in initiating mitotic exit and promoting chromosome segregation ensures that exit from mitosis is coupled to the completion of chromosome segregation.     

Spindle-independent condensation-mediated segregation of yeast ribosomal DNA in late anaphase

Mitotic cell division involves the equal segregation of all chromosomes during anaphase. The presence of ribosomal DNA (rDNA) repeats on the right arm of chromosome XII makes it the longest in the budding yeast genome. Previously, we identified a stage during yeast anaphase when rDNA is stretched across the mother and daughter cells. Here, we show that resolution of sister rDNAs is achieved by unzipping of the locus from its centromere-proximal to centromere-distal regions. We then demonstrate that during this stretched stage sister rDNA arrays are neither compacted nor segregated despite being largely resolved from each other. Surprisingly, we find that rDNA segregation after this period no longer requires spindles but instead involves Cdc14-dependent rDNA axial compaction. These results demonstrate that chromosome resolution is not simply a consequence of compacting chromosome arms and that overall rDNA compaction is necessary to mediate the segregation of the long arm of chromosome XII.     

Drosophila ribosomal RNA genes function as an X-Y pairing site during male meiosis

In Drosophila melanogaster males, the sex chromosomes pair during meiosis in the centric X heterochromatin and at the base of the short arm of the Y (YS), in the vicinity of the nucleolus organizers. X chromosomes deficient for the pairing region segregate randomly from the Y. In this report we show that a single ribosomal RNA (rRNA) gene stimulates X-Y pairing and disjunction when inserted onto a heterochromatically deficient X chromosome by P element-mediated transformation. We also show that insert-containing X chromosomes pair at the site of insertion, that autosomal rDNA inserts do not affect X-Y pairing or disjunction, and that the strength of an X pairing site is proportional to the dose of ectopic rRNA genes. These results demonstrate that rRNA genes can promote X-Y pairing and disjunction and imply that the nucleolus organizers function as X-Y pairing sites in wild-type Drosophila males.     

Isolation and characterization of Chinese hamster ribosomal DNA clones

We have examined the ribosomal RNA (rRNA) genes of the Chinese hamster ovary (CHO) cell line. A partial EcoRI library of genomic CHO DNA was prepared using lambda Charon-4A. We isolated two recombinants containing the region transcribed as 45S pre-rRNA and 13 kb of external spacer flanking 5' and 3' to the transcribed region. These sequences show restriction site homology with the vast majority of the genomic sequences complementary to rRNA. In addition to this form of rDNA, Southern blot analysis of EcoRI-cut CHO genomic DNA reveals numerous minor fragments ranging from 2 to 19 kb which are complementary to 18S rRNA. We isolated one clone which contains the 18S rRNA gene and sequences 5' which appear to contain length heterogeneity within the non-transcribed spacer region. We have nine additional cloned EcoRI fragments in which the homology with 18S rRNA is limited to a 0.9-kb EcoRI-HindIII fragment. This EcoRI-HindIII fragment is present in each of the cloned EcoRI fragments, and is flanked on both sides by apparently nonribosomal sequences which bear little restriction site homology with each other or the major cloned rDNA repeat.     

The structure of a single unit of ribosomal RNA gene (rDNA) including intergenic subrepeats in the Australian bulldog ant Myrmecia croslandi (Hymenoptera: Formicidae)

A complete single unit of a ribosomal RNA gene (rDNA) of M. croslandi was sequenced. The ends of the 18S, 5.8S and 28S rRNA genes were determined by using the sequences of D. melanogaster rDNAs as references. Each of the tandemly repeated rDNA units consists of coding and non-coding regions whose arrangement is the same as that of D. melanogaster rDNA. The intergenic spacer (IGS) contains, as in other species, a region with subrepeats, of which the sequences are different from those previously reported in other insect species. The length of IGSs was estimated to be 7-12 kb by genomic Southern hybridization, showing that an rDNA repeating unit of M. croslandi is 14-19 kb-long. The sequences of the coding regions are highly conserved, whereas IGS and ITS (internal transcribed spacer) sequences are not. We obtained clones with insertions of various sizes of R2 elements, the target sequence of which was found in the 28S rRNA coding region. A short segment in the IGS that follows the 3' end of the 28S rRNA gene was predicted to form a secondary structure with long stems.     

Cdc14 protein phosphatase and topoisomerase II mediate rDNA dynamics and nucleophagic degradation of nucleolar proteins after TORC1 inactivation

Nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase elicits nucleophagy degrading nucleolar proteins in budding yeast. After TORC1 inactivation, nucleolar proteins are relocated to sites proximal to the nucleus–vacuole junction (NVJ), where micronucleophagy occurs, whereas ribosomal DNA (rDNA encoding rRNA) escapes from the NVJ. Condensin-mediated rDNA condensation promotes the repositioning and nucleophagic degradation of nucleolar proteins. However, the molecular mechanism of TORC1 inactivation-induced chromosome condensation is still unknown. Here, we show that Cdc14 protein phosphatase and topoisomerase II (Topo II), which are engaged in rDNA condensation in mitosis, facilitate rDNA condensation after TORC1 inactivation. rDNA condensation after rapamycin treatment was compromised in cdc14-1 and top2-4 mutants. In addition, the repositioning of rDNA and nucleolar proteins and nucleophagic degradation of nucleolar proteins were impeded in these mutants. Furthermore, Cdc14 and Topo II were required for the survival of quiescent cells in prolonged nutrient-starved conditions. This study reveals that these factors are critical for starvation responses.     

Nucleolar segregation lags behind the rest of the genome and requires Cdc14p activation by the FEAR network

In order to transmit a full genetic complement cells must ensure that all chromosomes are accurately split and distributed during anaphase. Chromosome XII in S. cerevisiae contains the site of nucleolar assembly, a 1-2Mb array of rDNA genes named RDN1. Cdc14p is a conserved phosphatase, essential for anaphase progression and mitotic exit, which is kept inactive at the nucleolus until mitosis. In early anaphase, the FEAR network (Cdc Fourteen Early Anaphase Release) promotes the transient and partial release of Cdc14p from the nucleolus. The putative role of Cdc14p released by the FEAR network is thought to be the stimulation of full Cdc14p release by activation of the GTPase-driven signaling cascade (the Mitotic Exit Network or MEN) that ensures mitotic exit. Here, we show that nucleolar segregation is spatially separated and temporally delayed from the rest of the genome. Nucleolar segregation occurs during mid-anaphase and coincides with the FEAR release of Cdc14p. Inactivation of FEAR delays nucleolar segregation until late anaphase, demonstrating that one function of the FEAR network is to promote segregation of repetitive nucleolar chromatin during mid-anaphase.     

Cdc14 phosphatase induces rDNA condensation and resolves cohesin-independent cohesion during budding yeast anaphase

At anaphase onset, the protease separase triggers chromosome segregation by cleaving the chromosomal cohesin complex. Here, we show that cohesin destruction in metaphase is sufficient for segregation of much of the budding yeast genome, but not of the long arm of chromosome XII that contains the rDNA repeats. rDNA in metaphase, unlike most other sequences, remains in an undercondensed and topologically entangled state. Separase, concomitantly with cleaving cohesin, activates the phosphatase Cdc14. We find that Cdc14 exerts two effects on rDNA, both mediated by the condensin complex. Lengthwise condensation of rDNA shortens the chromosome XII arm sufficiently for segregation. This condensation depends on the aurora B kinase complex. Independently of condensation, Cdc14 induces condensin-dependent resolution of cohesin-independent rDNA linkage. Cdc14-dependent sister chromatid resolution at the rDNA could introduce a temporal order to chromosome segregation.