Many S. cerevisiae genes encode antisense transcripts some of which are unstable and degraded by the exosome component Rrp6. Loss of Rrp6 results in the accumulation of long PHO84 antisense RNAs and repression of sense transcription through PHO84 promoter deacetylation. We used single molecule resolution fluorescent in situ hybridization (smFISH) to investigate antisense-mediated transcription regulation. We show that PHO84 antisense RNA acts as a bimodal switch, where continuous low frequency antisense transcription represses sense expression within individual cells. Surprisingly, antisense RNAs do not accumulate at the PHO84 gene but are exported to the cytoplasm. Furthermore, loss of Rrp6, rather than stabilizing PHO84 antisense RNA, promotes antisense elongation by reducing its early transcription termination by Nrd1-Nab3-Sen1. These observations suggest that PHO84 silencing results from constant low frequency antisense transcription through the promoter rather than its static accumulation at the repressed gene.

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.

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Yra1 is an mRNA export adaptor involved in mRNA biogenesis and export in S . cerevisiae . Yra1 overexpression was recently shown to promote accumulation of DNA:RNA hybrids favoring DNA double strand breaks (DSB), cell senescence and telomere shortening, via an unknown mechanism. Yra1 was also identified at an HO-induced DSB and Yra1 depletion causes defects in DSB repair. Previous work from our laboratory showed that Yra1 ubiquitination by Tom1 is important for mRNA export. Here, we found that Yra1 is also ubiquitinated by the SUMO-targeted ubiquitin ligases Slx5-Slx8 implicated in the interaction of irreparable DSB with nuclear pores. We further show that Yra1 binds an HO-induced irreparable DSB in a process dependent on resection. Importantly, a Yra1 mutant lacking the evolutionarily conserved C-box is not recruited to an HO-induced irreparable DSB and becomes lethal under DSB induction in a HO-cut reparable system. Together, the data provide evidence that Yra1 plays a crucial role in DSB repair via homologous recombination. While Yra1 sumoylation and/or ubiquitination are dispensable, the Yra1 C-box region is essential in this process.

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Equal contribution + Corresponding author: Francoise.Stutz@unige.ch 1 ABSTRACT (168 words) 2 3Yra1 is an mRNA export adaptor involved in mRNA biogenesis and 4 export in S. cerevisiae. Yra1 overexpression was recently shown to promote 5 accumulation of DNA:RNA hybrids favoring DNA double strand breaks (DSB), 6 cell senescence and telomere shortening, via an unknown mechanism. Yra1 7 was also identified at an HO-induced DSB and Yra1 depletion causes defects 8 in DSB repair. Previous work from our laboratory showed that Yra1 9 ubiquitination by Tom1 is important for mRNA export. Interestingly, we found 10 that Yra1 is also ubiquitinated by the SUMO-targeted ubiquitin ligases Slx5-11 Slx8 implicated in the interaction of irreparable DSB with nuclear pores. Here 12 we show that Yra1 binds an HO-induced irreparable DSB. Importantly, a Yra1 13 mutant lacking the evolutionarily conserved C-box is not recruited to an HO-14 induced irreparable DSB and becomes lethal under DSB induction in a HO-15 cut reparable system. Together, the data provide evidence that Yra1 plays a 16 crucial role in DSB repair via homologous recombination. Unexpectedly, while 17 the Yra1 C-box is essential, Yra1 sumoylation and/or ubiquitination are 18 dispensable in this process. 19 20 21 Keywords: Yra1, HO endonuclease cut, genome instability, DSB repair, 22 homologous recombination. 49 export defect although this domain is not implicated in Mex67 interaction nor 50 RNA binding in vitro, suggesting that it may contribute to Yra1 function by 51 ensuring optimal folding of the protein. Loss of the highly conserved Yra1 C-52 box (yra1(1-210) mutant) does not cause an obvious poly(A)+ mRNA export 53 defect, but it is required for optimal growth (7). This observation is consistent 54 with the fact that the C-box does not play a major role in Mex67 or RNA 55 binding and suggests that this highly conserved 16 amino acids sequence 56 may be important for another aspect of Yra1 function. 57 Different layers of regulations have been shown to modulate Yra1 levels 58 and function in mRNA biogenesis. We have previously shown that Yra1 59 ubiquitination by the E3 ligase Tom1 displaces Yra1 from messenger 60 ribonucleoparticles (mRNPs) as a quality control signal for correctly processed 61 mRNP prior to export into the cytoplasm (8). Another important feature for Yra1 62 regulation is that the YRA1 gene harbors the second largest intron (776 nt) in 63 the S. cerevisiae genome, containing a non-canonical branchpoint sequence 64 (BS, gACUAAC) after a long first exon (300 nt). An excess of Yra1 protein 65 prevents YRA1 pre-mRNA splicing and promotes export of the unspliced 66 transcript into the cytoplasm where it is degraded by the 5' to 3' decay pathway 67 ( 4, 9). It has been reported previously that the yra1∆intron mutant shows Yra1 68 protein overexpression (7) that is toxic for cell growth (10, 11) and impairs 69 poly(A)+ RNA export (12, 13). The presence of the YRA1 intron is important to 70 maintain optimal Yra1 protein levels through Yra1 auto-regulation a...