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. 2009 Mar 12;458(7235):219-22.
doi: 10.1038/nature07652. Epub 2009 Jan 21.

Cdc14 inhibits transcription by RNA polymerase I during anaphase

Andrés Clemente-Blanco  1 , María Mayán-Santos, David A Schneider, Félix Machín, Adam Jarmuz, Herbert Tschochner, Luis Aragón
Affiliations

Cdc14 inhibits transcription by RNA polymerase I during anaphase

Andrés Clemente-Blanco et al. Nature. .

Abstract

Chromosome condensation and the global repression of gene transcription are features of mitosis in most eukaryotes. The logic behind this phenomenon is that chromosome condensation prevents the activity of RNA polymerases. In budding yeast, however, transcription was proposed to be continuous during mitosis. Here we show that Cdc14, a protein phosphatase required for nucleolar segregation and mitotic exit, inhibits transcription of yeast ribosomal genes (rDNA) during anaphase. The phosphatase activity of Cdc14 is required for RNA polymerase I (Pol I) inhibition in vitro and in vivo. Moreover Cdc14-dependent inhibition involves nucleolar exclusion of Pol I subunits. We demonstrate that transcription inhibition is necessary for complete chromosome disjunction, because ribosomal RNA (rRNA) transcripts block condensin binding to rDNA, and show that bypassing the role of Cdc14 in nucleolar segregation requires in vivo degradation of nascent transcripts. Our results show that transcription interferes with chromosome condensation, not the reverse. We conclude that budding yeast, like most eukaryotes, inhibit Pol I transcription before segregation as a prerequisite for chromosome condensation and faithful genome separation.

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Figures

Figure 1
Figure 1. Transcription is inhibited in budding yeast during anaphase
a, [3H]uracil incorporation into total RNA in wild-type yeast cells released from a G1 block. The mean (n = 3) and s.d. are shown. b, Primary rRNA transcript levels in cells released from a G1 block. qPCR using primers to the internal transcribed sequence 1 (ITS1) (Supplementary Fig. 1) were used to determine levels of intact 35S rRNA transcript. An average (n = 3) and s.d. are shown. c, ChIP analysis of Rpa43–9myc binding to ribosomal repeats in exponentially growing cells. An average (n = 2) and s.d. are shown. d, ChIP analysis of Rpa43–9myc binding to the 5′ end region of the 35S rRNA gene in wild-type yeast cells released from a G1 block. An average (n = 2) and s.d. are shown.
Figure 2
Figure 2. Cdc14 phosphatase inhibits rRNA transcription and prevents binding of Pol I subunits to ribosomal genes
a, Primary rRNA transcript levels in metaphase-arrested cells with expression of wild-type CDC14 or phosphatase-dead CDC14-C/S. b, Primary rRNA transcript levels upon expression of a stabilized form of cyclinB2 (CLB2-DK) in the presence of a wild-type CDC14 or temperature-sensitive mutant cdc14-1. c, Localization of Rpa43 and the nucleolar marker Cfi1 in CLB2-DK-expressing cells with wild-type CDC14 or cdc14-1. Scale bar 5 µm. d, ChIP analysis of Rpa43 binding to 35S DNA in cells before (left) and after (right) expression of CLB2-DK in the presence of a wild-type CDC14 or cdc14-1. An average (n = 2) and s.d. are shown.
Figure 3
Figure 3. Cdc14 inhibits RNA Pol I transcription in vitro
a, Multiround in vitro RNA Pol I transcription assays varying the relative amount of purified wild-type Cdc14 or phosphatase-dead Cdc14 (Cdc14-C/S) added to the reaction (concentration, 160 µgml−1 of Cdc14 or Cdc14-C/S). Addition of Cdc14, but not Cdc14-C/S, inhibits RNA Pol I transcription. An average (n = 2) and s.d. are shown. b, Multiround in vitro RNA Pol I transcription assays varying the relative amount of Cfi1 added (concentration as in ref. 19) to reactions containing wild-type Cdc14, phosphatase-dead Cdc14-C/S or buffer only. a.u., arbitrary units.
Figure 4
Figure 4. Condensin localization to rDNA and nucleolar segregation requires removal of rRNA transcripts
a, Localization of the condensin subunit Smc4 in cdc14-1 metaphase-arrested cells expressing the RntA RNase (+RntA) or not (−RntA). Smc4 locates throughout the nucleoplasm (nuclear) or at the nucleolus (nucleolar). Representative micrographs and quantification of the experiment are shown. Scale bar, 5 µm. Two hundred cells were scored per time point. b, rDNA segregation in the cohesin mutant mcd1-1 arrested in metaphase at 37 °C and expressing CDC14, RntA RNase, TopChlv or empty vector. Two hundred cells were scored per time point. c, rDNA segregation in the double cohesin condensin mutant mcd1-1/brn1-60 arrested in metaphase at 37 °C and expressing CDC14, RntA or empty vector. Two hundred cells were scored per time point. d, Schematic representation of CDC14-dependent rRNA transcription inhibition during yeast anaphase.
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