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. 2016 Nov 11;354(6313):aah5651.
doi: 10.1126/science.aah5651. Epub 2016 Oct 13.

RNA interference is essential for cellular quiescence

B Roche  1 , B Arcangioli  2 , R A Martienssen  3
Affiliations

RNA interference is essential for cellular quiescence

B Roche et al. Science. .

Abstract

Quiescent cells play a predominant role in most organisms. Here we identify RNA interference (RNAi) as a major requirement for quiescence (G0 phase of the cell cycle) in Schizosaccharomyces pombe RNAi mutants lose viability at G0 entry and are unable to maintain long-term quiescence. We identified suppressors of G0 defects in cells lacking Dicer (dcr1Δ), which mapped to genes involved in chromosome segregation, RNA polymerase-associated factors, and heterochromatin formation. We propose a model in which RNAi promotes the release of RNA polymerase in cycling and quiescent cells: (i) RNA polymerase II release mediates heterochromatin formation at centromeres, allowing proper chromosome segregation during mitotic growth and G0 entry, and (ii) RNA polymerase I release prevents heterochromatin formation at ribosomal DNA during quiescence maintenance. Our model may account for the codependency of RNAi and histone H3 lysine 9 methylation throughout eukaryotic evolution.

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Figures

Figure 1
Figure 1. RNAi mutants lose viability in G0
(A) Loss of viability at both G0-entry and during quiescence maintenance in prototroph dcr1Δ, rdp1Δ and ago1Δ mutants (n=5 biological replicates for wild-type, n=6 for dcr1Δ and rdp1Δ, n=7 for ago1Δ). ** indicates a statistically significant difference (p<0.01, t-test) between each mutant and wt for all time-points, and between time-points 24h and 15 days (B) Microscopic observation of DAPI-stained RNAi mutants reveals an increased proportion of cells retaining a rod-shape in early G0 (24h). After 15 days, while wild-type G0 cells look uniform, RNAi mutants display a variety of morphological defects and many dead refraction-negative and/or DAPI-negative cells. Upon G0-entry (C) RNAi mutants have a reduction in the increase in cell number found in wild type cells during the initial two divisions (24h G0; n=2; ** p<0.01), and (D) an increase in the number of cells staying rod-shaped (24h G0; n=2; * p<0.05, ** p<0.01). All error bars indicate standard deviation; n indicates biological replicates.
Figure 2
Figure 2. Design of a G0 suppressor screen
(A) Experimental design of the suppressor screen: a dcr1Δ population is alternated between the cell cycle (in rich medium, YES) and quiescence (in nitrogen-deprived medium, EMM-N) every 1 to 3 days. Spontaneous suppressors, by their relative fitness advantage compared to the parental strain, get enriched during the alternations. After up to 20 alternations, individual clones are isolated and re-assayed in G0 to check for suppression. (B) Summary of mutations present in 13 isolated dcr1Δ G0 suppressors showing three classes of mutants, in chromosome segregation, the CLRC/Rik1 complex and Swi6HP1, and RNA polymerase-associated factors. Each mutation in the list arose as an independent suppressor.
Figure 3
Figure 3. Quiescence maintenance of dcr1Δ results in rDNA heterochromatinization by H3K9me
(A) H3K9me2 ChIP-seq enrichment in dcr1Δ vs. wt cells. In G0 cells, but not cycling cells, dcr1Δ strongly accumulates H3K9me2 at the rDNA locus (n=2 biological replicates, cycling cell data from (31)). (B) Validation by H3K9me2 ChIP-qPCR in wild-type and dcr1Δ cycling and G0 cells shows a stronger rDNA H3K9me2 increase in G0 cells in dcr1Δ as compared to wild-type G0 cells (n≥3 biological replicates; ** p<0.01, t-test).
Figure 4
Figure 4. dcr1Δ mutants are defective in RNA polymerase I release in quiescence maintenance
(A) The main subunit of RNA polymerase I Nuc1RPA190 was tagged with a (Gly)6 linker and 3xFLAG. (B) Probe location for ChIP-qPCRs: 1: rDNA promoter, 2: 5′ETS, 3: 18S, 4: 3′ETS. (C) RNA polymerase I enrichment at the rDNA was assayed by ChIP-qPCR using anti-FLAG antibody in the nuc1-(Gly)6-FLAG background, showing an higher occupancy in wild-type than dcr1Δ mutants in cycling cells, due to the reduced growth rate of dcr1Δ, and (D) a similar occupancy in early G0 cells (n=2 biological replicates). (E, F) RNA polymerase I enrichment at 18S rDNA over time spent in G0 (2 to 8 days) shows a reduction in wild-type cells, but dcr1Δ cells fail to release RNA pol I (n≥2 biological replicates). (G) The class (iii) suppressors, dcr1Δ tbp1-D156Y and dcr1Δmed31-ins, but not the class (ii) suppressor dcr1Δclr4Δ, significantly decrease RNA polymerase I occupancy at rDNA in 8 days G0 cells. (n=2 biological replicates, * p<0.05, ** p<0.01, t-test) (H) The accumulation of H3K9me2 at rDNA repeats, assayed by ChIP-qPCR, is significantly reduced in class (iii) suppressors (n≥3 biological replicates, ** p<0.01, t-test).
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