Research Article

RNA interference is essential for cellular quiescence

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Science  11 Nov 2016:
Vol. 354, Issue 6313, aah5651
DOI: 10.1126/science.aah5651

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RNAi soothes the path to quiescence

Cells, such as those in stem cell niches and immunity memory cells, can exist in a nondividing, quiescent state, from which they can be aroused with the appropriate signal. RNA interference (RNAi) is an important epigenetic pathway in many organisms. Roche et al. found that in fission yeast, RNAi was an essential regulator of the quiescent state. RNAi promoted proper chromosome segregation during entry into quiescence. It also prevented inappropriate silencing of the ribosomal DNA during quiescence.

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Structured Abstract

INTRODUCTION

Quiescence is defined as the state of a nondividing (G0) cell that is still metabolically active and able to return to the cell cycle with full viability. Quiescent cells, from microbial resting structures to memory lymphocytes and stem cells, have predominant roles in the life cycles of many organisms. The ability of quiescent cells, given the appropriate environment or signals, to return to the cell cycle highlights their reversibility and plasticity. Despite their importance, the molecular mechanisms underlying quiescence entry, maintenance, and exit are not yet well understood.

RATIONALE

Quiescence can be viewed as a state of epigenetic plasticity. We therefore reasoned that specific epigenetic pathways may be involved in the control of the switch between quiescence and growth, as well as in the maintenance of the quiescent state. We investigated this proposition in the fission yeast Schizosaccharomyces pombe, a model organism for both epigenetics and quiescence. In this species, quiescence can be induced by a simple environmental signal, namely, nitrogen deprivation; wild-type cells retain viability for over 2 weeks and return to the cell cycle when put back in rich medium. S. pombe centromeres resemble those of higher eukaryotes, with heterochromatin at the pericentromeric regions marked by histone H3 lysine 9 (H3K9) methylation, and S. pombe possesses many conserved epigenetic pathways, such as RNA interference (RNAi).

RESULTS

In this study, we identify RNAi as a major requirement for quiescence. RNAi null mutants lose viability in both entry into and long-term maintenance of quiescence. In contrast, heterochromatin mutants do not show the maintenance defect, indicating that the mechanism by which RNAi regulates quiescence supersedes its known role in guiding heterochromatin formation at centromeres. To understand the molecular mechanism responsible for the G0 defect, we designed a genetic screen, based on iterative transitions between the cell cycle and quiescence, which allows the progressive enrichment and isolation of spontaneous suppressors of G0 defects. Parallelization of this screen can be used to obtain a large number of independent suppressors. We characterized 13 independent suppressors of G0 defects in cells lacking Dicer (dcr1Δ), which mapped to genes involved in chromosome segregation (class i), in heterochromatin formation [the silencing cryptic loci repression complex (CLRC) and Swi6HP1; (class ii)], and in RNA polymerase–associated factors (class iii). We found that class i suppressors rescue the mitotic defects caused by the loss of pericentromeric heterochromatin, and that a similar suppression can be observed by restoring this heterochromatin independently of RNAi. Proper segregation is important for mitosis at the transition between the cell cycle and quiescence. In contrast, class ii suppressors are especially important for quiescence maintenance. We found that during quiescence maintenance, dcr1Δ cells are defective in the release of RNA polymerase I (pol I). This leads to an accumulation of H3K9me at the ribosomal DNA (rDNA), which causes cell death, without the repeat recombination found in cycling cells. H3K9 methylation mutants or H3K9R histone substitution mutants bypass rDNA heterochromatinization and therefore restore the viability of RNAi mutants in G0. Class iii suppressors act upstream of this heterochromatinization by limiting the accumulation of RNA pol I at the rDNA.

CONCLUSION

We propose a model in which RNAi promotes RNA polymerase release in both cycling and quiescent cells: (i) RNA pol II release mediates heterochromatin formation at centromeres, allowing proper chromosome segregation during mitotic growth and G0 entry, and (ii) RNA pol I release prevents heterochromatin formation at rDNA during quiescence maintenance. The dual role of RNAi in the active cell cycle and in quiescence may therefore stem from a similar mechanism of RNA polymerase release, with a different consequence for heterochromatin formation depending on the genomic context and cellular state. Throughout eukaryotic evolution, RNAi and H3K9 methylation are always found together, indicating a codependency. Our model provides an explanation for this phenomenon, because the loss of RNAi would result in a strong selective pressure against H3K9 methylation in order to maintain rDNA epigenetic stability.

The dual role of RNA interference in the S and G0 (quiescence) phases of the cell cycle is based on RNA polymerase release.

The release of RNA pol II allows heterochromatin formation at centromeres, and the release of RNA pol I from rDNA avoids heterochromatin overaccumulation during quiescence maintenance. In the lower part of the figure, red and blue crooked lines represent small RNAs.

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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