RNA interference regulates the cell cycle checkpoint through the RNA export factor, Ptr1, in fission yeast

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Abstract

Ago1, an effector protein of RNA interference (RNAi), regulates heterochromatin silencing and cell cycle arrest in fission yeast. However, the mechanism by which Ago1 controls cell cycle checkpoint following hydroxyurea (HU) treatment has not been elucidated. In this study, we show that Ago1 and other RNAi factors control cell cycle checkpoint following HU treatment via a mechanism independent of silencing. While silencing requires dcr1+, the overexpression of ago1+ alleviated the cell cycle defect in dcr1Δ. Ago1 interacted with the mRNA export factor, Ptr1. The ptr1-1 mutation impaired cell cycle checkpoint but gene silencing was unaffected. Genetic analysis revealed that the regulation of cell cycle checkpoint by ago1+ is dependent on ptr1+. Nuclear accumulation of poly(A)+ RNAs was detected in mutants of ago1+ and ptr1+, suggesting there is a functional link between the cell cycle checkpoint and RNAi-mediated RNA quality control.

Highlights

► RNAi is linked to the cell cycle checkpoint in fission yeast. ► Ptr1 co-purifies with Ago1. ► The ptr1-1 mutation impairs the checkpoint but does not affect gene silencing. ► ago1+ and ptr1+ regulate the cell cycle checkpoint via the same pathway. ► Mutations in ago1+ and ptr1+ lead to the nuclear accumulation of poly(A)+ RNAs.

Introduction

The RNA interference (RNAi) is a conserved silencing mechanism mediated by small interfering RNAs (siRNAs) [1]. In fission yeast (Schizosaccharomyces pombe), the RNAi mechanism is involved in assembly of centromeric heterochromatin, which depends on the processing of centromeric noncoding RNAs into siRNAs by Dcr1 [2], [3]. Double-stranded siRNAs are processed to single-stranded siRNAs by two Ago1-containing complexes, the Argonaute siRNA chaperone complex (ARC) (consisting of Ago1, Arb1, and Arb2) and the RNA-induced transcriptional silencing complex (RITS) (consisting of Ago1, Chp1, and Tas3) [4], [5]. RITS associates with nascent transcripts and chromatin through siRNAs and methylation of histone H3 at Lys 9 (H3K9) [6]. This RNA binding of RITS recruits the Rdp1-containing RNA-directed RNA polymerase complex (RDRC) and the Clr4-Rik1 complex (CLRC), a H3K9 methylase complex, to facilitate siRNA amplification and H3K9 methylation, respectively [6], [7]. Swi6, a heterochromatin protein 1 (HP1) homolog expressed in fission yeast, is recruited following H3K9 methylation, causing the degradation of heterochromatin-associated transcripts [8], [9].

RNA degradation at heterochromatin is mediated by RNA surveillance factors [10], [11]. Recently, Clr4 and RITS were found to interact with Mlo3, a protein related to mRNA quality control and export, to suppress antisense RNA at heterochromatin and euchromatin regions [11]. In budding yeast, Tom1, a HECT-type ubiquitin ligase, ubiquitinates Yra1, a homolog of Mlo3/Aly/REF, to promote dissociation of mRNA ribonucleoprotein (mRNP) and mRNA degradation prior to mRNA export [12]. Ptr1, a homolog of Tom1 in fission yeast, is an RNA export factor [13]. Whether Ptr1 plays a role in RNA surveillance remains uncertain; however the ptr1-1 mutant exhibits nuclear accumulation of poly(A)+ RNAs similar to the phenotype observed in mutants with defects in RNA export and/or RNA surveillance [13].

Dcr1 and Ago1 are also involved in cell cycle arrest induced by hydroxyurea (HU) treatment [14], which inhibits DNA replication and triggers S-phase checkpoint [15], [16], [17]. In the previous study, cell cycle arrest was unaffected in the rdp1 deletion mutant, suggesting that Ago1 and Dcr1 have functionally diverged from Rdp1 to control cell cycle events [14]. However, the mechanism by which RNAi contributes to the cell cycle checkpoint has not been elucidated. In this study, we characterized RNAi components and provide evidence of a functional linkage between RNAi, RNA quality control and the cell cycle checkpoint.

Section snippets

Media, yeast strains and plasmids

Fission yeast media and genetic methods have been described previously [18]. The S. pombe strains used in this study are listed in Supplementary Table 1. Genotyping was carried out by Southern analysis and PCR analysis of both wild-type and mutant alleles using genomic DNA as the template. The plasmids used in this study were the control vector, pREP1, and the ago1+ plasmid, pREP1-3flag-ago1 [4].

Checkpoint assay

HU-induced cell cycle checkpoint in yeast was analyzed by calculating the septation index as

Involvement of RNAi components in the regulation of cell cycle checkpoint following HU treatment

In response to the ribonucleotide reductase inhibitor, HU, checkpoint kinases prevent passage through the S phase checkpoint by inhibiting Cdc2, a cyclin-dependent kinase important for cell cycle control [15], [16], [17]. In fission yeast, activation of the S phase checkpoint by HU treatment delays passage through S phase into mitosis and results in the accumulation of elongated cells without septum. Since a previous study proposed that cell cycle arrest can be controlled independently of rdp1+

Acknowledgments

We thank Drs. Danesh Moazed, Tomoko Ando and Tokio Tani for generating the yeast strains and plasmids. We also thank Dr. Yumiko Kurokawa and all members of the Division of Cytogenetics for helpful discussions. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T. I. and N. I.).

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    Present address: Genome Informatics Laboratory, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540, Japan.

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