Rnai Pathway Participates Into Chromosome Segregation In
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OPEN Citation: Cell Discovery (2015) 1, 15029; doi:10.1038/celldisc.2015.29 ARTICLE www.nature.com/celldisc RNAi pathway participates in chromosome segregation in mammalian cells Chuan Huang, Xiaolin Wang, Xu Liu, Shuhuan Cao, Ge Shan School of Life Sciences, CAS Key Laboratory of Brain Function and Disease, University of Science and Technology of China, Hefei, China The RNAi machinery is a mighty regulator in a myriad of life events. Despite lines of evidence that small RNAs and components of the RNAi pathway may be associated with structure and behavior of mitotic chromosomes in diverse organisms, a direct role of the RNAi pathway in mammalian mitotic chromosome segregation remains elusive. Here we report that Dicer and AGO2, two central components of the mammalian RNAi pathway, participate in the chromosome segregation. Knockdown of Dicer or AGO2 results in a higher incidence of chromosome lagging, and this effect is independent from microRNAs as examined with DGCR8 knockout cells. Further investigation has revealed that α-satellite RNA, a noncoding RNA derived from centromeric repeat region, is managed by AGO2 under the guidance of endogenous small interference RNAs (ASAT siRNAs) generated by Dicer. Furthermore, the slicer activity of AGO2 is essential for the chromosome segregation. Level and distribution of chromosome-associated α-satellite RNA have crucial regulatory effect on the localization of centromeric proteins such as centromere protein C1 (CENPC1). With these results, we also provide a paradigm in which the RNAi pathway participates in vital cellular events through the maintenance of level and distribution of noncoding RNAs in cells. Keywords: RNAi pathway; chromosome segregation; CENPC1; AGO2; Dicer; α-satellite RNA; ASAT siRNA Cell Discovery (2015) 1, 15029; doi:10.1038/celldisc.2015.29; published online 20 October 2015 Introduction The core centromere of mammalian chromosome is the region for the assembly of kinetochore, and it The centromere is a special region of chromosome consists of α-satellite repeats in human and minor needed for successful assembly of kinetochore and satellite repeats in mice. Centromere protein B chromosome attachment to spindle [1–4]. Centromeres (CENP-B) is a highly conserved centromeric DNA- consist of long stretch of species-specific DNA repeats binding protein recognizing and binding to a 17-bp in most eukaryotes, and it has been known for more sequence (CENP-B box) in the centromeric α-satellite than a decade that some transcripts are derived from DNA [5, 6]. In vertebrates and mammals, studies have centromeric repeat regions in multiple eukaryotic demonstrated that transcripts from the centromeric organisms investigated [2, 3]. Although the organiza- core associate with centromeric core proteins to serve tion of centromere DNA as repeats is largely conserved as integral components of the centromere and in eukaryotes, the repeat sequences, transcripts from kinetochore [2, 3, 7–10]. Several studies have shown the repeats and the molecular mechanisms underneath that RNA polymerase II-dependent α-satellite RNA is the centromere/kinetochore assembly are not that an integral part of centromere and exerts an important conserved among different species. function in the kinetochore assembly [7–9]. Centromeric α-satellite RNA associates directly with centromere protein C1 (CENPC1) and facilitates nucleoprotein assembly at the mitotic centromere Correspondence: Ge Shan [7, 11–13]. CENPC1 has both RNA-binding and Tel: +86-551-63606274; Fax: +86-551-63606274; E-mail: [email protected] DNA-binding capacity [7, 8]. This protein is essential Received 5 March 2015; accepted 10 September 2015 for the integrity and localization of kinetochore, and RNAi pathway participates in chromosome segregation 2 loss of CENPC1 induces chromosome lagging [14, 15]. Results In mice, centromeric noncoding RNA (ncRNAs) of 120 nucleotides (nt) associate with centromeres Dicer or AGO2 knockdown leads to defect in and accumulate under stress or differentiation condi- mammalian chromosome segregation tions [16]. Components of kinetochore are recruited to We started to investigate roles of the RNAi pathway minor satellite repeat RNA by CENP-A [17, 18]. in chromosome segregation by knocking down Dicer In mouse embryonic stem cells, loss of Dicer reduces or AGO2 with siRNAs. Dicer or AGO2 knockdown the level of small double-stranded RNA from cen- resulted in chromosome lagging in mitotic human tromeric repeats [19]. In a chicken–human hybrid cell RPE-1 and HeLa cells (Figure 1a and Supplementary line, loss of Dicer results in a higher level of transcripts Figure S1A–E). As a comparison and also a positive from α-satellite sequences [20]. control, knocking down of CENPC1 resulted in higher The RNA interference (RNAi)/microRNA (miRNA) incidence of chromosome lagging (Figure 1a and pathway in mammalian cells is mediated by a cascade of Supplementary Figure S1B), and this phenomenon was enzymatic reactions [21–23]. First, the large primary consistent with early studies about roles of CENPC1 miRNA transcripts are processed by a complex of [14, 15, 26]. Furthermore, the mRNA and protein DGCR8 and Drosha into miRNA precursors of ~ 70 nt levels of CENPC1 were not significantly changed when with a hairpin structure in the nucleus. miRNA pre- knocking down the Dicer or AGO2, respectively cursors are then transported into the cytoplasm and get (Figure 1b). cut by Dicer to give rise to mature miRNAs. Endo- Functions of Dicer and AGO2 are largely associated genous or exogenous double-stranded RNA or short with two classes of small RNA, miRNA and siRNA hairpin RNA can also be processed by Dicer to form [23]. To see whether miRNAs are important, we next small interference RNA (siRNA). Both miRNA and carried out AGO2 and Dicer knockdown experiments siRNA can be loaded into RNA-induced silencing in DGCR8 knockout MEF cells [27–30]. It is well complex (RISC) with its core component as AGO2. known that these cells lack functional miRNAs, as Inside the siRNA-programmed RISC, the siRNA trig- DGCR8 is a critical component of microprocessor for gers the degradation of targeted complementary RNA the production of pre-miRNA from pri-miRNA with the slicer activity of AGO2. Whereas in the [27–30] (Supplementary Figure S1F). In DGCR8 miRISC (miRNA-programmed RISC), miRNA generally knockout cells, AGO2 or Dicer knockdown caused a suppresses the translation or promotes the degradation higher incidence of chromosome lagging (Figure 1c of the target mRNA by incomplete complementation. and Supplementary Figure S1G). Previous results showed that small RNAs and Interestingly, knocking down the other Argonaute components of the RNAi pathway might be associated family members including AGO1, AGO3 and AGO4 with mitotic chromosome segregation [1–3, 24, 25], did not induce chromosome lagging (Supplementary although the mechanism of how the RNAi machinery Figure S2). It seems that Dicer, as well as AGO2 rather participates into this event remains elusive. In this than the other Argonautes, possesses unique function study, we set out to investigate whether the RNAi in mitotic chromosome segregation. pathway participates in mitotic chromosome Taken together, our results demonstrated that segregation directly. We found that knockdown of AGO2 and Dicer acted in mammalian mitotic chro- Dicer or AGO2 resulted in chromosome lagging. With mosome segregation. Their roles in this cellular event a DGCR8 knockout cell line, we demonstrated that may be independent from miRNAs. this effect was largely independent from miRNAs. The RNAi pathway under the guidance of endogenous AGO2 but not dicer is a chromosomal protein siRNAs kept the level of α-satellite RNA balanced for To investigate further roles of Dicer and AGO2 on precise centromere/kinetochore formation. In addition, mitotic chromosome segregation, we isolated human the slicer activity of AGO2 was essential, indicating mitotic metaphase chromosomes with gradient further that a siRNA rather than a miRNA pathway centrifugation (Supplementary Figure S3A) [31]. was involved. Our data suggest that AGO2 guided by Results from flow cytometry and immunostaining of endogenous siRNAs generated by Dicer controls chromosome marker (Phos-histone H3) indicated that the level and localization of chromosomal α-satellite the isolated chromosomes were of high purity RNA to direct the deposition of important centromeric (Supplementary Figure S3B and C). Chromosomes factors such as CENPC1 for mitotic chromosome isolated with gradient centrifugation were mostly used segregation in mammalian cells. for western blot analyses and RNA isolation in this Cell Discovery | www.nature.com/celldisc Chuan Huang et al. 3 research. Western blot analysis with chromosome AGO2 or Dicer antibodies on mitotic chromosomes. samples showed that AGO2 rather than Dicer was a Metaphase chromosome spreads were prepared by chromosomal protein (Figure 1d). For further con- dropping for immunostaining assay. This method was firmation, we applied an immunostaining assay with used for all chromosomal immunofluorescent (IF) 120% *** ** ** ** ** RPE-1 cell 100% Scramble siRNA AGO2 siRNA-1 DICER siRNA-1 CENPC1 siRNA-1 80% 60% Normal 40% Chromosome AGO2 siRNA-2 DICER siRNA-2 CENPC1 siRNA-2 20% lagging Percentage of cells 0% AGO2 siRNA-1 AGO2 siRNA-2 Scramble siRNADICER siRNA-1 DICER siRNA-2 CENPC1 siRNA-1CENPC1 siRNA-2 qPCR of CENPC1 mRNA Western blot 1.2 1 scrambleAGO2 siRNA siRNADICER siRNACENPC1 siRNA 0.8 Scramble siRNA CENPC1 0.6 AGO2 siRNA DICER siRNA 1.00~0.80 ~0.85 ~0.14 0.4 ** CENPC1 siRNA Relative RNA level GAPDH 0.2 (Normalized with GAPDH) 0 120% *** ** 100% 80% MEF (DGCR8-null) cell Scramble siRNA AGO2 siRNA DICER siRNA CENPC1 siRNA 60% Normal 40% Chromosome Percentage of cells lagging 20% 0% AGO2 siRNADICER siRNA Scramble siRNA CENPC1 siRNA WCL CYTO CHR NUC 1/6 1/6 DAPI AGO2 Merge AGO2 DICER DAPI DICER Merge Phos-Histone H3 HADC2 ACTB Cell Discovery | www.nature.com/celldisc RNAi pathway participates in chromosome segregation 4 staining and RNA fluorescence in situ hybridization lengths in an array of human cells [33, 34], and our (FISH) in this study, unless specified.