Aze A , Fragkos M , Bocquet S , Cau J , Méchali M .

	Fig. 1. Incubation with RNase A inhibits DNA replication but not pre-RC assembly. a Replication kinetics of LSEs incubated with RNase A before (pretreat), at the same time (0 min), and 10 or 15 min after addition of sperm nuclei. b M13 ssDNA replication is not affected by LSE pre-incubation with RNase A, c Scheme illustrating the steps leading to the assembly of the complexes involved in the licensing reaction (green) and in DNA replication activation (red). The blue circle highlights reactions occurring in the nucleus. d Sperm nuclei incubated in mock or RNase A-treated LSE for 60 min were fixed in the presence of DiOC6 (green) and DAPI (blue). Scale bar, 17 μm. The nuclear size of 125 nuclei per condition was measured based on the DAPI staining area (right panel). Red bar, mean value. A two-tailed Student’s t-test was performed to determine the p-values. e Histones from sperm nuclei were isolated by acid extraction before and 5 min after incubation in mock or RNase A-treated extracts. DNA-bound proteins were then analysed by gel electrophoresis and silver staining. f Time-course analysis of pre-RC assembly in HSE (chromatin purification followed by western blotting with specific antibodies)
	Fig. 2. The nuclear architecture is affected when assembled in RNA-depleted egg extracts. a Immunofluorescence analysis of lamin LIII accumulation (green) in a representative population of nuclei fixed after incubation in mock- or RNase A-treated X. laevis egg extracts for 60 min. DNA synthesis was monitored by dCTP-Cy3 incorporation (red), and DNA was stained with DAPI (blue). Scale bar, 17 μm. b Higher magnification of representative nuclei from the experiments described in a. Lamin LIII could not be detected in 70% of nuclei assembled in RNA-depleted egg extracts and its accumulation was abnormal in the other 30%. Scale bar, 17 μm. c Western blot analysis of nuclear extracts prepared from sperm nuclei at different time points during incubation in mock-depleted or RNA-depleted extracts
	Fig. 3. RNA is required for pre-IC assembly but is dispensable after pre-IC formation. a Time-course analysis of the recruitment of pre-IC and replication fork components to sperm chromatin in mock- and RNase A-treated LSEs. Histone H3 was used as a loading control. b Western blot showing the absence of DNA unwinding in RNase A-treated extracts. Sperm chromatin was purified and analysed 30 min after incubation in LSE. Incubation with 100 ng/μl aphidicolin (Aphi) resulted in RPA overloading following DNA polymerase inhibition and uncoupling from the MCM2-7 helicase activity. Geminin was used as negative control. c Chromatin transfer experiments showing that DNA replication is inhibited by RNA depletion before pre-IC formation in a reversible manner. Lanes A and B, chromatin was assembled in a mock-treated LSE for 40 min to allow pre-IC formation, and then transferred to an RNase A-treated extract or another mock extract (control). The graph shows the percentage of DNA replication at 120 min after chromatin transfer (n = 5), and the right panel shows the loading of replication proteins onto chromatin. Lanes C and D, the DNA replication defect is abolished when nuclei assembled in RNase A-treated extracts are transferred into mock-treated extracts C, but not after transfer into a new RNAse A-treated extract D. Error bar represents the standard deviation (SD). P-values were determined with the two-tailed Student’s t-test. n.s.: not significant. d RNA depletion blocks S-phase-dependent kinase activation. Lanes A and B, sperm nuclei were initially incubated in p21-treated LSE (for 30 min) and then transferred to mock- or RNase A-treated LSE, respectively. C: Chromatin assembled in an RNase A-treated LSE extract was transferred in a p21-treated extract. DNA replication levels (n = 3) and binding of replication factors to chromatin were monitored as described in a. Error bar shows the SD. A two-tailed Student’s t-test was performed to calculate the p-values
	Fig. 4. RNAs are required for nuclear import mediated by functional nuclear envelope assembly. a Sperm nuclei were incubated in mock- and RNA-depleted extracts. At the indicated time points, nuclei were purified and nuclear extracts analysed by western blotting. The anti-tubulin antibody was used to monitor cytoplasmic contamination during nuclear extract preparation. Addition to the extracts of wheat germ agglutinin (WGA), which inhibits nuclear import, blocks accumulation of factors associated with replication activation and elongation (CDC45 and PCNA, respectively). b Sperm nuclei were incubated in egg extracts supplemented with GFP-NLS (green) to monitor nuclear import. Its accumulation into nuclei (stained with DAPI) was monitored with an epifluorescence microscope. Scale bar, 15 μm. GFP-NLS level in each condition was assessed by western blotting analysis of total extracts. MCM3 was used as loading control. c Chromatin was assembled in mock- or RNase A-treated LSE (for 20 min) and then transferred to a mock- or RNAse A-treated LSE, or to a HSE, which lacks membranes. The graph shows the percentage of DNA replication at 120 min after transfer to the second extract (n = 3). Error bar shows the SD. P-values were determined by using a two-tailed Student’s t-test
	Fig. 5. RNAs are required for nuclear pore complex assembly and nuclear import. a Representative images of immunofluorescence analysis of nuclei fixed after incubation in mock- or RNase A-treated X. laevis egg extracts for 20 min. The mouse monoclonal antibody 414 (mAb414) recognises several nucleoporins (NUP358, 214, 153, 62); DNA was stained with DAPI (blue). Scale bar, 10 μm. b Western blot analysis of nuclear extracts after incubation in mock- or RNA-depleted egg extracts for the indicated time. c Sperm nuclei were incubated in mock- or RNA-depleted egg extracts. At the indicated time points, chromatin was isolated and immunoblotted with the indicated antibodies. d Immunofluorescence analysis of ELYS (green) and NUPs (red) expression in a representative population of nuclei assembled in mock- or RNase A-treated extracts for 60 min. Images were acquired with an ApoTome microscope. Scale bar, 15 μm. e Nuclear swelling and DNA decompaction are restored when nuclei are first incubated in RNase A-treated extracts and then transferred to mock-treated extracts (as explained in Fig. 3c). After transfer into the second extract, nuclei were fixed and stained with DAPI and DiOC6 to monitor decompaction and nuclear swelling. Scale bar, 10 µm
	Fig. 6. RNAs modulate the higher-order chromatin structure. a Micrococcal nuclease (MNase) digestion assays of nuclei that were assembled in mock- and RNA-depleted extracts for 15 min. Black arrows indicate the ladder of mono-, di- and tri-nucleosomes. b Immunofluorescence images of histone H2B in nuclei treated with a moderate salt concentration after incubation in mock- or RNA-depleted X. laevis egg extracts for 15 min. Scale bar, 17 μm. c Quantification of the chromatin perimeter-to-axis ratio in one representative experiment out of three. Mock-depleted extracts: n = 102 nuclei; RNase A-depleted extracts: n = 93 nuclei. A two-tailed Student t-test was performed to determine the p-value. d Quantification of three independent DNA replication assays (mean ± SD) in which sperm nuclei were incubated in mock or RNase A-treated HSE (for replication licensing) and then transferred to mock- or RNase A-treated NPE (for replication initiation). Nuclear membrane-free replication was monitored by autoradiography. A two-tailed Student’s t-test was performed to calculate the p-values; n.s., not significant. e Stepwise model of the major events leading to the replication initiation defect observed upon RNA depletion in egg extracts. Protamine removal and histone incorporation, two major steps during chromatin remodelling following fertilisation, occur normally both in the presence or absence of maternal RNAs. Newly formed chromatin in RNA-depleted extracts acquires a compact architectural organisation compared with control (mock-depleted) extracts. While licensing (pre-RC assembly) occurs normally, ELYS binding to condensed chromatin of nuclei assembled in RNA-depleted extracts is repressed. As a consequence, its accumulation at the nuclear rim is disturbed, and NPC assembly is altered. Nuclear pores are not incorporated in the NE membranes. Nuclear import is impaired and the accumulation of factors required for DNA replication activation is inhibited

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