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

Adachi, Study of the cell cycle-dependent assembly of the DNA pre-replication centres in Xenopus egg extracts. 1994, Pubmed, Xenbase

Adachi, Study of the cell cycle-dependent assembly of the DNA pre-replication centres in Xenopus egg extracts. 1994, Pubmed , Xenbase
Aze, Author Correction: RNAs coordinate nuclear envelope assembly and DNA replication through ELYS recruitment to chromatin. 2018, Pubmed
Barkley, MicroRNA-29a regulates the benzo[a]pyrene dihydrodiol epoxide-induced DNA damage response through Cdc7 kinase in lung cancer cells. 2013, Pubmed
Blow, A role for the nuclear envelope in controlling DNA replication within the cell cycle. 1988, Pubmed , Xenbase
Blow, Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. 1986, Pubmed , Xenbase
Byun, Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint. 2005, Pubmed , Xenbase
Caudron-Herger, Coding RNAs with a non-coding function: maintenance of open chromatin structure. 2011, Pubmed
Coué, Chromatin binding, nuclear localization and phosphorylation of Xenopus cdc21 are cell-cycle dependent and associated with the control of initiation of DNA replication. 1996, Pubmed , Xenbase
Dileep, Topologically associating domains and their long-range contacts are established during early G1 coincident with the establishment of the replication-timing program. 2015, Pubmed
Dimitrova, The spatial position and replication timing of chromosomal domains are both established in early G1 phase. 1999, Pubmed , Xenbase
Fragkos, DNA replication origin activation in space and time. 2015, Pubmed
Franz, MEL-28/ELYS is required for the recruitment of nucleoporins to chromatin and postmitotic nuclear pore complex assembly. 2007, Pubmed , Xenbase
Françon, A hypophosphorylated form of RPA34 is a specific component of pre-replication centers. 2004, Pubmed , Xenbase
Galy, MEL-28, a novel nuclear-envelope and kinetochore protein essential for zygotic nuclear-envelope assembly in C. elegans. 2006, Pubmed
Gilbert, Space and time in the nucleus: developmental control of replication timing and chromosome architecture. 2010, Pubmed
Gillespie, Scc2 couples replication licensing to sister chromatid cohesion in Xenopus egg extracts. 2004, Pubmed , Xenbase
Gillespie, ELYS/MEL-28 chromatin association coordinates nuclear pore complex assembly and replication licensing. 2007, Pubmed , Xenbase
Gillespie, Nucleoplasmin-mediated chromatin remodelling is required for Xenopus sperm nuclei to become licensed for DNA replication. 2000, Pubmed , Xenbase
Goldberg, Xenopus lamin B3 has a direct role in the assembly of a replication competent nucleus: evidence from cell-free egg extracts. 1995, Pubmed , Xenbase
Harper, The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. 1993, Pubmed
Heller, Eukaryotic origin-dependent DNA replication in vitro reveals sequential action of DDK and S-CDK kinases. 2011, Pubmed
Hutchison, The control of DNA replication in a cell-free extract that recapitulates a basic cell cycle in vitro. 1988, Pubmed , Xenbase
Ilves, Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins. 2010, Pubmed
Inoue, Nucleosome assembly is required for nuclear pore complex assembly in mouse zygotes. 2014, Pubmed
Jares, Xenopus cdc7 function is dependent on licensing but not on XORC, XCdc6, or CDK activity and is required for XCdc45 loading. 2000, Pubmed , Xenbase
Katagiri, Remodeling of sperm chromatin induced in egg extracts of amphibians. 1994, Pubmed , Xenbase
Lebofsky, DNA replication in nucleus-free Xenopus egg extracts. 2009, Pubmed , Xenbase
Lutzmann, MCM9 binds Cdt1 and is required for the assembly of prereplication complexes. 2008, Pubmed , Xenbase
Maiorano, MCM8 is an MCM2-7-related protein that functions as a DNA helicase during replication elongation and not initiation. 2005, Pubmed , Xenbase
Masumoto, S-Cdk-dependent phosphorylation of Sld2 essential for chromosomal DNA replication in budding yeast. 2002, Pubmed
Meier, The role of lamin LIII in nuclear assembly and DNA replication, in cell-free extracts of Xenopus eggs. 1991, Pubmed , Xenbase
Moore, The two steps of nuclear import, targeting to the nuclear envelope and translocation through the nuclear pore, require different cytosolic factors. 1992, Pubmed , Xenbase
Moussaoui, A phosphate-binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site. 2007, Pubmed
Méchali, DNA synthesis in a cell-free system from Xenopus eggs: priming and elongation on single-stranded DNA in vitro. 1982, Pubmed , Xenbase
Naumova, Organization of the mitotic chromosome. 2013, Pubmed
Newport, A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription. 1982, Pubmed , Xenbase
Newport, Nuclear reconstitution in vitro: stages of assembly around protein-free DNA. 1987, Pubmed , Xenbase
Philpott, Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin. 1991, Pubmed , Xenbase
Rasala, ELYS is a dual nucleoporin/kinetochore protein required for nuclear pore assembly and proper cell division. 2006, Pubmed , Xenbase
Rasala, Capture of AT-rich chromatin by ELYS recruits POM121 and NDC1 to initiate nuclear pore assembly. 2008, Pubmed , Xenbase
Rodríguez-Campos, RNA is an integral component of chromatin that contributes to its structural organization. 2007, Pubmed
Schooley, Building a nuclear envelope at the end of mitosis: coordinating membrane reorganization, nuclear pore complex assembly, and chromatin de-condensation. 2012, Pubmed
Schooley, The lysine demethylase LSD1 is required for nuclear envelope formation at the end of mitosis. 2015, Pubmed , Xenbase
Schubert, Changes in higher order structures of chromatin by RNP complexes. 2013, Pubmed
Schubert, Df31 protein and snoRNAs maintain accessible higher-order structures of chromatin. 2012, Pubmed
Sheehan, Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs. 1988, Pubmed , Xenbase
Sonneville, Both Chromosome Decondensation and Condensation Are Dependent on DNA Replication in C. elegans Embryos. 2015, Pubmed
Suntharalingam, Peering through the pore: nuclear pore complex structure, assembly, and function. 2003, Pubmed
Takahashi, Cdc7-Drf1 is a developmentally regulated protein kinase required for the initiation of vertebrate DNA replication. 2005, Pubmed , Xenbase
Tanaka, CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast. 2007, Pubmed
Thomson, The radial positioning of chromatin is not inherited through mitosis but is established de novo in early G1. 2004, Pubmed
Waga, The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. 1994, Pubmed
Walter, Regulated chromosomal DNA replication in the absence of a nucleus. 1998, Pubmed , Xenbase
Walter, Chromosome order in HeLa cells changes during mitosis and early G1, but is stably maintained during subsequent interphase stages. 2003, Pubmed
Walter, Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase alpha. 2000, Pubmed , Xenbase
Yan, An analysis of the regulation of DNA synthesis by cdk2, Cip1, and licensing factor. 1995, Pubmed , Xenbase
Zegerman, Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast. 2007, Pubmed
Zhang, Dynamic interaction of Y RNAs with chromatin and initiation proteins during human DNA replication. 2011, Pubmed
Zierhut, Nucleosomal regulation of chromatin composition and nuclear assembly revealed by histone depletion. 2014, Pubmed , Xenbase
van Wolfswinkel, The role of small non-coding RNAs in genome stability and chromatin organization. 2010, Pubmed
von Holt, Isolation and characterization of histones. 1989, Pubmed