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