Abstract
Upon fertilisation, the sperm pronucleus acquires the competence to replicate the genome through a cascade of events that link chromatin remodelling to nuclear envelope formation. The factors involved have been partially identified and are poorly characterised. Here, using Xenopus laevis egg extracts we show that RNAs are required for proper nuclear envelope assembly following sperm DNA decondensation. Although chromatin remodelling and pre-replication complex formation occur normally, RNA-depleted extracts show a defect in pre-RC activation. The nuclear processes affected by RNA-depletion included ELYS recruitment, which accounts for the deficiency in nuclear pore complex assembly. This results in failure in chromatin relaxation as well as in the import and proper nuclear concentration of the S-phase kinases necessary for DNA replication activation. Our results highlight a translation-independent RNA function necessary for the parental genome progression towards the early embryonic cell cycle programme.
Highlights
nuclear envelope (NE) assembly in Xenopus laevis egg extracts mimics events occurring after fertilisation, when nucleoplasmin-dependent decondensation of sperm chromatin occurs rapidly and results in the replacement of sperm protamines by maternal histones stored in the egg[8]
To investigate whether RNAs are involved in the processes leading to pronucleus formation and activation of DNA replication following fertilisation in metazoans, we used the in vitro system derived from X. laevis low-speed egg extracts (LSE), where nuclear formation and semi-conservative DNA replication of the added demembranated sperm nuclei occur in the absence of both transcription and translation[23,24]
Our results were obtained using X. laevis egg extracts that are transcriptionally quiescent and in which translation is dispensable for DNA synthesis
Summary
NE assembly in Xenopus laevis egg extracts mimics events occurring after fertilisation, when nucleoplasmin-dependent decondensation of sperm chromatin occurs rapidly and results in the replacement of sperm protamines by maternal histones stored in the egg[8]. As ELYS interacts with lamins and MCM2−79-11, it could be one of the main factors associated with the NE to coordinate chromatin compaction up to chromosome decondensation and assembly of the proteins involved in S-phase activation. This in vitro system is transcriptionindependent[23] and reproduces most of the events linked to chromatin reorganisation and DNA replication activation during early development This is possible because X. laevis eggs contain large amounts of the components necessary for these processes because they are stored during oogenesis. We found that when sperm nuclei were incubated in RNA-depleted X. laevis egg extracts, DNA replication was strongly inhibited This defect was not owing to alterations in pre-RC formation, but was linked to a defect in the activation of DNA replication origins. By investigating the molecular mechanisms responsible for this replication defect, we found that RNAs are required to favour ELYS accessibility to chromatin and for NPC assembly, two precursor steps before DNA replication
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