Direct non transcriptional role of NF-Y in DNA replication

NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication

Etude du rôle de la protéine INT6 dans la dégradation des ARN par la voie du "Nonsense Mediated mRNA Decay" (NMD) et dans la traduction et la dégradation des ARN histones

Several observations show that the human protein INT6 has a tumour suppressor activity.It has been demonstrated that in humans the expression of the int6 gene is reduced in about 30% of non-small cell lung cancers and that this under-expression is linked with a bad prognosis. Yeast two hybrid screening experiments using INT6 as bait have led to the identication ofa protein named SLIP1 (SLBP Interacting Protein 1). An effect of SLIP1 on the translation of histone messenger RNAs has been shown. My work indicates that INT6 – through its interaction with SLIP1 – plays a role in the controlof the stability and translation of histone mRNAs. INT6 knockdown induces a reduction of the level of endogenous histones without affecting that of their mRNAs. My studies, by revealing a new histone translation mechanism in which INT6 plays a direct role, establish a connection between – on one side – the known functions of this protein in translation and its control and – on the other – the known oncogenic effects of its alteration. The study of the function of INT6 in human cells by RNA interference resulted in inhibition of the degradation of mRNAs containing a premature termination codon by the Nonsense Mediated mRNA Decay pathway (NMD). Since we identified the HTLV-1 protein Tax as an interactor of INT6, and since some features of HTLV-1 mRNAs suggest that they may be potential NMD targets, we have studied the effect of Tax on the NMD pathway.We have observed that Tax significantly stabilizes NMD targets.We have demonstrated that the interference of Tax with the NMD pathway is mediated through – first – the perturbation of the interaction between INT6 and the NMD factor UPF1 and – second – through direct interaction between Tax and phosphorylated UPF1.Through its effect on NMD, Tax may favor viral replication at a post-transcriptional level as well as enable cellular tolerance for some mutations resulting from the established mutagenic activity of Tax.Différentes observations montrent que la protéine INT6 humaine possède une activité suppresseur de tumeurs. Il a été démontré que chez l’homme le gène int6 était sous-exprimé dans environ 30% des cancers du poumon non à petits cellules et que cette sous-expression était un facteur de mauvais pronostic. Des expériences de criblage double hybride avec INT6 comme appât ont identifié une protéine nommée SLIP1 (SLBP Interacting Protein 1). Un effet de SLIP1 sur la traduction des ARN messager des histones a été montré. Les travaux que j’ai menés indiquent qu’INT6 en interagissant avec SLIP intervient dans le contrôle de la stabilité et de la traduction des ARNs codant pour les histones. Un knockdown d’INT6 provoque une baise des niveaux des histones endogènes sans avoir un effet au niveau d’ARN. Mes études, en révélant un nouveau mécanisme de dans lequel INT6 joue un rôle direct, permettent ainsi de faire le lien entre – d’une part – les fonctions connues de cette protéine dans la traduction et son contrôle et – d’autre part – les effets oncogéniques connus de son altération. Par ailleurs, l’étude de la fonction d’INT6 dans les cellules humaines réalisée par ARN interférence montre une inhibition de la dégradation des ARNm possédant un codon stop prématuré par la voie du Nonsense Mediated mRNA Decay (NMD). Nous avons étudié son action par rapport aux ARNs HTLV-1. Nous avons observé une stabilisation significative des cibles de NMD. Ceci démontre que la protéine Tax interfère avec cette voie de dégradation des ARN d’une part en empêchant l’interaction entre UPF1 et INT6 et d’autre part en interagissant lui-même avec la protéine UPF1 phosphorylée. En agissant sur le NMD, Tax intervient à un niveau post transcriptionel qui pourrait avantager la réplication virale et aussi permettre la tolérance cellulaire aux mutations liées à l'effet mutagénique établi de Tax

To translate or to degrade? The role of INT6 in histone mRNA translation and Nonsense Mediated mRNA Decay

Différentes observations montrent que la protéine INT6 humaine possède une activité suppresseur de tumeurs. Il a été démontré que chez l’homme le gène int6 était sous-exprimé dans environ 30% des cancers du poumon non à petits cellules et que cette sous-expression était un facteur de mauvais pronostic. Des expériences de criblage double hybride avec INT6 comme appât ont identifié une protéine nommée SLIP1 (SLBP Interacting Protein 1). Un effet de SLIP1 sur la traduction des ARN messager des histones a été montré. Les travaux que j’ai menés indiquent qu’INT6 en interagissant avec SLIP intervient dans le contrôle de la stabilité et de la traduction des ARNs codant pour les histones. Un knockdown d’INT6 provoque une baise des niveaux des histones endogènes sans avoir un effet au niveau d’ARN. Mes études, en révélant un nouveau mécanisme de dans lequel INT6 joue un rôle direct, permettent ainsi de faire le lien entre – d’une part – les fonctions connues de cette protéine dans la traduction et son contrôle et – d’autre part – les effets oncogéniques connus de son altération. Par ailleurs, l’étude de la fonction d’INT6 dans les cellules humaines réalisée par ARN interférence montre une inhibition de la dégradation des ARNm possédant un codon stop prématuré par la voie du Nonsense Mediated mRNA Decay (NMD). Nous avons étudié son action par rapport aux ARNs HTLV-1. Nous avons observé une stabilisation significative des cibles de NMD. Ceci démontre que la protéine Tax interfère avec cette voie de dégradation des ARN d’une part en empêchant l’interaction entre UPF1 et INT6 et d’autre part en interagissant lui-même avec la protéine UPF1 phosphorylée. En agissant sur le NMD, Tax intervient à un niveau post transcriptionel qui pourrait avantager la réplication virale et aussi permettre la tolérance cellulaire aux mutations liées à l'effet mutagénique établi de Tax.Several observations show that the human protein INT6 has a tumour suppressor activity.It has been demonstrated that in humans the expression of the int6 gene is reduced in about 30% of non-small cell lung cancers and that this under-expression is linked with a bad prognosis. Yeast two hybrid screening experiments using INT6 as bait have led to the identication ofa protein named SLIP1 (SLBP Interacting Protein 1). An effect of SLIP1 on the translation of histone messenger RNAs has been shown. My work indicates that INT6 – through its interaction with SLIP1 – plays a role in the controlof the stability and translation of histone mRNAs. INT6 knockdown induces a reduction of the level of endogenous histones without affecting that of their mRNAs. My studies, by revealing a new histone translation mechanism in which INT6 plays a direct role, establish a connection between – on one side – the known functions of this protein in translation and its control and – on the other – the known oncogenic effects of its alteration. The study of the function of INT6 in human cells by RNA interference resulted in inhibition of the degradation of mRNAs containing a premature termination codon by the Nonsense Mediated mRNA Decay pathway (NMD). Since we identified the HTLV-1 protein Tax as an interactor of INT6, and since some features of HTLV-1 mRNAs suggest that they may be potential NMD targets, we have studied the effect of Tax on the NMD pathway.We have observed that Tax significantly stabilizes NMD targets.We have demonstrated that the interference of Tax with the NMD pathway is mediated through – first – the perturbation of the interaction between INT6 and the NMD factor UPF1 and – second – through direct interaction between Tax and phosphorylated UPF1.Through its effect on NMD, Tax may favor viral replication at a post-transcriptional level as well as enable cellular tolerance for some mutations resulting from the established mutagenic activity of Tax

To translate or to degrade? The role of INT6 in histone mRNA translation and Nonsense Mediated mRNA Decay

Différentes observations montrent que la protéine INT6 humaine possède une activité suppresseur de tumeurs. Il a été démontré que chez l homme le gène int6 était sous-exprimé dans environ 30% des cancers du poumon non à petits cellules et que cette sous-expression était un facteur de mauvais pronostic. Des expériences de criblage double hybride avec INT6 comme appât ont identifié une protéine nommée SLIP1 (SLBP Interacting Protein 1). Un effet de SLIP1 sur la traduction des ARN messager des histones a été montré. Les travaux que j ai menés indiquent qu INT6 en interagissant avec SLIP intervient dans le contrôle de la stabilité et de la traduction des ARNs codant pour les histones. Un knockdown d INT6 provoque une baise des niveaux des histones endogènes sans avoir un effet au niveau d ARN. Mes études, en révélant un nouveau mécanisme de dans lequel INT6 joue un rôle direct, permettent ainsi de faire le lien entre d une part les fonctions connues de cette protéine dans la traduction et son contrôle et d autre part les effets oncogéniques connus de son altération. Par ailleurs, l étude de la fonction d INT6 dans les cellules humaines réalisée par ARN interférence montre une inhibition de la dégradation des ARNm possédant un codon stop prématuré par la voie du Nonsense Mediated mRNA Decay (NMD). Nous avons étudié son action par rapport aux ARNs HTLV-1. Nous avons observé une stabilisation significative des cibles de NMD. Ceci démontre que la protéine Tax interfère avec cette voie de dégradation des ARN d une part en empêchant l interaction entre UPF1 et INT6 et d autre part en interagissant lui-même avec la protéine UPF1 phosphorylée. En agissant sur le NMD, Tax intervient à un niveau post transcriptionel qui pourrait avantager la réplication virale et aussi permettre la tolérance cellulaire aux mutations liées à l'effet mutagénique établi de Tax.Several observations show that the human protein INT6 has a tumour suppressor activity.It has been demonstrated that in humans the expression of the int6 gene is reduced in about 30% of non-small cell lung cancers and that this under-expression is linked with a bad prognosis. Yeast two hybrid screening experiments using INT6 as bait have led to the identication ofa protein named SLIP1 (SLBP Interacting Protein 1). An effect of SLIP1 on the translation of histone messenger RNAs has been shown. My work indicates that INT6 through its interaction with SLIP1 plays a role in the controlof the stability and translation of histone mRNAs. INT6 knockdown induces a reduction of the level of endogenous histones without affecting that of their mRNAs. My studies, by revealing a new histone translation mechanism in which INT6 plays a direct role, establish a connection between on one side the known functions of this protein in translation and its control and on the other the known oncogenic effects of its alteration. The study of the function of INT6 in human cells by RNA interference resulted in inhibition of the degradation of mRNAs containing a premature termination codon by the Nonsense Mediated mRNA Decay pathway (NMD). Since we identified the HTLV-1 protein Tax as an interactor of INT6, and since some features of HTLV-1 mRNAs suggest that they may be potential NMD targets, we have studied the effect of Tax on the NMD pathway.We have observed that Tax significantly stabilizes NMD targets.We have demonstrated that the interference of Tax with the NMD pathway is mediated through first the perturbation of the interaction between INT6 and the NMD factor UPF1 and second through direct interaction between Tax and phosphorylated UPF1.Through its effect on NMD, Tax may favor viral replication at a post-transcriptional level as well as enable cellular tolerance for some mutations resulting from the established mutagenic activity of Tax.LYON-ENS Sciences (693872304) / SudocSudocFranceF

INT6 interacts with MIF4GD/SLIP1 and is necessary for efficient histone mRNA translation.

International audienceThe INT6/EIF3E protein has been implicated in mouse and human breast carcinogenesis. This subunit of the eIF3 translation initiation factor that includes a PCI domain exhibits specific features such as presence in the nucleus and ability to interact with other important cellular protein complexes like the 26S proteasome and the COP9 signalosome. It has been previously shown that INT6 was not essential for bulk translation, and this protein is considered to regulate expression of specific mRNAs. Based on the results of a two-hybrid screen performed with INT6 as bait, we characterize in this article the MIF4GD/SLIP1 protein as an interactor of this eIF3 subunit. MIF4GD was previously shown to associate with SLBP, which binds the stem-loop located at the 3' end of the histone mRNAs, and to be necessary for efficient translation of these cell cycle-regulated mRNAs that lack a poly(A) tail. In line with the interaction of both proteins, we show using the RNA interference approach that INT6 is also essential to S-phase histone mRNA translation. This was observed by analyzing expression of endogenous histones and by testing heterologous constructs placing the luciferase reporter gene under the control of the stem-loop element of various histone genes. With such a reporter plasmid, silencing and overexpression of INT6 exerted opposite effects. In agreement with these results, INT6 and MIF4GD were observed to colocalize in cytoplasmic foci. We conclude from these data that INT6, by establishing interactions with MIF4GD and SLBP, plays an important role in translation of poly(A) minus histone mRNAs

Cell Cycle Synchronization in Xenopus Egg Extracts

Many important discoveries in cell cycle research have been made using cell-free extracts prepared from the eggs of the South African clawed frog Xenopus laevis . These extracts effi ciently support the key nuclear functions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. Here, we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei. We detail how these extracts can be used to study the key transitions of the eukaryotic cell cycle and describe conditions under which these transitions can be manipulated by addition of drugs that either retard or advance passage. In addition, we describe in detail essential techniques that provide a practical starting point for investigating the function of proteins involved in the operation of the eukaryotic cell cycle

A non transcriptional role for NF-Y in DNA replication

The heterotrimeric transcription factor NF-Y, composed by NF-YA, NF-YB and NF-YC subunits, is a key transcriptional regulator of cell cycle progression. Using data generated by ENCODE, we identified a striking overlap between loci bound by NF-Y-and ORC2, hinting at a possible role of NF-Y in DNA replication. NF-YA knock-down leads to replication defects and the activation an intra-S checkpoint. We investigated the role of NF-Y in DNA replication by using the Xenopus cell-free system. NF-Y subunits were found to be recruited to chromatin during DNA replication. Both immunodepletion of NF-YA or NF-YB and overexpression of a dominant-negative NF-YA mutant lead to a clear decrease in DNA synthesis. In mammalian cells, NF-Y colocalizes and directly interacts with DNA replication proteins. Nascent strand abundance assay in NF-YA inactivated cells corroborates that NF-Y participates to the DNA replication process. Our data highlight that, in addition to its transcriptional activity in controlling cell proliferation, NF-Y plays a key role in the non transcriptional control of DNA replication

Direct non transcriptional role of the CCAAT-factor NF-Y in DNA replication

The heterotrimeric transcription factor NF-Y plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation. The expression levels of the sequence specific subunit NF-YA increase at the onset of S phase, and NF-YA loss in primary and tumor mammalian cells triggers defects in S-phase progression. Here, we show that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Using a Xenopus cell-free system, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication. Spontaneous DNA damage occurs in NF-YA-deficient cells, while NF-YA overexpression reduces the sensitivity to replication stress, suggesting a role for NF-Y in replication stress response and genome maintenance. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific transcription factors required for proper and efficient DNA replication

Direct non transcriptional role of NF-Y in DNA replication

NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication