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.

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 efficiently 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.

The human T-lymphotropic virus type 1 tax protein inhibits nonsense-mediated mRNA decay by interacting with INT6/EIF3E and UPF1.

In this report, we analyzed whether the degradation of mRNAs by the nonsense-mediated mRNA decay (NMD) pathway was affected in human T-lymphotropic virus type 1 (HTLV-1)-infected cells. This pathway was indeed strongly inhibited in C91PL, HUT102, and MT2 cells, and such an effect was also observed by the sole expression of the Tax protein in Jurkat and HeLa cells. In line with this activity, Tax binds INT6/EIF3E (here called INT6), which is a subunit of the translation initiation factor eukaryotic initiation factor 3 (eIF3) required for efficient NMD, as well as the NMD core factor upstream frameshift protein 1 (UPF1). It was also observed that Tax expression alters the morphology of processing bodies (P-bodies), the cytoplasmic structures which concentrate RNA degradation factors. The presence of UPF1 in these subcellular compartments was increased by Tax, whereas that of INT6 was decreased. In line with these effects, the level of the phosphorylated form of UPF1 was increased in the presence of Tax. Analysis of several mutants of the viral protein showed that the interaction with INT6 is necessary for NMD inhibition. The alteration of mRNA stability was observed to affect viral transcripts, such as that coding for the HTLV-1 basic leucine zipper factor (HBZ), and also several cellular mRNAs sensitive to the NMD pathway. Our data indicate that the effect of Tax on viral and cellular gene expression is not restricted to transcriptional control but can also involve posttranscriptional regulation.

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

The 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.