Different dynamics in nuclear entry of subunits of the repair/transcription factor TFIIH

Abstract

We report here the different ways in which four subunits of the basal transcription/repair factor TFIIH (XPB, XPD, p62 and p44) and the damage recognition XPC repair protein can enter the nucleus. We examined their nuclear localization by transiently expressing the gene products tagged with the enhanced green fluorescent protein (EGFP) in transfected 3T3 cells. In agreement with the identification of more than one putative nuclear localization signal (NLS) in their protein sequences, XPB, XPC, p62 and p44 chimeras were rapidly sorted to the nucleus. In contrast, the XPD-EGFP chimeras appeared mainly localized in the cytoplasm, with a minor fraction of transfectants showing the EGFP-based fluorescence also in the nucleus. The ability of the XPD chimeras to enter the nucleus was confirmed by western blotting on fractionated cell extracts and by functional complementation of the repair defect in the UV5 rodent cells, mutated in the XPD homologous gene. By deletion mutagenesis, we were unable to identify any sequence specific for nuclear localization. In particular, deletion of the putative NLS failed to affect subcellular localization and, conversely, the C-terminal part of XPD containing the putative NLS showed no specific nuclear accumulation. These findings suggest that the nuclear entry of XPD depends on its complexation with other proteins in the cytoplasm, possibly other components of the TFIIH complex.

TFIIH trafficking and its nuclear assembly during earlyDrosophilaembryo development

“…In mammalian cells, nuclear entry of a fused GFP-XPD protein is independent of fused GFP-XPB protein and XPD nuclear transport is concluded to be different from that of other TFIIH subunits (Santagati et al, 2001). However, our results suggest that in early fly development, XPD must be assembled with other TFIIH subunits to enter the nuclei.…”

NSs Protein of Rift Valley Fever Virus Promotes Posttranslational Downregulation of the TFIIH Subunit p62

“…NSs is distributed throughout the cell during RVFV infection (52); however, its ability to suppress transcription has been suggested to require nuclear localization and the formation of filamentous structures inside the nucleus (2). The localization of p62 is predominantly nuclear (24,42), and it is thought that, immediately after translation, it is translocated into the nucleus, where it assembles with the other subunits of TFIIH to form the functional holocomplex (43). Le May et al suggested that RVFV NSs sequesters p44 subunits before the assembly of TFIIH but does not interact with p44 when already associated with XPD within TFIIH (27).…”

Identification and Functional Characterization of a Ku-binding Motif in Aprataxin Polynucleotide Kinase/Phosphatase-like Factor (APLF)

“…Overall, our data are consistent with APLF being engaged in basal interactions with Ku, which may both stabilize APLF and contribute to its nuclear retention. There are other reported examples of small molecular weight and NLS-deficient repair proteins that rely primarily on interactions with NLS-containing proteins, which are involved in their respective repair pathway, to bring them into the nucleus, often in a DNA damage-dependent manner (36,37). This may represent a level of spatiotemporal regulation that is more efficient than simple diffusion.…”