Cadmium binding by the F-box domain induces p97-mediated SCF complex disassembly to activate stress response programs.

The F-box domain is a highly conserved structural motif that defines the largest class of ubiquitin ligases, Skp1/Cullin1/F-box protein (SCF) complexes. The only known function of the F-box motif is to form the protein interaction surface with Skp1. Here we show that the F-box domain can function as an environmental sensor. We demonstrate that the F-box domain of Met30 is a cadmium sensor that blocks the activity of the SCFMet30 ubiquitin ligase during cadmium stress. Several highly conserved cysteine residues within the Met30 F-box contribute to binding of cadmium with a KD of 8 µM. Binding induces a conformational change that allows for Met30 autoubiquitylation, which in turn leads to recruitment of the segregase Cdc48/p97/VCP followed by active SCFMet30 disassembly. The resulting inactivation of SCFMet30 protects cells from cadmium stress. Our results show that F-box domains participate in regulation of SCF ligases beyond formation of the Skp1 binding interface.

Discovery of compounds that reactivate p53 mutants in vitro and in vivo.

The tumor suppressor p53 is the most frequently mutated protein in human cancer. The majority of these mutations are missense mutations in the DNA binding domain of p53. Restoring p53 tumor suppressor function could have a major impact on the therapy for a wide range of cancers. Here we report a virtual screening approach that identified several small molecules with p53 reactivation activities. The UCI-LC0023 compound series was studied in detail and was shown to bind p53, induce a conformational change in mutant p53, restore the ability of p53 hotspot mutants to associate with chromatin, reestablish sequence-specific DNA binding of a p53 mutant in a reconstituted in vitro system, induce p53-dependent transcription programs, and prevent progression of tumors carrying mutant p53, but not p53null or p53WT alleles. Our study demonstrates feasibility of a computation-guided approach to identify small molecule corrector drugs for p53 hotspot mutations.

An F-Box Protein, Mdm30, Interacts with TREX Subunit Sub2 To Regulate Cellular Abundance Cotranscriptionally in Orchestrating mRNA Export Independently of Splicing and Mitochondrial Function.

Although an F-box protein, Mdm30, is found to regulate ubiquitylation of the Sub2 component of TREX (transcription-export) complex for proteasomal degradation in stimulation of mRNA export, it remains unknown whether such ubiquitin-proteasome system (UPS) regulation of Sub2 occurs cotranscriptionally via its interaction with Mdm30. Further, it is unclear whether impaired UPS regulation of Sub2 in the absence of Mdm30 alters mRNA export via splicing defects of export factors and/or mitochondrial dynamics/function, since Sub2 controls mRNA splicing and Mdm30 regulates mitochondrial aggregation. Here, we show that Mdm30 interacts with Sub2, and temporary shutdown of Mdm30 enhances Sub2's abundance and impairs mRNA export. Likewise, Sub2's abundance is increased following transcriptional inhibition. These results support Mdm30's direct role in regulation of Sub2's cellular abundance in a transcription-dependent manner. Consistently, the chromatin-bound Sub2 level is increased in the absence of Mdm30. Further, we find that Mdm30 does not facilitate splicing of export factors. Moreover, Mdm30 does not have a dramatic effect on mitochondrial respiration/function, and mRNA export occurs in the absence of Fzo1, which is required for mitochondrial dynamics/respiration. Collective results reveal that Mdm30 interacts with Sub2 for proteasomal degradation in a transcription-dependent manner to promote mRNA export independently of splicing or mitochondrial function, thus advancing our understanding of mRNA export.

A new regulatory pathway of mRNA export by an F-box protein, Mdm30.

Mdm30, an F-box protein in yeast, has been recently shown to promote mRNA export. However, it remains unknown how Mdm30 facilitates mRNA export. Here, we show that Mdm30 targets the Sub2 component of the TREX (Transcription/Export) complex for ubiquitylation and subsequent proteasomal degradation. Such a targeted degradation of Sub2 enhances the recruitment of the mRNA export adaptor, Yra1, to the active genes to promote mRNA export. Together, these results elucidate that Mdm30 promotes mRNA export by lowering Sub2's stability and consequently enhancing Yra1 recruitment, thus illuminating new regulatory mechanisms of mRNA export by Mdm30.

The mRNA cap-binding complex stimulates the formation of pre-initiation complex at the promoter via its interaction with Mot1p in vivo.

The cap-binding complex (CBC) binds to the cap structure of mRNA to protect it from exonucleases as well as to regulate downstream post-transcriptional events, translational initiation and nonsense-mediated mRNA decay. However, its role in regulation of the upstream transcriptional events such as initiation or elongation remains unknown. Here, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation assay in conjunction with transcriptional, mutational and co-immunoprecipitational analyses, we show that CBC is recruited to the body of yeast gene, and then stimulates the formation of pre-initiation complex (PIC) at several yeast promoters through its interaction with Mot1p (modifier of transcription). Mot1p is recruited to these promoters, and enhances the PIC formation. We find that CBC promotes the recruitment of Mot1p which subsequently stimulates PIC formation at these promoters. Furthermore, the formation of PIC is essential for recruitment of CBC. Thus, our study presents an interesting observation that an mRNA binding factor exhibits a reciprocal synergistic effect on formation of PIC (and hence transcriptional initiation) at the promoter, revealing a new pathway of eukaryotic gene regulation in vivo.

Rad26p, a transcription-coupled repair factor, is recruited to the site of DNA lesion in an elongating RNA polymerase II-dependent manner in vivo.

Rad26p, a yeast homologue of human Cockayne syndrome B with an ATPase activity, plays a pivotal role in stimulating DNA repair at the coding sequences of active genes. On the other hand, DNA repair at inactive genes or silent areas of the genome is not regulated by Rad26p. However, how Rad26p recognizes DNA lesions at the actively transcribing genes to facilitate DNA repair is not clearly understood in vivo. Here, we show that Rad26p associates with the coding sequences of genes in a transcription-dependent manner, but independently of DNA lesions induced by 4-nitroquinoline-1-oxide in Saccharomyces cerevisiae. Further, histone H3 lysine 36 methylation that occurs at the active coding sequence stimulates the recruitment of Rad26p. Intriguingly, we find that Rad26p is recruited to the site of DNA lesion in an elongating RNA polymerase II-dependent manner. However, Rad26p does not recognize DNA lesions in the absence of active transcription. Together, these results provide an important insight as to how Rad26p is delivered to the damage sites at the active, but not inactive, genes to stimulate repair in vivo, shedding much light on the early steps of transcription-coupled repair in living eukaryotic cells.