RsfA (YbeB) Proteins Are Conserved Ribosomal Silencing Factors

The YbeB (DUF143) family of uncharacterized proteins is encoded by almost all bacterial and eukaryotic genomes but not archaea. While they have been shown to be associated with ribosomes, their molecular function remains unclear. Here we show that YbeB is a ribosomal silencing factor (RsfA) in the stationary growth phase and during the transition from rich to poor media. A knock-out of the rsfA gene shows two strong phenotypes: (i) the viability of the mutant cells are sharply impaired during stationary phase (as shown by viability competition assays), and (ii) during transition from rich to poor media the mutant cells adapt slowly and show a growth block of more than 10 hours (as shown by growth competition assays). RsfA silences translation by binding to the L14 protein of the large ribosomal subunit and, as a consequence, impairs subunit joining (as shown by molecular modeling, reporter gene analysis, in vitro translation assays, and sucrose gradient analysis). This particular interaction is conserved in all species tested, including Escherichia coli, Treponema pallidum, Streptococcus pneumoniae, Synechocystis PCC 6803, as well as human mitochondria and maize chloroplasts (as demonstrated by yeast two-hybrid tests, pull-downs, and mutagenesis). RsfA is unrelated to the eukaryotic ribosomal anti-association/60S-assembly factor eIF6, which also binds to L14, and is the first such factor in bacteria and organelles. RsfA helps cells to adapt to slow-growth/stationary phase conditions by down-regulating protein synthesis, one of the most energy-consuming processes in both bacterial and eukaryotic cells

Requirement for Interaction of PI3-Kinase p110α with RAS in Lung Tumor Maintenance

SummaryRAS proteins directly activate PI3-kinases. Mice bearing a germline mutation in the RAS binding domain of the p110α subunit of PI3-kinse are resistant to the development of RAS-driven tumors. However, it is unknown whether interaction of RAS with PI3-kinase is required in established tumors. The need for RAS interaction with p110α in the maintenance of mutant Kras-driven lung tumors was explored using an inducible mouse model. In established tumors, removal of the ability of p110α to interact with RAS causes long-term tumor stasis and partial regression. This is a tumor cell-autonomous effect, which is improved significantly by combination with MEK inhibition. Total removal of p110α expression or activity has comparable effects, albeit with greater toxicities

DARPins detect the formation of hetero-tetramers of p63 and p73 in epithelial tissues and in squamous cell carcinoma

The two p53 homologues p63 and p73 regulate transcriptional programs in epithelial tissues and several cell types in these tissues express both proteins. All members of the p53 family form tetramers in their active state through a dedicated oligomerization domain that structurally assembles as a dimer of dimers. The oligomerization domain of p63 and p73 share a high sequence identity, but the p53 oligomerization domain is more divergent and it lacks a functionally important C-terminal helix present in the other two family members. Based on these structural differences, p53 does not hetero-oligomerize with p63 or p73. In contrast, p63 and p73 form hetero-oligomers of all possible stoichiometries, with the hetero-tetramer built from a p63 dimer and a p73 dimer being thermodynamically more stable than the two homo-tetramers. This predicts that in cells expressing both proteins a p63$_{2}$/p73$_{2}$ hetero-tetramer is formed. So far, the tools to investigate the biological function of this hetero-tetramer have been missing. Here we report the generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) that bind with high affinity and selectivity to the p63$_{2}$/p73$_{2}$ hetero-tetramer. Using these DARPins we were able to confirm experimentally the existence of this hetero-tetramer in epithelial mouse and human tissues and show that its level increases in squamous cell carcinoma

Arginine methylation of the c-Jun coactivator RACO-1 is required for c-Jun/AP-1 activation

c-Jun, the major component of the AP-1 transcription factor complex, has important functions in cellular proliferation and oncogenic transformation. The RING domain-containing protein RACO-1 functions as a c-Jun coactivator that molecularly links growth factor signalling to AP-1 transactivation. Here we demonstrate that RACO-1 is present as a nuclear dimer and that c-Jun specifically interacts with dimeric RACO-1. Moreover, RACO-1 is identified as a substrate of the arginine methyltransferase PRMT1, which methylates RACO-1 on two arginine residues. Arginine methylation of RACO-1 promotes a conformational change that stabilises RACO-1 by facilitating K63-linked ubiquitin chain formation, and enables RACO-1 dimerisation and c-Jun interaction. Abrogation of PRMT1 function impairs AP-1 activity and results in decreased expression of a large percentage of c-Jun target genes. These results demonstrate that arginine methylation of RACO-1 is required for efficient transcriptional activation by c-Jun/AP-1 and thus identify PRMT1 as an important regulator of c-Jun/AP-1 function

Mitglied der Hermann von Helmholtz-Gemeinschaft

urn:nbn:de:0005-074943 The transcriptional co-activator function of the LIM-domain protein nTrip6 Zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat

The LIM Domain Protein nTRIP6 Recruits the Mediator Complex to AP-1-Regulated Promoters

Several LIM domain proteins regulate transcription. They are thought to act through their LIM protein-protein interaction domains as adaptors for the recruitment of transcriptional co-regulators. An intriguing example is nTRIP6, the nuclear isoform of the focal adhesion protein TRIP6. nTRIP6 interacts with AP-1 and enhances its transcriptional activity. nTRIP6 is also essential for the transrepression of AP-1 by the glucocorticoid receptor (GR), by mediating GR tethering to promoter-bound AP-1. Here we report on the molecular mechanism by which nTRIP6 exerts these effects. Both the LIM domains and the pre-LIM region of nTRIP6 are necessary for its co-activator function for AP-1. Discrete domains within the pre-LIM region mediate the dimerization of nTRIP6 at the promoter, which enables the recruitment of the Mediator complex subunits THRAP3 and Med1. This recruitment is blocked by GR, through a competition between GR and THRAP3 for the interaction with the LIM domains of nTRIP6. Thus, nTRIP6 both positively and negatively regulates transcription by orchestrating the recruitment of the Mediator complex to AP-1-regulated promoters