Project description:During the last decade several examples of coordination between gene transcription and mRNA degradation have been reported. mRNA imprinting by Rpb4 and 7 subunits of RNA polymerase II (RNAPII) and by the Ccr4-Not complex allows controlling its fate during transcription. Transcription regulation by mRNA degradation factors like Xrn1 constitutes a feedback loop that contributes to mRNA homeostasis. Mechanistic details of these phenomena are unclear. Most studies involve measurement of mRNA decay rates, usually by stressing procedures such as transcriptional shut-off or incorporation of modified nucleotides that can lead to biased results. In this work we have used the easily repressible yeast GAL1 gene to perform a genetic analysis of mRNA synthesis and degradation under physiological conditions. We combined this experimental approach with computational multi-agent modelling, testing different possibilities of Xrn1 and Ccr4-Not action in gene transcription. This double strategy brought us to conclude that Xrn1 regulates RNAPII backtracking in a Ccr4-independent manner. We validated this conclusion measuring TFIIS genome-wide recruitment to elongating RNAPII molecules. We found that xrn1∆ and ccr4∆ exhibited very different patterns of TFIIS/RNPAII which confirmed their differential role in controlling transcription elongation.
Project description:N-terminal acetylation of proteins is a key modification in eukaryotes; however, our understanding of its biological function in plants is limited. Naa50 is the catalytic subunit of the protein N-terminal acetyltransferase NatE complex. We previously showed that the absence of Naa50 led to sterility in Arabidopsis thaliana. In the present study, we show that a lack of Naa50 in Arabidopsis resulted in collapsed and sterile pollen grains. Further study revealed that the mutation of Naa50 accelerated programmed cell death in the tapetum. Expression pattern analysis showed that Naa50 was specifically expressed in tapetal cells from anthers at stages 9–11 of pollen development, when tapetal programmed cell death occurs. Reciprocal cross analyses indicated that the male sterility of naa50 plants was due to sporophytic effects. Transcriptome sequencing of closed buds showed that the deletion of Naa50 resulted in up-regulation of the cysteine protease-coding gene CEP1 and impaired the expression of several genes that function in pollen wall deposition and pollen mitosis. Our findings suggest that Naa50 regulates cell degradation in the tapetum during anther development and plays an important role in pollen development by affecting several pathways.
Project description:Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in a variety of cancers. The ubiquitin ligase Cbl regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. We used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes in the absence and presence of EGF stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by the EGFR inhibitor erlotinib. CRISPR knockout of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and trafficking to the early endosome. FLOT2 interacts with both Cbl and EGFR. Downregulation of EGFR upon FLOT2 loss is Cbl-dependent, as co-knockdown of Cbl and Cbl-b restored EGFR levels. Stable overexpression of FLOT2 in HeLa cells decreased EGF-stimulated EGFR phosphorylation and ubiquitination. Overexpression of wild type (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induces EGFR-dependent proliferation and anchorage-independent cell growth. Lastly, FLOT2 knockout increases tumor formation and tumor volume in nude mice and NSG mice, respectively. These data demonstrate that FLOT2 negatively regulates EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation. FLOT2 negatively regulates proliferation in vitro and in vivo.
Project description:Epithelial-Mesenchymal Transition (EMT) is essential for tissue patterning and organization. It involves both regulation of cell motility and alterations in the composition and organization of the extracellular matrix (ECM); a complex environment of proteoglycans and fibrous proteins that promotes tissue homeostasis, regulates signaling in response to chemical and biomechanical stimuli and is often dysregulated in diseases such as cancer and fibrosis. Here, we demonstrate that Basonuclin-2 (BNC2), a mesenchymal-expressed gene that has been widely associated with cancer and developmental defects by genome-wide association study (GWAS), is a novel regulator of ECM composition and degradation. We find that at endogenous levels, BNC2 controls the expression of specific collagens, matrix metalloproteases and other matrisomal components in breast cancer cells, and in fibroblasts that are primarily responsible for the deposition and processing of the ECM within the tumour microenvironment. In so doing, BNC2 modulates the motile and invasive properties of cancers which likely explains the association of high BNC2 expression with increasing cancer grade and poor patient prognosis.