Elucidation of the translation initiation factor interaction network of Haloferax volcanii reveals coupling of transcription and translation in haloarchaea
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ABSTRACT: Translation is an important step in gene expression. Initiation of translation is rate-limiting, and it is 13 phylogenetically more diverse than elongation or termination. Bacteria contain only three initiation 14 factors. In stark contrast, eukaryotes contain more than 10 (subunits of) initiation factors (eIFs). The 15 genomes of archaea contain many genes that are annotated to encode archaeal homologs of 16 eukaryotic initiation factors (aIFs). However, experimental characterization of aIFs is scarce and 17 mostly restricted to very few species. To broaden the view, the protein-protein interaction network of 18 aIFs in the halophilic archaeon Haloferax volcanii has been characterized. To this end, tagged 19 versions of 14 aIFs were overproduced, affinity isolated, and the co-isolated binding partners were 20 identified by peptide mass fingerprinting.
Project description:Using protein microarrays, derived from 642 His-tag proteins, we could distinguish sera from breast-nodule positive patients and healthy control individuals. Each Protein microarray was divided in to 4 sub-arrays. Each protein was spotted in duplicates in each sub-array. For evaluation 24 malignant, 16 benign breast cancer serum samples and 20 healthy control serum samples were used.
Project description:Fragile-X Syndrome (FXS) is a multi-organ disease leading to mental retardation, macro-orchidism in males, and premature ovarian insufficiency in female carriers. FXS is also a prominent monogenic disease associated with autism spectrum disorders (ASD). FXS is typically caused by the loss of FRAGILE X-MENTAL RETARDATION 1 (FMR1) expression, which encodes for the RNA-binding protein (RBP), FMR1 (or FMRP). We report the discovery of the RNA recognition elements (RREs), binding sites, and mRNA targets for wild-type and I304N mutant FMRP isoforms as well as its paralogs, FXR1 and FXR2. RRE frequency, ratio, and distribution determine target mRNA association with FMRP. Among highly-enriched targets, we identified many genes involved in ASD and demonstrate that FMRP can affect their protein levels in cell culture, mice, and human brain. Unexpectedly, we discovered that these targets are also dysregulated in Fmr1-/- mouse ovaries, showing signs of premature follicular overdevelopment. These results indicate that FMRP targets shared signaling pathways across different cellular contexts. As it is become increasingly appreciated that signaling pathways are important to FXS and ASD, our results here provide an invaluable molecular guide towards the pursuit of novel therapeutic targets for these devastating neurological disorders. PAR-CLIP profiling for wild-type and I304N mutant FMRP isoforms as well as paralogs, FXR1 and FXR2.
Project description:Protein Ser/Thr kinase CK2 is involved in a myriad of cellular processes including cell growth and proliferation by phosphorylating hundreds of substrates, yet the regulation process of CK2 function is poorly understood. The CK2 catalytic subunit, CK2α, is phosphorylated at Thr344 and phosphorylation on the C-terminal tail of CK2α is required for interaction with Pin1 protein. The substrate selectivity for protein kinase CK2α was examined by performing kinase assays on protein microarrays spotted with 17,000 human proteins. Semisynthetic CK2α proteins were prepared to contain an unmodified C-terminal tail or phospho-Thr (pThr) at T344. These semisynthetic proteins were used to determine if the phosphorylation-dependent interaction of CK2α with Pin1 can modulate the substrate selectivity for CK2. The different semisynthetic CK2α proteins (unmodified and pThr344) were tested alone and in the presence of the recombinant Pin1 protein. Pin1 has been shown to interaction with CK2α only when CK2α is phoshorylated on its C-terminal site (including Thr344). In the study presented here, kinase assays were performed using two different semisynthetic CK2α proteins: unmodified C-terminal tail and phospho-Thr (pThr) at 344. The semisynthetic proteins were each tested alone and in the presence of the recombinant Pin1 protein. There were four different kinase conditions and each condition was performed in duplicate.
Project description:SrcR388A/Y527F or SrcD386N/Y527F stably transfected SYF cells were serum-starved overnight and then treated with imidazole 5 mM for 1 h or 12 h. RNeasy Miniprep column (Qiagen) was used to prepare total RNA from each treatment group. All samples were run in commercial arrays from Affymetrix,
Project description:Protein Ser/Thr kinase CK2 is involved in a myriad of cellular processes including cell growth and proliferation by phosphorylating hundreds of substrates, yet the regulation process of CK2 function is poorly understood. The CK2 catalytic subunit, CK2α, is modified by O-GlcNAc on Ser347 proximal to a Cdk1 phosphorylation site at Thr344 on the same protein. The substrate selectivity for protein kinase CK2 was examined by performing kinase assays on protein microarrays spotted with 17,000 human proteins. Semisynthetic CK2α proteins were prepared to contain an unmodified C-terminal tail, S-GlcNAc-Serine at S347, or Pfa (non-hyrdolyzeable phosphomimic) at T344. These semisynthetic proteins were used to determine if the posttranslational modifications on CK2 alpha modulate the substrate selectivity for this pleiotropic kinase. The different semisynthetic CK2α proteins were tested alone and in the presence of the regulatory subunit CK2β since it is known that the CK2α subunit is active both in its isolated state and in the heterotetrameric state formed in the presence of the regulatory beta subunit. The CK2β subunit has been shown to modulate CK2 activity with some substrates and not others. In the study presented here, kinase assays were performed using three different semisynthetic CK2 alpha proteins: unmodified C-terminal tail; S-GlcNAc-Ser at 347; and Pfa (phosphomimic) at 344. The semisynthetic proteins were each tested alone and in the presence of the regualatory CK2 beta subunit. There were six different kinase conditions and each condition was performed in duplicate and one no kinase control was performed to eliminate autophorylated proteins.
Project description:To obtain an insight into the in vivo dynamics of RNA polymerase (RNAP) on the B. subtilis genome, we analyzed the distribution of ?A and ? subunits of RNAP and the NusA elongation factor on the genome in exponentially growing cells, using the ChAP (Chromatin Affinity Precipitation)-chip method. In contrast to E. coli RNAP, which often accumulates at the promoter-proximal region, B. subtilis RNAP is evenly distributed from the promoter to the coding sequences in the majority of genes. This finding suggests that B. subtilis RNAP recruited to the promoter promptly translocates away from the promoter to form the elongation complex. We detected RNAP accumulation in the promoter-proximal regions of some genes, most of which are attributed to transcription attenuation systems in the leader region. Our findings suggest that the differences in RNAP behavior during initiation and early elongation steps between E. coli and B. subtilis result in distinct strategies for post-initiation control of transcription. The E. coli mechanism involves trapping at the promoter and promoter-proximal pausing of RNAP in addition to transcription attenuation, whereas transcription attenuation in leader sequences is mainly employed in B. subtilis. Wild-type strain, Bacillus subtilis 168, was also used for RNA and genomic DNA extraction and analysis - RNA data was divided by genome DNA data to normalize (1) PCR bias and (2) copy number of RNA molecule per genome for multi-copy genome of exponentially growing bacteria.
Project description:SAGA is a highly conserved transcriptional co-activator complex involved in multiple steps of transcription with activities that function both pre and post initiation. Loss of individual subunits results in developmental defects, suggesting a role in development. To better understand the roles of SAGA functions in developmental gene expression and it's relationship with RNA polymerase II, we examined its composition, binding profile, and the effects of subunit loss on gene expression in two distinct cell types in late stage Drosophila embryos: muscle and neurons. Chromatin IP of FLAG-tagged SAGA subunit Ada2b, and RNA polymerase II (antibody 4H8), was performed in neuronal (elav+) or muscle (mef2+) cells isolated by FACS from late stage embryos, and compared to whole cell extracts (input). The control constists of an IP performed in neuronal cells from a non-tagged strain.
Project description:His6-Cdc48a and associated proteins purified from H. volcanii. A) SDS- PAGE of proteins purified by Ni2+ -chromatography. H. volcanii carrying empty vector control (H1209-pJAM202c, lane 1) and ectopically expressing His6-Cdc48a (H1209- pJAM1409, lane 2). Red bars indicate regions of gel excised. B) Proteins identified in gel slices by LC-MS/MS analysis. Criteria for protein inclusion: FDR < 0.01%, normalized total spectra minus control > 50, protein threshold > 99% and 2 peptide minimum. Based on spectral counting, Cdc48a and DUF111 were most abundant in regions (a) and (b), respectively, as indicated. DHS1, deoxyhypusine synthase. DUF111, Cdc48a gene neighbor of unknown function. TnaA, tryptophanase; Rps3, ribosomal protein S3; n.d., not detected.
Project description:An important question for the use of the mouse as a model for studying human disease is the degree of functional conservation of genetic control pathways from human to mouse. The human and mouse placenta show structural similarities but there have been no systematic attempt to assess their molecular similarities or differences. We built a comprehensive database of protein and microarray data for the highly vascular exchange region micro-dissected from the human and mouse placenta near-term. Abnormalities in this region are associated with two of the most common and serious complications of human pregnancy, maternal preeclampsia (PE) and fetal intrauterine growth restriction (IUGR), each disorder affecting ~5% of all pregnancies. Over 7,000 orthologs were detected with 70% co-expressed and over 80% of genes known to cause placental phenotypes in mouse were co-expressed. These genes form a tight protein-protein interaction network with novel candidate genes likely to be important in placental structure and/or function. The entire data is available as a web-accessible database to guide the informed development of mouse models to study human disease This experiment is now fully represented in NCBI Peptidome database with accession PSE115; http://www.ncbi.nlm.nih.gov/peptidome/search/index.shtml?acc=PSE115 Microdissection of human villous trees and mouse placental labyrinth. Tissues were split for microarray and protein analysis. For protein analysis samples were first fractionated by differential sucrose gradients into mitochrondria, cytosol, microsomes and nuclei. Mitochrondira and neuclei were each extracted by two different methods for soluble and insoluble material. Each subcellular fraction for each tissue was analysed in quintuplet by 9 step 2 dimensional LC/MSMS. This generated a total of 270 mzXML files for each tissue.