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Identification of human proteins whose binding to the N6-methyladenosine-containing hairpin from 28S rRNA is modulated by methylation

ABSTRACT: We have recently found that the human methyltransferase ZCCHC4 is responsible for introducing the single m6A modification in 28S rRNA. The project is aimed at further elucidating the functional consequences of such methylation. To this end, protein mass spectrometry is utilized to identify proteins that bind preferentially to the methylated or unmethylated form of the m6A containing hairpin sequence in 28S rRNA. Specifically, this is done by incubating a HeLa cell extract with an methylated or unmethylated RNA oligonucleotide containing a biotin affinity tag, which is used to pull-down the RNA with associated proteins.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cervix Epithelium, Hela Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: Pascal Jansen

LAB HEAD: Michiel Vermeulen

PROVIDER: PXD013354 | Pride | 2019-11-22

REPOSITORIES: Pride

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A chromatin-regulated biphasic circuit coordinates IL1B-mediated inflammation

Project description:Inflammation is characterized by a biphasic cycle consisting initially of a pro-inflammatory phase which is subsequently resolved by anti-inflammatory processes. The coordination of these two disparate states needs to be highly controlled, suggesting that the regulation of the cytokines that drive these processes are intimately linked. Interleukin-1 beta (IL1B) is a master regulator of pro-inflammation and is encoded within the same topologically associated domain (TAD) as interleukin-37 (IL37). IL37 has recently emerged as a powerful anti-inflammatory cytokine which diametrically opposes the function of IL1B. Within this TAD, we identified a novel long non-coding RNA called AMANZI which negatively regulates IL1B expression and trained immunity through the induction of IL37 transcription. We found that the activation of IL37 occurs through the formation of a dynamic long-range chromatin contact that leads to the temporal delay of anti-inflammatory responses. The common variant rs16944 present in AMANZI augments this regulatory circuit, predisposing individuals to enhanced pro-inflammation or immunosuppression. Our work illuminates a chromatin-mediated biphasic circuit coordinating expression of IL1B and IL37, thereby regulating two functionally opposed states of inflammation from within a single TAD.

2023-10-27 | PXD045971 | Pride

Identification of RNA binding proteins that interact with AU-rich elements of Lif 3`UTR

Project description:Leukemia inhibitory factor (LIF) is an important endogenous factor for photoreceptor protection. Lif is known to be upregulated both in induced and inherited disease models of photoreceptor degeneration only in a subset of Muller cells. Since Lif is induced in Muller cells in response to photoreceptor injury, we studied its regulation in Muller cells. We have recently identified several AU-rich elements (AREs) within the 3′ untranslated region (UTR) of Lif . We also showed that as a result of these elements Lif encodes a highly unstable mRNA. mRNAs containing AREs in the 3`UTR undergo rapid ARE-mediated mRNA decay in the cytoplasm which is mediated by the RNA–binding proteins. In order to identify the RNA-binding proteins that are involved in the regulation of Lif transcripts, we will use the biotin-labelled RNA. By using this methodology, we will isolate the related RNA binding proteins. We plan to analyze the isolated proteins by mass spectrometry, and compare the mass spectrometry results of different regions with or without AREs . Once we identify the specific proteins that bind only to AREs, we will do the functional characterization of these proteins by cotransfecting the constructs with AREs and the siRNA that inhibits the expression of related RNA binding protein.

2016-07-20 | PXD001463 | Pride

Identification of mRNA processing complexes bound to histone H2A.2 3’ untranslated region

Project description:Processing of 3’ untranslated region (3’-UTR) of precursor messenger RNAs (pre-mRNA) is a fundamental step in mRNA maturation, where the mRNA transcript is cleaved at a specific site, located downstream the open reading frame of the encoded gene. The position of the cleavage site is determined by cis-acting RNA sequence elements, which are recognized with high specificity by large 3’ end processing multiprotein complexes. Two main categories of metazoan 3’ pre-mRNA processing have been identified so far, describing elaboration of either canonical mRNAs – occurring during all phases of the cell cycle and containing a characteristic Poly-Adenylation Signal (PAS) element – or histone mRNAs – prevalently occurring during the S phase and containing a Histone Downstream Element (HDE). In this work, we isolate both human endogenous canonical and histone 3’ pre-mRNA processing complexes from human nuclear extracts by using a pre-mRNA containing the 3’UTR sequence of replication-dependent histone H2A.2. This H2A.2 pre-mRNA (H2A_4m), containing both an HDE sequence and a PAS element in a native overlaid position, was modified with a 5’ photocleavable biotin tag, and with modification of few bases to allow complex assembly and purification by affinity chromatography on streptavidin-agarose beads, elution by UV irradiation and subsequent mass spectrometric identification of bound 3’ mRNA processing factors.

2022-12-09 | PXD034984 | Pride

Aubergine protein interactors in 0-2 hour-Drosophila embryos

Project description:To identify Aub function in mRNA regulation in the Drosophila embryo, we have performed mass spectrometry analysis of Aub interactors, following immunoprecipitation of GFP-Aub in 0-2 hour-embryos. Immunoprecipitation of GFP alone was used as negative control. Because Aub accumulates at high levels in the germ plasm, GFP-Aub immunoprecipitation was also performed in oskar mutant embryos that do not assemble the germ plasm. Proteins coprecipitating with GFP-Aub were similar in wild-type and oskar mutant embryos. Translation factors were enriched among proteins coprecipitating with Aub.

2020-04-27 | PXD016399 | Pride

Transcription profiling of Vicia narbonensis (legume) and Pisum sativum (pea) expressing a bacterial phosphoenol pyruvate carboxylase during seed maturation

Project description:In order to change assimilate partitioning during seed maturation a bacterial phosphoenolpyruvate carboxylase (PEPC) has been expressed in V. narbonensis under control of the LegB4 promoter. Transgenic bean seeds accumulate app. 20% more protein and have higher seed weight. Physiological and biochemical analyses indicate a shift of metabolic fluxes from sugars/starch to organic acids/free amino acids suggesting increased anaplerotic carbon flow. This indicates that the availability of carbon acceptors limits seed protein synthesis. Gene expression was analysed in growing embryos using macro-arrays containing 5,548 seed-specific pea genes. Radioactive labeled cDNA probes were prepared from RNA isolated from embryo of developing seeds of wild type and transgenic PEPC plants of V. narbonensis (15, 17, 19, 21, 25, and 30 DAP) and hybridized to cDNA macroarrays.

2007-11-14 | E-IPKG-2 | biostudies-arrayexpress

Transcription profiling of pea seed development

Project description:SnRK1 (sucrose-non-fermenting-1-related) protein kinases are involved in the regulation of plant metabolism controlling both gene expression and phosphorylation. The aim of the study was to investigate the role of SnRK1 in pea seed development. To study the effect of SnRK1 deficiency, transgenic pea plants were generated carrying a gene for VfSnRK1 in antisense orientation under control of seed specific vicilin promoter. Selected transgenic lines were characterized with decreased levels of PsSnRK1 mRNA and reduced up to 71% phosphorylation activity. Antisense inhibition of SnRK1 resulted in reduced seeds fresh weight, defect of pollen development. To dissect the SnRK1-antisense phenotype at the molecular level, a search for genes with differential expression patterns in transgenic plant versus wild type seeds has been performed using cDNA macroarray analysis. Radioactive labeled cDNA probes were prepared from RNA isolated from embryo of developing seeds of wild type (11, 13, 15, 17, 19, and 21 DAP) and transgenic SnRK1-antisense plant (13, 15, 17 and 19 DAP), which correspond to the transition phase of seed development, and hybridized to cDNA macroarrays.

2013-01-30 | E-IPKG-1 | biostudies-arrayexpress

Identification of human mRNA processing complexes bound to histone H2A.2 3’ untranslated region

2022-12-09 | PXD027636 | Pride

Inverted chromatin immunoprecipitation identifies Tcf1 as a negative regulator of Foxp3 expression

Project description:Regulatory T (Treg) cells are important regulators of the immune system and have versatile functions for the homeostasis and repair of tissues. They express the forkhead box transcription factor Foxp3 as a lineage-defining protein. In mature Treg cells, the Foxp3 core promoter is unmethylated indicating that this area could harbor a transcription factor complex to initiate or repress gene expression, respectively. We used an unbiased method to identify Foxp3-promoter-binding transcription factors (TFs) by inverted chromatin immunoprecipitation (IP) followed by quantitative mass spectrometry. We identified several candidate factors which showed Foxp3-promoter suppressive capacity, one of which was T-cell factor 1 (Tcf1). Using viral overexpression and CRISPR/Cas knockout studies, we identified Tcf1 as a repressor of Foxp3 expression in primary conventional CD4 T cells (Tconv). In Tcf1-deficient animals, increased levels of Foxp3intermediateCD25negative T cells were identified in secondary lymphoid tissues, implicating that Tcf1 protects Foxp3-negative T cells from inadvertent Foxp3 expression.

2020-05-26 | PXD018764 | Pride

Cisplatin induces the rearrangement of RBPs on CDKN1B/p27 mRNA

Project description:Cisplatin (CP) is a chemotherapeutic drug that is used to cure different types of cancer. CP induces DNA damage and leads to cell cycle arrest. The cyclin-dependent kinase inhibitor 1B (CDKN1B), also termed p27, plays an important role in the drug response ; and increased levels of p27 correlated with CP resistance. In HEK293 cells, we observed that p27 mRNAs levels increased whereas protein level drastically decreased in cells treated with CP; suggesting post-transcriptional regulatory events. To further understand the underlying mechanisms, we applied a biochemical approach combined with mass-spectrometry to systematically identify the RNA-binding proteins (RBPs) that are bound to the 3’UTR of p27 mRNAs in CP-treated versus non-treated cells in vivo. We found that 24 proteins, most of them known RBPs such HuR, hNRNPD, changed their association with p27 mRNA upon CP treatement. Furthermore, knock-down of a subset of the identified RBPs led to the inhibition of the CP-induced increase of p27 mRNA levels. In conclusion, these results highlight substantial rearrangement between RBPs and p27 mRNA upon CP treatment and corroborate the importance of post-transcriptional control in cellular drug response.

2019-09-27 | PXD008498 | Pride

RNA targets of FASTKD2 in Human HEK293 cells

Project description:A mutation within FASTKD2 causes a rare form of Mendelian mitochondrial encephalomyopathy. To investigate whether and how RNA binding of FASTKD2 contributes to the disease phenotype, we identified the RNA targets of FASTKD2 by iCLIP.

2015-11-09 | E-MTAB-4010 | biostudies-arrayexpress

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