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Identification of proteins specifically binding to ac4C RNA probes

ABSTRACT: As a newly identified mRNA modification, the regulation of ac4C remains largely unexplored. RNA-binding proteins (RBPs) that specifically binds to ac4C modification and mediate downstream cellular activities (readers) have not been reported yet. We synthesized acetylated and non-acetylated RNA probes by in vitro transcription. The sequences of the probes were segments of FUS and 18s rRNA, which contain ac4C sites as reported. A biotin-RNA pulldown assay and mass spectrometry were performed with HEK 293T cell lysates.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Yuting Xiang

LAB HEAD: Xiaoyan Liang

PROVIDER: PXD027014 | Pride | 2021-07-20

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20200601-001-PR1-20040011-SET2-18s.wiff	Wiff
	20200601-001-PR1-20040011-SET2-FUS.wiff	Wiff
	20200601-001-PR1-20040011-SET2-ac18s.wiff	Wiff
	20200601-001-PR1-20040011-SET2-acFUS.wiff	Wiff
	Ident_Quant_SummaryResult.xlsx	Xlsx

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Project description:Clavulanic acid (CLV) and amoxicillin, frequently administered in combination, can be independently involved in allergic reactions. Protein haptenation with β-lactams is considered necessary to activate the immune system. The aim of this study was to assess the suitability of biotinylated analogues of CLV as probes to study protein haptenation by this β-lactam. Two synthetic approaches afforded the labelling of CLV through esterification of its carboxylic group with a biotin moiety, via either direct binding (CLV-B) or tetraethylenglycol linker (CLV-TEG-B). The second analogue offered advantages as solubility in aqueous solution and potential lower steric hindrance for both intended interactions, with the protein and with avidin. NMR reactivity studies showed that both CLV and CLV-TEG-B reacts through β-lactam ring opening by aliphatic amino nitrogen, however with different stability of resulting conjugates. Unlike CLV conjugates, that promoted the decomposition of clavulanate fragment, the conjugates obtained with the CLV-TEG-B remained linked, as a whole structure including biotin, to nucleophile and showed a better stability. This was a desired key feature to allow CLV-TEG-B conjugated protein detection at great sensitivity. We have used biotin detection and mass spectrometry (MS) to detect the haptenation of human serum albumin (HSA) and human serum proteins. MS of conjugates showed that HSA could be modified by CLV-TEG-B. Remarkably, HSA preincubation with CLV excess only reduced moderately the incorporation of CLV-TEG-B, which could be attributed to different protein interferences. The CLV-TEG-B fragment with opened β-lactam was detected bound to the 404-430HSA peptide of the treated protein. Incubation of human serum with CLV-TEG-B resulted in the haptenation of several proteins that were identified by 2D-electrophoresis and peptide mass fingerprinting as HSA, haptoglobin, and heavy and light chains of immunoglobulins. Taken together, our results show that tagged-CLV keeps some of the CLV features. Moreover, although we observe a different behaviour in the conjugate stability and in the site of protein modification, the similar reactivity indicates that it could constitute a valuable tool to identify protein targets for haptenation by CLV with high sensitivity to get insights into the activation of the immune system by CLV and mechanisms involved in β-lactams allergy.

Project description:The hippocampal proteins obtained from both exercised (moderate-intensity running for 6 weeks) and sedentary mice were separately analyzed for Liquid chromatography–tandem mass spectrometry (LC-MS/MS) identification with three replications. Acetylome data identified 3876 acetyl sites and 1764 acetylated proteins, which constituted 31% of the whole hippocampal proteome (totally 5725 proteins identified). In addition, 1305 proteins with 2769 acetyl sites were quantified. When defining the cutoffs for the fold change in abundance of 1.5 and P value < 0.05, 272 acetyl sites on 252 proteins were differentially regulated between exercise and control mice. Analysis of the subcellular localization showed that these differentially acetylated proteins (DAPs) exert different functions in multiple compartments, including the cytoplasm, mitochondria, nucleus and plasma membrane. The 19 up-regulated acetylated proteins mainly resided in mitochondria, while the 253 down-regulated DAPs were most abundant in cytoplasm, and the annotation of molecular functions were significantly enriched in catalytic activity and binding, respectively. Gene Ontology (GO) analysis showed that DAPs were primarily correlated with cytoskeleton, myelin sheath and axons in the cellular component category; and actin cytoskeleton organization, regulation of protein polymerization or depolymerization, and hexose/glucose metabolic process were significantly enriched in the biological process category. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DAPs were involved in central carbon metabolism, neurodegeneration diseases, synapses, and various signaling pathways. Among them, chronic exercise induced significant alterations in phototransduction, carbon-related metabolism (carbohydrate digestion and absorption, glycolysis/gluconeogenesis, fructose and mannose metabolism, and pyruvate metabolism) and Hippo signaling pathway. Meanwhile, 21 proteins were significantly expressed, which were enriched in the pathway of complement and coagulation cascades.

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Identification of proteins specifically binding to ac4C RNA probes

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