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Dithiothreitol (DTT) acts as a specific, UV-inducible cross-linker in elucidation of protein-RNA interactions

ABSTRACT: In the present paper, we introduce dithiothreitol (DTT) as a potent protein-RNA cross-linker. To prove this three model systems, a) a small synthetic peptide from smB'/B protein incubated with U1snRNA oligonucleotide, b) in vitro reconstituted 15.5K protein with U4 snRNA oligonucleotide and c) native ribonucleoprotein-complexes (RNPs) from S. cerevisiae were used . All protein-RNA complexes were UV irradiated and heteroconjugates cross-linked enriched prior to LC-MS analysis. Our results unambiguously show that DTT covalently participates in cysteine-uracil crosslinks which is observable as a mass increments of 152 Da upon mass spectrometric analysis.

INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive

ORGANISM(S): Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Uzma Zaman

LAB HEAD: Henning Urlaub

PROVIDER: PXD002392 | Pride | 2022-03-01

REPOSITORIES: Pride

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Dithiothreitol (DTT) acts as a specific, UV-inducible cross-linker in elucidation of protein-RNA interactions

Project description:In the present paper, we introduce dithiothreitol (DTT) as a potent protein-RNA cross-linker. To prove this three model systems, a) a small synthetic peptide from smB'/B protein incubated with U1snRNA oligonucleotide, b) in vitro reconstituted 15.5K protein with U4 snRNA oligonucleotide and c) native ribonucleoprotein-complexes (RNPs) from S. cerevisiae were used . All protein-RNA complexes were UV irradiated and heteroconjugates cross-linked enriched prior to LC-MS analysis. Our results unambiguously show that DTT covalently participates in cysteine-uracil crosslinks which is observable as a mass increments of 152 Da upon mass spectrometric analysis.

2015-10-13 | PXD002656 | Pride

Photo-cross-linking mass spectrometry for single amino acid assignment of RNA-binding sites in RNA-binding proteins

Project description:Mass spectrometry data of peptide–RNA cross-links derived from complex biological systems. Yeast (pre-)mRNPs were isolated by TAP tag purification of Cbp20; human protein–RNA complexes were assembled on an aptamer tagged pre-mRNA. Isolated complexes were UV irradiated. For each of the aforementioned experiments, a non-irradiated control was prepared. Yeast RBPs cross-linked in vivo after labeling with 4-thio-uridine (4SU) were isolated with oligo d(T). All samples were enriched for cross-linked heteroconjugates prior to LC-MS analysis.

2014-08-07 | PXD000513 | Pride

MS-based analysis of enzymatic methylation of an in vitro-transcribed lncRNA

Project description:The field of epitranscriptomics is growing in importance, with chemical modification of RNA being associated with a wide variety of biological phenomena. Mass spectrometry (MS) enables the identification of modified RNA residues within their sequence contexts, by using analogous approaches to shotgun proteomics. We have developed a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), as part of the OpenMS software framework. NASE allows the reliable identification of (modified) RNA sequences from LC-MS/MS data in a high-throughput fashion. For this validation dataset, we prepared two samples of an in vitro-transcribed yeast lncRNA (NME1, 340 nt long), one of which was treated with an RNA methyltransferase (NCL1) catalyzing the 5-methylcytidine (m5C) modification. These samples were subsequently digested with an RNA endonuclease (RNase) to generate oligonucleotide sequences of a length amenable to mass spectrometry.

2020-03-31 | PXD016308 | Pride

MS-based analysis of enzymatic methylation of an in vitro-transcribed lncRNA

2018-12-31 | PXD012095 | Pride

Extended DNA threading through a dual-engine motor module in the activating signal co-integrator complex

Project description:Activating signal co-integrator complex (ASCC) supports diverse genome maintenance and gene expression processes. Its ASCC3 subunit is an unconventional nucleic acid helicase, harboring tandem Ski2-like NTPase/helicase cassettes crucial for ASCC functions. Presently, the molecular mechanisms underlying ASCC3 helicase activity and regulation remain unresolved. Here, we present cryogenic electron microscopy, DNA-protein cross-linking/mass spectrometry as well as in vitro and cellular functional analyses of the ASCC3-ASC1/TRIP4 sub-module of ASCC. Unlike the related spliceosomal SNRNP200 RNA helicase, ASCC3 can thread substrates through both helicase cassettes. ASC1 docks on ASCC3 via a zinc finger domain and stimulates the helicase by positioning a C-terminal ASC1-homology domain next to the C-terminal helicase cassette of ASCC3, likely assisting the DNA exit. ASC1 binds ASCC3 mutually exclusively with the dealkylase, ALKBH3, directing ASCC for specific ASCC-dependent processes. Our findings define ASCC3-ASC1/TRIP4 as a tunable motor module of ASCC that encompasses two cooperating ATPase/helicase units functionally expanded by ASC1/TRIP4.

2023-03-06 | PXD036106 | Pride

Identification of VE-cadherin associated proteins regulated through the lncRNA LASSIE

Project description:The shear stress-regulated lncRNA LASSIE interacts with cytoskeletal proteins as shown by RNA-antisense purification and subsequent mass spectrometry analysis using an anti-LASSIE and a non-targeting 3’ Desthiobiotin-TEG labeled 2’ O-Me-RNA. As this analysis resulted in high protein binding to the non-targeting control oligonucleotide, the RNA antisense purification was repeated in HUVECs treated with control and anti-LASSIE siRNA. Mass spectrometry analysis confirmed binding of LASSIE to the Intermediate filament protein nestin, as nestin enrichment was decreased in the absence of LASSIE. Additional functional analyses demonstrate a role of LASSIE in shear stress sensing and barrier function in endothelial cells, by stabilizing endothelial cell junctions through connection to the cytoskeleton.

2021-09-09 | PXD018734 | Pride

Liver proteome upon in vivo N-acetylgalactosamine-conjugated antisense oligonucleotide silencing of ceramide synthase 2

Project description:Mouse liver proteome was investigated upon in vivo mouse treatment with a N-acetylgalactosamine-conjugated antisense oligonucleotide engineered to silence ceramide synthase 2 specifically in hepatocytes in vivo. The data is a part of a study on the involvement of ceramide enzymatic machinery in cardiovasular disorders and its potential as a target for the disease treatment.

2023-05-10 | PXD025957 | Pride

LC-MS/MS analysis of human long ribosomal RNA

Project description:The field of epitranscriptomics is growing in importance, with chemical modification of RNA being associated with a wide variety of biological phenomena. Mass spectrometry (MS) enables the identification of modified RNA residues within their sequence contexts, by using analogous approaches to shotgun proteomics. We have developed a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), as part of the OpenMS software framework. NASE allows the reliable identification of (modified) RNA sequences from LC-MS/MS data in a high-throughput fashion. For this validation dataset, we generated samples of human long ribosomal RNA from a cellular extract. The samples were RNase-treated prior to nanoflow LC-MS/MS analysis.

2020-03-31 | PXD016323 | Pride

LC-MS/MS analysis of an intact synthetic microRNA

Project description:The field of epitranscriptomics is growing in importance, with chemical modification of RNA being associated with a wide variety of biological phenomena. Mass spectrometry (MS) enables the identification of modified RNA residues within their sequence contexts, by using analogous approaches to shotgun proteomics. We have developed a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), as part of the OpenMS software framework. NASE allows the reliable identification of (modified) RNA sequences from LC-MS/MS data in a high-throughput fashion. For this validation dataset, oligonucleotides with the sequence of mature Drosophila let-7 microRNA, 21 nt in length, were produced synthetically in unmodified and modified (2’-O-methylated at the 3’ uridine) forms. We characterised a 1:1 mixture of both forms of this RNA.

2019-01-10 | PXD012094 | Pride

LC-MS/MS analysis of human cellular tRNA

Project description:The field of epitranscriptomics is growing in importance, with chemical modification of RNA being associated with a wide variety of biological phenomena. Mass spectrometry (MS) enables the identification of modified RNA residues within their sequence contexts, by using analogous approaches to shotgun proteomics. We have developed a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), as part of the OpenMS software framework. NASE allows the reliable identification of (modified) RNA sequences from LC-MS/MS data in a high-throughput fashion. For this validation dataset, we generated a sample of human total tRNA from a cellular extract - a complex mixture of highly modified RNAs. This sample was RNase-treated prior to nanoflow LC-MS/MS analysis.

2018-12-31 | PXD012097 | Pride

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