Hybrid structural modeling of protein-RNA complexes using crosslinking of segmentally isotope labeled RNA and tandem mass spectrometry
ABSTRACT: To study protein-RNA interactions at single amino acid and single nucleotide resolution we developed a new approach, termed cross-linking of segmentally isotope labeled RNA and tandem mass spectrometry (CLIR-MS/MS). The method was developed using the PTBP1-EMCV IRES complex as a model system and additionally applied to the U1 snRNP complex. Data from both complexes are included in this submission.
Ribonucleoproteins (RNPs) are key regulators of cellular function. We established an efficient approach, crosslinking of segmentally isotope-labeled RNA and tandem mass spectrometry (CLIR-MS/MS), to localize protein-RNA interactions simultaneously at amino acid and nucleotide resolution. The approach was tested on polypyrimidine tract binding protein 1 and U1 small nuclear RNP. Our method provides distance restraints to support integrative atomic-scale structural modeling and to gain mechanistic ...[more]
Project description:Chemical cross-linking coupled to mass spectrometry was used to study the A. thaliana DDR complex consisting of DMS3, RMD1 and a peptide from the interaction region of DRD1. Cross-linking was performed using different cross-linking chemistries: disuccinimidyl suberate (DSS) and a combination of adipic acid dihydrazide (ADH) and the coupling reagent, DMTMM.
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.
Project description:Chemical cross-linking coupled to mass spectrometry was used to study the architecture and conformational state(s) of the Drosophila melanogaster origin replication complex (ORC). Two versions of the ORC were subjected to cross-linking with the amine-reactive reagents, disuccinimidyl suberate (DSS). One was a complex with truncated subunits (“core complex”), one with full-length sequences of all six subunits (“full-length complex”).
Project description:Chemical cross-linking coupled to mass spectrometry was used to study the complex between the ribosomal protein, eS26A, and the escortin, Tsr2. Cross-linking was performed using different cross-linking chemistries: (1) disuccinimidyl suberate (DSS); (2) a combination of adipic acid dihydrazide (ADH) and the coupling reagent, DMTMM; (3) a combination of pimelic acid dihydrazide (PDH) and the coupling reagent, DMTMM.
Project description:Chemical cross-linking coupled to mass spectrometry was used to study the complex between the HMCV pentameric complex (gH/gL/UL128/UL130/UL131A) and its receptor, neuropilin-2 (NRP2). Cross-linking was performed using either disuccinimidyl suberate (DSS) or a combination of pimelic acid dihydrazide (PDH) and the coupling reagent, DMTMM.
Project description:Our ChIP resuls suggested that coilin association with U3, snRNA and histone genes might be dependent on coilin-RNA interaction. We used iCLIP of coilin-GFP expressed in HeLa and P19 cell lines at endogenous levels to identify coilin RNA targets and investigate RNA-binding specificity. P19 cells expressing GFP fused to a nuclear localization signal (GFP-NLS) was used as a negative control. iCLIP results revealed that coilin binds several classes of ncRNA including snRNAs, U3 snoRNA and scaRNAs. Interestlignly the majority of coilin targets were intronic snoRNAs, suggesting a novel role of CBs in snoRNA biogenesis. 5 biological replicates from P19 and 2 biological replicates from HeLa cells after UV-crosslinking. Negative control samples prepared from GFP-NLS fusion protein are stored uder accession E-MTAB-747.
Project description:The goal of this study was to investigate the role of intragenic CTCF in alternative pre-mRNA splicing through a combined CTCF-ChIP-seq and RNA-seq approach. CTCF depletion led to decreased inclusion of weak upstream exons. CTCF ChIP-seq was performed in BJAB and BL41 B cell lines and normalized relative to Rabbit Ig control IP-seq reads. RNA-seq was performed in BJAB and BL41 cells transduced with shRNA against CTCF or RFP as a control, and in untransduced cells as well.
Project description:Fanconi Anemia (FA) is a rare genetic disorder characterized by an increased susceptibility to squamous cell cancers. Fifteen FA genes are known, and the encoded proteins cooperate in a common DNA repair pathway. A critical step is the monoubiquitination of the FANCD2 protein, and cells from most FA patients are deficient in this step. How monoubiquitinated FANCD2 suppresses squamous cell cancers is unknown. Here we show that Fancd2-deficient mice are prone to Ras oncogene-driven skin carcinogenesis, while Usp1-deficient mice, expressing elevated cellular levels of Fancd2-Ub, are resistant to skin tumors. Moreover, Fancd2-Ub activates the transcription of the tumor suppressor TAp63, thereby promoting cellular senescence and blocking skin tumorigenesis. For FA patients, the reduction of FANCD2-Ub and TAp63 protein levels may account for their susceptibility to squamous cell neoplasia. Taken together, Usp1 inhibition may be a useful strategy for upregulating TAp63 and preventing or treating squamous cell cancers in the general non-FA population. Examination of FANCD2 binding after UV treatment in 293T cells
Project description:To elucidate the Nodal transcriptional network that governs endoderm formation, we used ChIP-Seq to identify genomic targets for SMAD2/3, SMAD3, SMAD4, FOXH1 and the active and repressive chromatin marks, H3K4me3 and H3K27me3, in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that while SMAD2/3, SMAD4 and FOXH1 target binding is highly dynamic, there is an optimal signature for driving endoderm commitment. Initially, this signature is marked by both H3K4me3 and H3K27me3 as a very broad bivalent domain in hESCs. Within the first 24 hours, at a few select promoters, SMAD2/3 accumulation coincides with H3K27me3 depletion so that these loci become selectively monovalent marked only by H3K4me3. The correlation between SMAD2/3 binding, monovalent formation and transcriptional activation suggests a mechanism by which SMAD proteins coordinate with chromatin at critical promoters to drive endoderm specification. Examination of 2 different histone modifications and 4 different transcription factor associations in 2 cell types. For transcription factor analysis, three biological replicate ChIPs were pooled from each antibody, as well as input controls, for both hESCs and derived endoderm. For histone modifications, two biological replicates for H3K4me3 and three for H3K27me3 were used.