Project description:FAM60A, traditionally linked to chromatin remodeling within the Sin3/HDAC complex, has emerged as a critical regulator in RNA splicing. Employing an integrative approach that combines immunological assays, CRISPR/Cas9 technology, comprehensive genomics, proteomics, and advanced cross-linking mass spectrometry (XL-MS), supplemented by sophisticated 3D molecular modeling, our study challenges and extends the existing understanding of FAM60A's functional dynamics. Contravening previous perceptions, our findings elucidate that FAM60A does not engage directly with SIN3A, but rather establishes direct interactions with SAP30 and HDAC1, redefining its relationship with the Sin3/HDAC complex. These interactions, deciphered through detailed 3D structural analysis backed by cross-linking constraints, signify a complex architectural role of FAM60A within chromatin remodeling processes. Moreover, our research unveils FAM60A's pivotal role in RNA processing, particularly in splicing regulation. Through extensive molecular interactions with a diverse array of mRNA-binding proteins and principal spliceosome components, FAM60A emerges as a key regulator of RNA splicing. This expanded role delineates its influence on gene expression regulation, spotlighting its capacity to modulate critical cellular processes. In sum, this study unveils FAM60A's key role in gene regulation and RNA splicing, suggesting new paths for cellular and therapeutic research.
Project description:Cross-linking/mass spectrometry was used to study inter-domain interactions in the multifunctional AROM complex from Chaetomium thermophilum.
Project description:UV cross-linking and immunoprecipitation (CLIP) and individual-nucleotide resolution CLIP (iCLIP) are the most frequently used methods to study protein-RNA interactions in the intact cells and tissues, but their relative advantages or inherent biases have not been evaluated. To benchmark CLIP and iCLIP method, we performed iCLIP with Nova protein, which is the most extensively studied protein by CLIP. Further, we assessed UV-C-induced cross-linking preferences, by exploiting the UV-independent formation of covalent RNA cross-links of the mutant RNA methylase NSUN2.
Project description:Chemical cross-linking coupled to mass spectrometry using the amine-reactive DSS reagent was used to study the interactions between the RNA helicase Prp43 and its interactors, Pxr1 and Tma23, in Saccharomyces cerevisiae.
Project description:This study aims to study binding events between the Zika virus RNA genome and endogenous transcripts during infection of a human cell line. We develop a novel psolaren-based cross-linking technique to preserve interactions between mRNA and the Zika genome. Interacting RNA molecules are ligated together prior to reversal of the cross-links and selection for Zika-containing fragments. Reverse transcription and paired-end high-throughput sequencing then allow us to identify the interacting transcripts. We generated three batches of libraries, where all samples in each batch were generated from the same pool of RNA. Within each batch, we have the livefire sample, where the protocol was performed as described above; a reverse-control sample, where the protocol was performed by reversing the cross-links prior to ligation; and a no-cross-link control, where the protocol was performed without any cross-linking. The latter two samples represent negative controls where no genuine interactions should be observed.
Project description:Structural study of the polyA signal recognition by the human CPSF using X-ray crystallography, cross-linking MS, pull-downs and fluorescence polarisation assays.
Project description:Cross-linking MS data from the yeast Mediator complex. Cross-linking was performed using either BS3 or 1:1 mix of d0:d12 DSS. Includes unfractionated, SEC enriched, high pH reverse phase fractionated, DDA and inclusion list generated files.
Project description:The neuronal SNARE complex assembles from vesicular Synaptobrevin-2 as well as Syntaxin-1 and SNAP25 anchored to the presynaptic membrane. It mediates fusion of synaptic vesicles with the presynaptic plasma membrane resulting in exocytosis of neurotransmitters into the synaptic cleft. While the general sequence of SNARE complex formation is well-established, our knowledge on possible intermediates is still incomplete. We, therefore, follow the step-wise assembly of the SNARE complex and target individual SNAREs, binary sub-complexes, the ternary SNARE complex as well as interactions with Complexin-1. Using native mass spectrometry, we identify the stoichiometry of preferred sub-complexes and monitor oligomerisation of various assemblies. Importantly, we find that interactions with Complexin-1 reduce self-association of the ternary SNARE complex. Chemical cross-linking provides detailed insights into these interactions suggesting a role during membrane fusion. Taken together, we unravel possible intermediates and preferred sub-complexes and compile a road map of SNARE complex assembly including regulation by Complexin-1.
Project description:Chemical cross-linking coupled to mass spectrometry was used to study the complex between the nucleolar factor, Puf6, and the yeast 60S ribosome. Disuccinimidyl suberate (DSS) was used as the cross-linking reagent.