Dissecting RNA Selectivity Mediated by Tandem RNA-Binding Domains
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PROVIDER: PRJNA1176144 | ENA |
REPOSITORIES: ENA
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Project description:RNA-protein interactions are pivotal to proper gene regulation. Many RNA-binding proteins possess multiple RNA-binding domains; however, how these domains interplay to specify and regulate RNA targets remains poorly understood. Here, we investigate three multi-domain proteins, Musashi-1, Musashi-2, and Unkempt, three factors which share a high degree of RNA specificity. We use a combination of massively parallel in vitro assays with random or naturally derived RNA sequences and find that individual domains within a protein can have differing affinities, specificities, and spacing preferences. Further, we emphasize that while all three proteins have overlapping motif specificities, non-overlapping sequences may allow for target discrimination. We carry out large scale competition assays between these proteins and determine how individual protein specificities and affinities influence competitive binding. Integration of in vivo binding and regulation with in vitro specificities shows that target selection involves a combination of the protein intrinsic specificities described here, but cellular context is critical to drive these proteins to motifs in specific transcript regions. Finally, evolutionarily conserved RNA regions display evidence of binding multiple RBPs in vivo, and these RNA regions recapitulate this trend with the highest affinity in vitro. We highlight the importance of understanding features of complex RNA-protein interactions and how protein-target discrimination can be established.
2025-01-31 | GSE280076 | GEO
Project description:Dissecting the functions of mycobacteria RbpA structural domains
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2011-10-24 | E-GEOD-33151 | biostudies-arrayexpress
Project description:RNA polymerase (RNAP) binding protein RbpA contributes to the formation of stable RNAP-promoter open complexes (RPo) and is essential for viability in mycobacteria. Based on structural and biochemical data, four domains have been identified in the RbpA protein: a N-terminal tail (NTT) domain of unknown function, a core domain (CD) that contacts the RNAP β’ subunit in a recently solved crystal structure, a basic linker (BL) that binds DNA, and a s-interaction domain (SID) that binds group I and group II s-factors. However, limited in vivo studies have been performed in mycobacteria and how the individual structural domains of RbpA contribute to RbpA function and mycobacterial gene expression remains mostly unknown. We dissected the roles of the RbpA structural domains in mycobacteria using a panel of rbpA mutants that target individual RbpA domains. The function of each RbpA domain was required for Mycobacterium tuberculosis viability and optimal growth in Mycobacterium smegmatis. We determined that the RbpA SID is both necessary and sufficient for RbpA interaction with the RNAP holoenzyme, indicating that the primary function of the CD is not solely association with the RNAP. We show that RbpA BL and SID are required for stabilization of RPo complexes at the ribosomal RNA rrnAP3 promoter in vitro, while the NTT and CD are dispensable. Finally, we determine that the NTT and CD impact gene expression of a distinct set of genes from that affected by the BL and SID activities. Our findings highlight specific outcomes for the activities of the individual functional domains in RbpA.
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Project description:In the current work, we present a comprehensive coverage of the RNA-binding protein (RBP) interactome of Drosophila at four levels of resolution. The highest level tackled in our study represents the RNA-protein complexes, which we address by combining formaldehyde crosslinking of Drosophila S2 cells followed by polyA+ pulldown using oligo-dT beads. On the second level of resolution we identified direct RBPs in the cell using UV crosslinking. Given the paucity of information available on the RNA-binding regions in the Drosophila RBPs that we uncovered, we developed the CAPRI methodology to dissect RNA binding domains (RBDs) on a systematic level. The CAPRI technique provided the final two levels of resolution of our study. The protocol utilised FASP to enable isolation of peptides crosslinked to RNA and peptides adjacent to these crosslinked sites. The CAPRI pipeline mapped both the peptides in proximity to RNA and the precise amino acids contacting RNA. The RNA crosslinked peptide analysis was made by de novo peptide analysis software, PEAKS (Bioinformatics Solutions Inc). In a separate analysis, we also applied FA crosslinking to RBD capture and showed it to be a viable complementary method to the CAPRI workflow. We next applied CAPRI interactome capture to human cells in order to analyse the evolutionary conservation of newly identified RNA binding domains between the two species. These interactome capture techniques and the datasets acquired using them are discussed in detail in the following sections. In summary, we present a comprehensive resource for the study of RNA-protein interactions at a high throughput scale.
2019-07-02 | PXD013338 | Pride
Project description:U1 small nuclear ribonucleoprotein 70 kDa (U1-70K) and other proteins of the spliceosome complex are mislocalized to cytoplasmic aggregates in Alzheimer’s disease (AD) brain, yet understanding of the specific mechanisms that cause their aggregation is limited. Many of the RNA binding proteins (RBPs) that aggregate in neurodegenerative diseases, including TDP-43 and FUS, self-assemble into RNA granules through disordered low complexity (LC) domains. We report here that a LC domain within U1-70K, that is comprised of tandem arrays of basic (R/K) and acidic (D/E) residues, shares many of the same properties of the Q/N-rich prion-like LC domains found in TDP-43 and FUS. These properties include the ability to self-assemble into oligomers, and to form nuclear granules in cells. To analyze the functional roles of the U1-70K LC domains, we performed co-immunoprecipitation of recombinant U1-70K and deletions lacking the C-terminal LC domain(s) followed by quantitative proteomics. Using a network-driven approach, functional classes of U1-70K interacting proteins were identified that showed a varying dependency on the U1-70K LC domain(s) for their interaction. We characterize a family of structurally homologous RBPs containing U1-70K LC1-like domains including LUC7L3 and RBM25, which require their respective LC1-like domains for reciprocal interactions with U1-70K and for participation in nuclear RNA granules. Finally, we also show that the LC1 domain of U1-70K can interact with Tau from AD brain supporting a hypothesis that mixed charge structural motifs on U1-70K and other RBPs could mediate cooperative interactions with pathological tau isoforms
2018-06-21 | PXD008260 | Pride
Project description:Tandem repeat sequencing in XLID
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Project description:The RNA interactomes of HeLa and HEK293 cells jointly comprise 1106 RNA-binding proteins (RBPs), with almost half of these lacking well-defined RNA-binding domains (RBDs), suggesting the existence of numerous unknown RNA-binding architectures. Here, we report on “RBDmap”, a new method built on interactome capture, to comprehensively identify the RBDs of native RBPs in HeLa cells. Making use of in vivo UV-crosslinking of RBPs to polyadenylated RNAs, capture on oligo(dT) magnetic beads, sequential proteolytic digestion and LC-MS/MS combined with a sophisticated scoring algorithm, RBDmap “re-discoveres” many known RNA-binding sites (e.g. RRM, KH) of numerous well characterized RBPs and suggests novel RBDs.
2016-08-08 | PXD000883 | Pride