Project description:We report the application of NGS for high-throughput profiling of transcripts assoicated with the ribonuleoprotein (RNP) formed on Vg1 mRNA in oocytes of Xenopus Laevis.
Project description:RNAs interact with networks of proteins to form complexes (RNPs) that govern many biological processes, but inter-protein networks on RNA are currently impossible to examine in a comprehensive way. We developed a live-cell RNP-MaP (RNP network analysis by mutational profiling) chemical probing strategy for mapping simultaneous binding by and cooperative interactions among multiple proteins with single RNA molecules at nucleotide resolution. RNP-MaP revealed that two structurally related, but sequence-divergent noncoding RNAs, RNase P and RMRP, share nearly identical RNP networks and, further, that protein-mediated structural communication identifies function-critical network hubs in these RNAs. RNP-MaP identified previously unknown protein interaction networks within the XIST long noncoding RNA that are conserved between mouse and human RNAs and defined silencing, compartmentalization and splicing communities of proteins whose binding sites are networked together on XIST. The XIST E region contains a dense network of protein interactions, and including PTBP1, MATR3, and TIA1 proteins , which RNP-MaP revealed to each bind the XIST E region via two distinct interaction modes.; Depletion of PTBP1 and MATR3 caused native XIST particles to disperse and disappear in a human cell line. the The highly networked XIST E region was sufficient to mediate XIST RNA-like foci formation in cells. RNP-MaP enables discovery and prioritization of in-cell protein interaction networks critical for function in long RNAs, in the absence of pre-existing knowledge about protein binding sites.
Project description:RNA binding proteins (RBPs) control varied processes, including RNA splicing, stability, transport, and translation. Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease, however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation was combined with mass spectrometry (irCLIP-RNP) to identify RNA-dependent associated proteins (RDAPs) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodeling in response to epidermal growth factor (EGF), uncovering EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP (Re-CLIP) method was also developed. Re-CLIP confirmed binding relationships seen in irCLIP-RNP and identified HNRNPC and UPF1 RBP co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.
Project description:RNA binding proteins (RBPs) control varied processes, including RNA splicing, stability, transport, and translation. Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease, however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation was combined with mass spectrometry (irCLIP-RNP) to identify RNA-dependent associated proteins (RDAPs) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodeling in response to epidermal growth factor (EGF), uncovering EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP (Re-CLIP) method was also developed. Re-CLIP confirmed binding relationships seen in irCLIP-RNP and identified HNRNPC and UPF1 RBP co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.
Project description:C. elegans GLD-2 forms an active PAP with multiple RNA-binding partners to regulate diverse aspects of germline and early embryonic development. One GLD-2 partner, RNP-8, was previously shown to influence oocyte fate specification. To identify transcripts selectively associated with both GLD-2 and RNP-8, we employ a genomic approach using the method of RNA immunoprecipitation followed by microarray analysis (RIP-chip). We used microarrays to identify mRNAs selectively associated with either GLD-2 or RNP-8.
Project description:C. elegans GLD-2 forms an active PAP with multiple RNA-binding partners to regulate diverse aspects of germline and early embryonic development. One GLD-2 partner, RNP-8, was previously shown to influence oocyte fate specification. To identify transcripts selectively associated with both GLD-2 and RNP-8, we employ a genomic approach using the method of RNA immunoprecipitation followed by microarray analysis (RIP-chip). We used microarrays to identify mRNAs selectively associated with either GLD-2 or RNP-8. Worm extracts were prepared from synchronized adult C. elegans (15 h after L4 stage). For GLD-2 IP, an immoblized anti-GLD-2 antibody was then used to purify the GLD-2 complexes from either wild-type (N2) or gld-2(RNAi) worm extracts. RNA was then extracted from the pellets and analyzed on C.elegans Affymetrix genechip. Four biological replicates were performed, each sample processed in parallel. For RNP-8 IP, an immoblized anti-RNP-8 antibody was then used to purify the RNP-8 complexes from either wild-type (N2) or rnp-8(q784) worm extracts and three biological replicates were performed. For wt or gld-2(RNAi) samples, total RNA was extracted from worm extracts and hybridized on C.elegans Affymetrix genechip.
Project description:UV cross-linking and immunoprecipiatation with high throughput sequencing of cytoplasmic SLBP RNP Polyclonal α-SLBP antibody was used to immunoprecipitate SLBP RNP from polyribosomal fractions of HeLa S3 lysates. Mock immunoprecipitation were also performed and serve as a negative control.
Project description:UV cross-linking and immunoprecipiatation with high throughput sequencing of cytoplasmic SLBP RNP Polyclonal α-SLBP antibody was used to immunoprecipitate UV-crosslinked SLBP RNP from cytoplasmic HeLa lysates. Mock immunoprecipitation were also performed and serve as a negative control.