Project description:RNA-protein interactions underlie a wide range of cellular processes. Improved methods are needed to systematically map RNA-protein interactions in living cells in an unbiased manner. We used two approaches to target the engineered peroxidase APEX2 to specific cellular RNAs for RNA-centered proximity biotinylation of protein interaction partners. Both an MS2-MCP system and an engineered CRISPR-Cas13 system were used to deliver APEX2 to the human telomerase RNA hTR with high specificity. One-minute proximity biotinylation captured candidate binding partners for hTR, including more than a dozen proteins not previously linked to hTR. We validated the interaction between hTR and the N 6-methyladenosine (m6A) demethylase ALKBH5 and showed that ALKBH5 is able to erase the m6A modification on endogenous hTR. ALKBH5 also modulates telomerase complex assembly and activity. MS2- and Cas13-targeted APEX2 may facilitate the discovery of novel RNA-protein interactions in living cells.
Project description:We sought to map RBM6 interactome using ascorbate peroxidase (APEX2)-based proximity labelling combined with mass spectrometry. Toward this end, we used CRISPR-cas9 methodology to establish MCF10A cell line expressing APEX2 fused to the N-terminal of RBM6, hereafter called MCF10AAPEX2-RBM6. The peroxidase activity of APEX2-RBM6 fusion was confirmed. Next, RBM6 proximal proteins that were biotinylated by APEX2 activation using hydrogen peroxidase (H2O2) in the presence of biotin-phenol were isolated and subjected to mass spectrometry. We identified 173 (P-value<0.05) RBM6 proximity-interaction partners.
Project description:We developed a method using site-specific genetic code expansion strategy in conjunction with APEX2-mediated proximity labeling to decipher interactors of hPTMs in living cells via quantitative proteomics. The method allows site-specific incorporation of a PTM-modified amino acid into histones that fused to APEX2, facilitating the labeling of the PTM-modified histones to binding proteins in vivo. Thus, this system will be a promising alternative strategy for interpreting the interactomes of hPTMs in cellular contexts.
Project description:We established an APEX proximity labeling strategy in coupled with mass spectrometry to identify interacting proteins of RNF214. In this approach, we first fused an engineered ascorbate peroxidase (APEX2) to either N-terminus or C-terminus of RNF214, expressed these two fusion proteins in HLF, an HCC cell line, near the endogenous level, and generated short-lived radicals around the APEX2-RNF214 fusion proteins to label biotin on nearby interactive proteins by adding hydrogen peroxide (H2O2) and biotin-phenol (also called biotin-tyramide) transiently. Biotinylated proteins were then isolated using Streptavidin resin for protein identification by mass spectrometry.
Project description:We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.
Project description:Using APEX2, an engineered peroxidase, as an imaging tag, we successfully located iPSC-derived cardiomyocytes (iPSC-CMs) engrafted in post-myocardial infarction mouse hearts for more than 6 months. APEX2 caused clear contrast in X-ray microscopy and electron microscopy, and relatively well organized sarcomere structures were formed in iPSC-CMs. Electron microscopic tomography further found the development of T-tubules and dyads in APEX2-labelled cells, which are the signature characteristics of the maturation of cardiomyocytes.
Project description:The subject of the current study is the finding of possible molecular partners of Drosophila EcR receptor. Two labelling enzymes (BioID2 and APEX2) were fused to EcR or Usp to biotin label the surrounding proteins. All fused proteins were expressed using the Act5C promoter in Drosophila S2 cells. To ensure functionality of the generated proteins, we verified their ability to bind EcR and Usp sites in the Drosophila genome with the ChIP-Seq. Our results demonstrate that EcR and Usp fusions can be recruited to genomic sites endogenous for the EcR/Usp proteins. Conversely, unfused BioID2 and APEX2 enzymes do not bind to EcR/Usp sites. A more in-depth study was conducted to clarify the association of EcR/Usp with one of the detected proteins, CP190, a well-described cofactor of Drosophila insulators. ChIP-Seq experiments revealed an enrichment of CP190 binding on transcription start and end sites of 20E-dependent genes but not at EcR/Usp-bound enhancers.
Project description:In this project we used an unbiased approach to identify the proteins that interact with NDUFS4 in the mouse podocytes using the APEX2 proximity labeling system. An immortalized murine podocyte was permanently transduced with a doxycycline (DOX) inducible NDUFS4-APEX2 chimeric construct. The cells were treated with DOX for 72 h to induce NDUFS4-APEX2 expression, followed by H2O2 activation of APEX2 mediated biotinylation of neighboring proteins. Biotinylated proteins were pull down by streptavidin beads and analyzed by LC-MS/MS. Cells without DOX induction or with DOX but without H2O2 activation were used as controls
Project description:Proximity labeling approach to identify protein inside nuclear envelope blebs arising in Torsin-deficient HeLa cells. WT and TorsinKO cells were engineered to express MLF2-APEX2 fusion. APEX reaction was carried out via 1 mM H2O2 for 1 minute in the presence of biotin phenol. Biotinylated protein were captured via streptavidin beads. Each APEX reaction was accompanied by an untreated (no H2O2) control.