ArreSTick motif controls ß-arrestin-binding stability and extends phosphorylation-dependent ß-arrestin interactions to non-receptor proteins.

The binding and function of ß-arrestins are regulated by specific phosphorylation motifs present in G protein-coupled receptors (GPCRs). However, the exact arrangement of phosphorylated amino acids responsible for establishing a stable interaction remains unclear. We employ a 1D sequence convolution model trained on GPCRs with established ß-arrestin-binding properties. With this approach, amino acid motifs characteristic of GPCRs that form stable interactions with ß-arrestins can be identified, a pattern that we name "arreSTick." Intriguingly, the arreSTick pattern is also present in numerous non-receptor proteins. Using proximity biotinylation assay and mass spectrometry analysis, we demonstrate that the arreSTick motif controls the interaction between many non-receptor proteins and ß-arrestin2. The HIV-1 Tat-specific factor 1 (HTSF1 or HTATSF1), a nuclear transcription factor, contains the arreSTick pattern, and its subcellular localization is influenced by ß-arrestin2. Our findings unveil a broader role for ß-arrestins in phosphorylation-dependent interactions, extending beyond GPCRs to encompass non-receptor proteins as well.

Modulation of GPR133 (ADGRD1) signaling by its intracellular interaction partner extended synaptotagmin 1.

GPR133 (ADGRD1) is an adhesion G-protein-coupled receptor that signals through Gαs/cyclic AMP (cAMP) and is required for the growth of glioblastoma (GBM), an aggressive brain malignancy. The regulation of GPR133 signaling is incompletely understood. Here, we use proximity biotinylation proteomics to identify ESYT1, a Ca2+-dependent mediator of endoplasmic reticulum-plasma membrane bridge formation, as an intracellular interactor of GPR133. ESYT1 knockdown or knockout increases GPR133 signaling, while its overexpression has the opposite effect, without altering GPR133 levels in the plasma membrane. The GPR133-ESYT1 interaction requires the Ca2+-sensing C2C domain of ESYT1. Thapsigargin-mediated increases in cytosolic Ca2+ relieve signaling-suppressive effects of ESYT1 by promoting ESYT1-GPR133 dissociation. ESYT1 knockdown or knockout in GBM slows tumor growth, suggesting tumorigenic functions of ESYT1. Our findings demonstrate a mechanism for the modulation of GPR133 signaling by increased cytosolic Ca2+, which reduces the signaling-suppressive interaction between GPR133 and ESYT1 to raise cAMP levels.

Interaction of ROMK2 channel with lipid kinases DGKE and AGK: Potential channel activation by localized anionic lipid synthesis.

In this study, we utilized enzyme-catalyzed proximity labeling with the engineered promiscuous biotin ligase Turbo-ID to identify the proxisome of the ROMK2 channel. This channel resides in various cellular membrane compartments of the cell including the plasma membrane, endoplasmic reticulum and mitochondria. Within mitochondria, ROMK2 has been suggested as a pore-forming subunit of mitochondrial ATP-regulated potassium channel (mitoKATP). We found that ROMK2 proxisome in addition to previously known protein partners included two lipid kinases: acylglycerol kinase (AGK) and diacylglycerol kinase ε (DGKE), which are localized in mitochondria and the endoplasmic reticulum, respectively. Through co-immunoprecipitation, we confirmed that these two kinases are present in complexes with ROMK2 channels. Additionally, we found that the products of AGK and DGKE, lysophosphatidic acid (LPA) and phosphatidic acid (PA), stimulated the activity of ROMK2 channels in artificial lipid bilayers. Our molecular docking studies revealed the presence of acidic lipid binding sites in the ROMK2 channel, similar to those previously identified in Kir2 channels. Based on these findings, we propose a model wherein localized lipid synthesis, mediated by channel-bound lipid kinases, contributes to the regulation of ROMK2 activity within distinct intracellular compartments, such as mitochondria and the endoplasmic reticulum.

Unveiling the role of PUS7-mediated pseudouridylation in host protein interactions specific for the SARS-CoV-2 RNA genome.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive single-stranded RNA virus, engages in complex interactions with host cell proteins throughout its life cycle. While these interactions enable the host to recognize and inhibit viral replication, they also facilitate essential viral processes such as transcription, translation, and replication. Many aspects of these virus-host interactions remain poorly understood. Here, we employed the catRAPID algorithm and utilized the RNA-protein interaction detection coupled with mass spectrometry technology to predict and validate the host proteins that specifically bind to the highly structured 5' and 3' terminal regions of the SARS-CoV-2 RNA. Among the interactions identified, we prioritized pseudouridine synthase PUS7, which binds to both ends of the viral RNA. Using nanopore direct RNA sequencing, we discovered that the viral RNA undergoes extensive post-transcriptional modifications. Modified consensus regions for PUS7 were identified at both terminal regions of the SARS-CoV-2 RNA, including one in the viral transcription regulatory sequence leader. Collectively, our findings offer insights into host protein interactions with the SARS-CoV-2 UTRs and highlight the likely significance of pseudouridine synthases and other post-transcriptional modifications in the viral life cycle. This new knowledge enhances our understanding of virus-host dynamics and could inform the development of targeted therapeutic strategies.

Hepatitis C virus alters the morphology and function of peroxisomes.

Despite the introduction of effective treatments for hepatitis C in clinics, issues remain regarding the liver disease induced by chronic hepatitis C virus (HCV) infection. HCV is known to disturb the metabolism of infected cells, especially lipid metabolism and redox balance, but the mechanisms leading to HCV-induced pathogenesis are still poorly understood. In an APEX2-based proximity biotinylation screen, we identified ACBD5, a peroxisome membrane protein, as located in the vicinity of HCV replication complexes. Confocal microscopy confirmed the relocation of peroxisomes near HCV replication complexes and indicated that their morphology and number are altered in approximately 30% of infected Huh-7 cells. Peroxisomes are small versatile organelles involved among other functions in lipid metabolism and ROS regulation. To determine their importance in the HCV life cycle, we generated Huh-7 cells devoid of peroxisomes by inactivating the PEX5 and PEX3 genes using CRISPR/Cas9 and found that the absence of peroxisomes had no impact on replication kinetics or infectious titers of HCV strains JFH1 and DBN3a. The impact of HCV on peroxisomal functions was assessed using sub-genomic replicons. An increase of ROS was measured in peroxisomes of replicon-containing cells, correlated with a significant decrease of catalase activity with the DBN3a strain. In contrast, HCV replication had little to no impact on cytoplasmic and mitochondrial ROS, suggesting that the redox balance of peroxisomes is specifically impaired in cells replicating HCV. Our study provides evidence that peroxisome function and morphology are altered in HCV-infected cells.

Proteomic investigation of neural stem cell to oligodendrocyte precursor cell differentiation reveals phosphorylation-dependent Dclk1 processing.

Oligodendrocytes are generated via a two-step mechanism from pluripotent neural stem cells (NSCs): after differentiation of NSCs to oligodendrocyte precursor/NG2 cells (OPCs), they further develop into mature oligodendrocytes. The first step of this differentiation process is only incompletely understood. In this study, we utilized the neurosphere assay to investigate NSC to OPC differentiation in a time course-dependent manner by mass spectrometry-based (phospho-) proteomics. We identify doublecortin-like kinase 1 (Dclk1) as one of the most prominently regulated proteins in both datasets, and show that it undergoes a gradual transition between its short/long isoform during NSC to OPC differentiation. This is regulated by phosphorylation of its SP-rich region, resulting in inhibition of proteolytic Dclk1 long cleavage, and therefore Dclk1 short generation. Through interactome analyses of different Dclk1 isoforms by proximity biotinylation, we characterize their individual putative interaction partners and substrates. All data are available via ProteomeXchange with identifier PXD040652.

Identification and Characterization of Proteins That Are Involved in RTP1S-Dependent Transport of Olfactory Receptors.

Olfaction is mediated via olfactory receptors (ORs) that are expressed on the cilia membrane of olfactory sensory neurons in the olfactory epithelium. The functional expression of most ORs requires the assistance of receptor-transporting proteins (RTPs). We examined the interactome of RTP1S and OR via proximity biotinylation. Deubiquitinating protein VCIP135, the F-actin-capping protein sub-unit alpha-2, and insulin-like growth factor 2 mRNA-binding protein 2 were biotinylated via AirID fused with OR, RTP1S-AirID biotinylated heat shock protein A6 (HSPA6), and double-stranded RNA-binding protein Staufen homolog 2 (STAU2). Co-expression of HSPA6 partially enhanced the surface expression of Olfr544. The surface expression of Olfr544 increased by 50-80%. This effect was also observed when RTP1S was co-expressed. Almost identical results were obtained from the co-expression of STAU2. The interactions of HSPA6 and STAU2 with RTP1S were examined using a NanoBit assay. The results show that the RTP1S N-terminus interacted with the C-terminal domain of HSP6A and the N-terminal domain of STAU2. In contrast, OR did not significantly interact with STAU2 and HSPA6. Thus, HSP6A and STAU2 appear to be involved in the process of OR traffic through interaction with RTP1S.

Stabilization of 14-3-3 protein-protein interactions with Fusicoccin-A decreases alpha-synuclein dependent cell-autonomous death in neuronal and mouse models.

Synucleinopathies are a group of neurodegenerative diseases without effective treatment characterized by the abnormal aggregation of alpha-synuclein (aSyn) protein. Changes in levels or in the amino acid sequence of aSyn (by duplication/triplication of the aSyn gene or point mutations in the encoding region) cause familial cases of synucleinopathies. However, the specific molecular mechanisms of aSyn-dependent toxicity remain unclear. Increased aSyn protein levels or pathological mutations may favor abnormal protein-protein interactions (PPIs) that could either promote neuronal death or belong to a coping response program against neurotoxicity. Therefore, the identification and modulation of aSyn-dependent PPIs can provide new therapeutic targets for these diseases. To identify aSyn-dependent PPIs we performed a proximity biotinylation assay based on the promiscuous biotinylase BioID2. When expressed as a fusion protein, BioID2 biotinylates by proximity stable and transient interacting partners, allowing their identification by streptavidin affinity purification and mass spectrometry. The aSyn interactome was analyzed using BioID2-tagged wild-type (WT) and pathological mutant E46K aSyn versions in HEK293 cells. We found the 14-3-3 epsilon isoform as a common protein interactor for WT and E46K aSyn. 14-3-3 epsilon correlates with aSyn protein levels in brain regions of a transgenic mouse model overexpressing WT human aSyn. Using a neuronal model in which aSyn cell-autonomous toxicity is quantitatively scored by longitudinal survival analysis, we found that stabilization of 14-3-3 protein-proteins interactions with Fusicoccin-A (FC-A) decreases aSyn-dependent toxicity. Furthermore, FC-A treatment protects dopaminergic neuronal somas in the substantia nigra of a Parkinson's disease mouse model. Based on these results, we propose that the stabilization of 14-3-3 epsilon interaction with aSyn might reduce aSyn toxicity, and highlight FC-A as a potential therapeutic compound for synucleinopathies.

Peroxidase proximity selection to identify aptamers targeting a subcellular location.

The efficient and specific delivery of functional cargos such as small-molecule drugs, proteins, or nucleic acids across lipid membranes and into subcellular compartments is a significant unmet need in nanomedicine and molecular biology. Systematic Evolution of Ligands by EXponential enrichment (SELEX) exploits vast combinatorial nucleic acid libraries to identify short, nonimmunogenic single-stranded DNA molecules (aptamers) capable of recognizing specific targets based on their 3D structures and molecular interactions. While SELEX has previously been applied to identify aptamers that bind specific cell types or gain cellular uptake, selection of aptamers capable of carrying cargos to specific subcellular compartments is challenging. Here, we describe peroxidase proximity selection (PPS), a generalizable subcellular SELEX approach. We implement local expression of engineered ascorbate peroxidase APEX2 to biotinylate naked DNA aptamers capable of gaining access to the cytoplasm of living cells without assistance. We discovered DNA aptamers that are preferentially taken up into endosomes by macropinocytosis, with a fraction apparently accessing APEX2 in the cytoplasm. One of these selected aptamers is capable of endosomal delivery of an IgG antibody.

Rapid detection of calmodulin/target interaction via the proximity biotinylation method.

Calmodulin (CaM) is known to function as a central signal transducer in calcium-mediated intracellular pathways. In this study, a fusion molecule of a recently developed proximity biotinylation enzyme (AirID) with rat CaM (AirID-CaM) was expressed and purified to near homogeneity using an E. coli expression system to examine the physical interactions between CaM and its target proteins by converting the interaction to biotinylation of CaM targets under nondenatured conditions. AirID-CaM catalyzed a Ca2+-dependent biotinylation of a target protein kinase (Ca2+/CaM-dependent protein kinase kinase α/1, CaMKKα/1) in vitro, which was suppressed by the addition of excess amounts of CaM, and AirID alone did not catalyze the biotinylation of CaMKKα/1, indicating that the biotinylation of CaMKKα/1 by AirID-CaM likely occurs in an interaction-dependent manner. Furthermore, we also observed the Ca2+-dependent biotinylation of GST-CaMKIα and GST-CaMKIV by AirID-CaM, suggesting that AirID-CaM can be useful for the rapid detection of CaM/target interactions with relatively high sensitivity.

Identifying Protein-Protein Interactions by Proximity Biotinylation with AirID and splitAirID.

Proteins frequently function in high-order complexes. Defining protein-protein interactions is essential to acquiring a full understanding of their activity and regulation. Proximity biotinylation has emerged as a highly specific approach to capture transient and stable interactions in living cells or organisms. Proximity biotinylation exploits promiscuous biotinylating enzymes fused to a bait protein, resulting in the biotinylation of adjacent endogenous proteins. Biotinylated interactors are purified under very strict conditions and identified by mass spectrometry to obtain a high-confidence list of candidate binding partners. AirID is a recently described biotin ligase specifically engineered for proximity labeling. This protocol details proximity biotinylation by AirID, using protein complexes that form during a type I interferon response as an example. It covers the construction and validation of AirID fusion proteins and the enrichment and identification of biotinylated interactors. We describe a variation on the protocol using splitAirID. In this case, AirID is split into two inactive fragments and ligase activity is only restored upon dimerization of the bait proteins. This permits selective detection of proteins that interact with homo- or heterodimeric forms of the bait. The protocol considers design strategies, optimization, and the properties of different biotin ligases to identify optimal conditions for each experimental question. We also discuss common pitfalls and how to troubleshoot them. These approaches allow proximity biotinylation to be a powerful tool for defining protein interactomes. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Construction and functional validation of AirID fusion proteins Alternate Protocol: Construction and functional validation of splitAirID fusion proteins Support Protocol: Western blot for biotinylated proteins Basic Protocol 2: Biotinylation, enrichment, and identification of protein interactors.

In-Cell Chemical Crosslinking Identifies Hotspots for SQSTM-1/p62-IκBα Interaction That Underscore a Critical Role of p62 in Limiting NF-κB Activation Through IκBα Stabilization.

We have previously documented that in liver cells, the multifunctional protein scaffold p62/SQSTM1 is closely associated with IκBα, an inhibitor of the transcriptional activator NF-κB. Such an intimate p62-IκBα association we now document leads to a marked 18-fold proteolytic IκBα-stabilization, enabling its nuclear entry and termination of the NF-κB-activation cycle. In p62-/--cells, such termination is abrogated resulting in the nuclear persistence and prolonged activation of NF-κB following inflammatory stimuli. Utilizing various approaches both classic (structural deletion, site-directed mutagenesis) as well as novel (in-cell chemical crosslinking), coupled with proteomic analyses, we have defined the precise structural hotspots of p62-IκBα association. Accordingly, we have identified such IκBα hotspots to reside around N-terminal (K38, K47, and K67) and C-terminal (K238/C239) residues in its fifth ankyrin repeat domain. These sites interact with two hotspots in p62: One in its PB-1 subdomain around K13, and the other comprised of a positively charged patch (R183/R186/K187/K189) between its ZZ- and TB-subdomains. APEX proximity analyses upon IκBα-cotransfection of cells with and without p62 have enabled the characterization of the p62 influence on IκBα-protein-protein interactions. Interestingly, consistent with p62's capacity to proteolytically stabilize IκBα, its presence greatly impaired IκBα's interactions with various 20S/26S proteasomal subunits. Furthermore, consistent with p62 interaction with IκBα on an interface opposite to that of its NF-κB-interacting interface, p62 failed to significantly affect IκBα-NF-κB interactions. These collective findings together with the known dynamic p62 nucleocytoplasmic shuttling leads us to speculate that it may be involved in "piggy-back" nuclear transport of IκBα following its NF-κB-elicited transcriptional activation and de novo synthesis, required for termination of the NF-κB-activation cycle. Consequently, mice carrying a liver-specific deletion of p62-residues 68 to 252 reveal age-dependent-enhanced liver inflammation. Our findings reveal yet another mode of p62-mediated pathophysiologically relevant regulation of NF-κB.

Fibril treatment changes protein interactions of tau and α-synuclein in human neurons.

In several neurodegenerative disorders, the neuronal proteins tau and α-synuclein adopt aggregation-prone conformations capable of replicating within and between cells. To better understand how these conformational changes drive neuropathology, we compared the interactomes of tau and α-synuclein in the presence or the absence of recombinant fibril seeds. Human embryonic stem cells with an inducible neurogenin-2 transgene were differentiated into glutamatergic neurons expressing (1) WT 0N4R tau, (2) mutant (P301L) 0N4R tau, (3) WT α-synuclein, or (4) mutant (A53T) α-synuclein, each genetically fused to a promiscuous biotin ligase (BioID2). Neurons expressing unfused BioID2 served as controls. After treatment with fibrils or PBS, interacting proteins were labeled with biotin in situ and quantified using mass spectrometry via tandem mass tag labeling. By comparing interactions in mutant versus WT neurons and in fibril- versus PBS-treated neurons, we observed changes in protein interactions that are likely relevant to disease progression. We identified 45 shared interactors, suggesting that tau and α-synuclein function within some of the same pathways. Potential loci of shared interactions include microtubules, Wnt signaling complexes, and RNA granules. Following fibril treatment, physiological interactions decreased, whereas other interactions, including those between tau and 14-3-3 η, increased. We confirmed that 14-3-3 proteins, which are known to colocalize with protein aggregates during neurodegeneration, can promote or inhibit tau aggregation in vitro depending on the specific combination of 14-3-3 isoform and tau sequence.

Proximity-dependent biotinylation technologies for mapping RNA-protein interactions in live cells.

Proximity ligation technologies are extremely powerful tools for unveiling RNA-protein interactions occurring at different stages in living cells. These approaches mainly rely on the inducible activity of enzymes (biotin ligases or peroxidases) that promiscuously biotinylate macromolecules within a 20 nm range. These enzymes can be either fused to an RNA binding protein or tethered to any RNA of interest and expressed in living cells to biotinylate the amino acids and nucleic acids of binding partners in proximity. The biotinylated molecules can then be easily affinity purified under denaturing conditions and analyzed by mass spectrometry or next generation sequencing. These approaches have been widely used in recent years, providing a potent instrument to map the molecular interactions of specific RNA-binding proteins as well as RNA transcripts occurring in mammalian cells. In addition, they permit the identification of transient interactions as well as interactions among low expressed molecules that are often missed by standard affinity purification strategies. This review will provide a brief overview of the currently available proximity ligation methods, highlighting both their strengths and shortcomings. Furthermore, it will bring further insights to the way these technologies could be further used to characterize post-transcriptional modifications that are known to regulate RNA-protein interactions.

A New Monoclonal Antibody Enables BAR Analysis of Subcellular Importin ß1 Interactomes.

Importin ß1 (KPNB1) is a nucleocytoplasmic transport factor with critical roles in both cytoplasmic and nucleocytoplasmic transport, hence there is keen interest in the characterization of its subcellular interactomes. We found limited efficiency of BioID in the detection of importin complex cargos and therefore generated a highly specific and sensitive anti-KPNB1 monoclonal antibody to enable biotinylation by antibody recognition analysis of importin ß1 interactomes. The monoclonal antibody recognizes an epitope comprising residues 301-320 of human KPBN1 and strikingly is highly specific for cytoplasmic KPNB1 in diverse applications, with little reaction with KPNB1 in the nucleus. Biotinylation by antibody recognition with this novel antibody revealed numerous new interactors of importin ß1, expanding the KPNB1 interactome to cytoplasmic and signaling complexes that highlight potential new functions for the importins complex beyond nucleocytoplasmic transport. Data are available via ProteomeXchange with identifier PXD032728.

Mislocalization of protein kinase A drives pathology in Cushing's syndrome.

Mutations in the catalytic subunit of protein kinase A (PKAc) drive the stress hormone disorder adrenal Cushing's syndrome. We define mechanisms of action for the PKAc-L205R and W196R variants. Proximity proteomic techniques demonstrate that both Cushing's mutants are excluded from A kinase-anchoring protein (AKAP)-signaling islands, whereas live-cell photoactivation microscopy reveals that these kinase mutants indiscriminately diffuse throughout the cell. Only cAMP analog drugs that displace native PKAc from AKAPs enhance cortisol release. Rescue experiments that incorporate PKAc mutants into AKAP complexes abolish cortisol overproduction, indicating that kinase anchoring restores normal endocrine function. Analyses of adrenal-specific PKAc-W196R knockin mice and Cushing's syndrome patient tissue reveal defective signaling mechanisms of the disease. Surprisingly each Cushing's mutant engages a different mitogenic-signaling pathway, with upregulation of YAP/TAZ by PKAc-L205R and ERK kinase activation by PKAc-W196R. Thus, aberrant spatiotemporal regulation of each Cushing's variant promotes the transmission of distinct downstream pathogenic signals.

Comprehensive Interactome Mapping of Nuclear Receptors Using Proximity Biotinylation.

Nuclear receptors, including hormone receptors, perform their cellular activities by modulating their protein-protein interactions. They engage with specific ligands and translocate to the nucleus, where they bind the DNA and activate extensive transcriptional programs. Therefore, gaining a comprehensive overview of the protein-protein interactions they establish requires methods that function effectively throughout the cell with fast dynamics and high reproducibility. Focusing on estrogen receptor alpha (ESR1), the founding member of the nuclear receptor family, this chapter describes a new lentiviral system that allows the expression of TurboID-hemagglutinin (HA)-2 × Strep tagged proteins in mammalian cells to perform fast proximity biotinylation assays. Key validation steps for these reagents and their use in interactome mapping experiments in two distinct breast cancer cell lines are described. Our protocol enabled the quantification of ESR1 interactome generated by cellular contexts that were hormone-sensitive or not.

A simple method for labeling proteins and antibodies with biotin using the proximity biotinylation enzyme TurboID.

Proteins and antibodies labeled with biotin have been widely used for protein analysis, enzyme immunoassays, and diagnoses. Presently, they are prepared using either a chemical reaction involving a biotin N-hydroxysuccinimide (NHS) ester compound or by enzymatic biotin ligation using a combination of a biotinylation-peptide tag and Escherichia coli BirA. However, these methods are relatively complicated. Recently BirA was improved to TurboID, a highly active enzyme for proximity labeling with biotin. Here, we demonstrate a novel simple biotin labeling method for proteins and antibodies using TurboID. Purified TurboID was mixed with a protein or an antibody in the presence of biotin and ATP in the general biochemical buffer condition, followed by biotin labeling. Biotin labeling sites by TurboID were found on the surface of green fluorescent protein. Biotin labeling of IκBα by TurboID indicated its binding to RelA. Furthermore, TurboID-dependent biotin labeling of monoclonal antibodies from rabbits and mice could be directly used for immunoblotting detection of specific proteins without the purification step. These results indicate that TurboID provides a very useful and simple method for biotin labeling of functional proteins.

Mapping histone modification-dependent protein interactions with chemical proteomics.

Post-translational modifications (PTMs) of histones play essential roles in chromatin function and epigenetic regulation. Determining the interaction partners of these modifications is crucial to understanding transcriptional processes related to diverse developmental and pathological cues. We discuss how chemical proteomics can be applied to the simultaneous and global exploration of these interaction networks.

Hybridization-proximity labeling reveals spatially ordered interactions of nuclear RNA compartments.

The ability of RNAs to form specific contacts with other macromolecules provides an important mechanism for subcellular compartmentalization. Here we describe a suite of hybridization-proximity (HyPro) labeling technologies for unbiased discovery of proteins (HyPro-MS) and transcripts (HyPro-seq) associated with RNAs of interest in genetically unperturbed cells. As a proof of principle, we show that HyPro-MS and HyPro-seq can identify both known and previously unexplored spatial neighbors of the noncoding RNAs 45S, NEAT1, and PNCTR expressed at markedly different levels. Notably, HyPro-seq uncovers an extensive repertoire of incompletely processed, adenosine-to-inosine-edited transcripts accumulating at the interface between their encoding chromosomal regions and the NEAT1-containing paraspeckle compartment. At least some of these targets require NEAT1 for their optimal expression. Overall, this study provides a versatile toolkit for dissecting RNA interactomes in diverse biomedical contexts and expands our understanding of the functional architecture of the mammalian nucleus.