The Small Non-coding Vault RNA1-1 Acts as a Riboregulator of Autophagy.

Vault RNAs (vtRNA) are small non-coding RNAs transcribed by RNA polymerase III found in many eukaryotes. Although they have been linked to drug resistance, apoptosis, and viral replication, their molecular functions remain unclear. Here, we show that vault RNAs directly bind the autophagy receptor sequestosome-1/p62 in human and murine cells. Overexpression of human vtRNA1-1 inhibits, while its antisense LNA-mediated knockdown enhances p62-dependent autophagy. Starvation of cells reduces the steady-state and p62-bound levels of vault RNA1-1 and induces autophagy. Mechanistically, p62 mutants that fail to bind vtRNAs display increased p62 homo-oligomerization and augmented interaction with autophagic effectors. Thus, vtRNA1-1 directly regulates selective autophagy by binding p62 and interference with oligomerization, a critical step of p62 function. Our data uncover a striking example of the potential of RNA to control protein functions directly, as previously recognized for protein-protein interactions and post-translational modifications.

Comprehensive Identification of RNA-Binding Domains in Human Cells.

Mammalian cells harbor more than a thousand RNA-binding proteins (RBPs), with half of these employing unknown modes of RNA binding. We developed RBDmap to determine the RNA-binding sites of native RBPs on a proteome-wide scale. We identified 1,174 binding sites within 529 HeLa cell RBPs, discovering numerous RNA-binding domains (RBDs). Catalytic centers or protein-protein interaction domains are in close relationship with RNA-binding sites, invoking possible effector roles of RNA in the control of protein function. Nearly half of the RNA-binding sites map to intrinsically disordered regions, uncovering unstructured domains as prevalent partners in protein-RNA interactions. RNA-binding sites represent hot spots for defined posttranslational modifications such as lysine acetylation and tyrosine phosphorylation, suggesting metabolic and signal-dependent regulation of RBP function. RBDs display a high degree of evolutionary conservation and incidence of Mendelian mutations, suggestive of important functional roles. RBDmap thus yields profound insights into native protein-RNA interactions in living cells.

In Planta Determination of the mRNA-Binding Proteome of Arabidopsis Etiolated Seedlings.

RNA binding proteins (RBPs) control the fate and expression of a transcriptome. Despite this fundamental importance, our understanding of plant RBPs is rudimentary, being mainly derived via bioinformatic extrapolation from other kingdoms. Here, we adapted the mRNA-protein interactome capture method to investigate the RNA binding proteome in planta. From Arabidopsis thaliana etiolated seedlings, we captured more than 700 proteins, including 300 with high confidence that we have defined as the At-RBP set. Approximately 75% of these At-RBPs are bioinformatically linked with RNA biology, containing a diversity of canonical RNA binding domains (RBDs). As no prior experimental RNA binding evidence exists for the majority of these proteins, their capture now authenticates them as RBPs. Moreover, we identified protein families harboring emerging and potentially novel RBDs, including WHIRLY, LIM, ALBA, DUF1296, and YTH domain-containing proteins, the latter being homologous to animal RNA methylation readers. Other At-RBP set proteins include major signaling proteins, cytoskeleton-associated proteins, membrane transporters, and enzymes, suggesting the scope and function of RNA-protein interactions within a plant cell is much broader than previously appreciated. Therefore, our foundation data set has provided an unbiased insight into the RNA binding proteome of plants, on which future investigations into plant RBPs can be based.

FASTKD2 is an RNA-binding protein required for mitochondrial RNA processing and translation.

Mitochondrial RNA processing is an essential step for the synthesis of the components of the electron transport chain in all eukaryotic organisms, yet several aspects of mitochondrial RNA biogenesis and regulation are not sufficiently understood. RNA interactome capture identified several disease-relevant RNA-binding proteins (RBPs) with noncanonical RNA-binding architectures, including all six members of the FASTK (FAS-activated serine/threonine kinase) family of proteins. A mutation within one of these newly assigned FASTK RBPs, FASTKD2, causes a rare form of Mendelian mitochondrial encephalomyopathy. To investigate whether RNA binding of FASTKD2 contributes to the disease phenotype, we identified the RNA targets of FASTKD2 by iCLIP. FASTKD2 interacts with a defined set of mitochondrial transcripts including 16S ribosomal RNA (RNR2) and NADH dehydrogenase subunit 6 (ND6) messenger RNA. CRISPR-mediated deletion of FASTKD2 leads to aberrant processing and expression of RNR2 and ND6 mRNA that encodes a subunit of the respiratory complex I. Metabolic phenotyping of FASTKD2-deficient cells reveals impaired cellular respiration with reduced activities of all respiratory complexes. This work identifies key aspects of the molecular network of a previously uncharacterized, disease-relevant RNA-binding protein, FASTKD2, by a combination of genomic, molecular, and metabolic analyses.

A versatile assay for RNA-binding proteins in living cells.

RNA-binding proteins (RBPs) control RNA fate from synthesis to decay. Since their cellular expression levels frequently do not reflect their in vivo activity, methods are needed to assess the steady state RNA-binding activity of RBPs as well as their responses to stimuli. While electrophoresis mobility shift assays (EMSA) have been used for such determinations, their results serve at best as proxies for the RBP activities in living cells. Here, we describe a quantitative dual fluorescence method to analyze protein-mRNA interactions in vivo. Known or candidate RBPs are fused to fluorescent proteins (eGFP, YFP), expressed in cells, cross-linked in vivo to RNA by ultraviolet light irradiation, and immunoprecipitated, after lysis, with a single chain antibody fragment directed against eGFP (GFP-binding protein, GBP). Polyadenylated RNA-binding activity of fusion proteins is assessed by hybridization with an oligo(DT) probe coupled with a red fluorophore. Since UV light is directly applied to living cells, the assay can be used to monitor dynamic changes in RNA-binding activities in response to biological or pharmacological stimuli. Notably, immunoprecipitation and hybridization can also be performed with commercially available GBP-coupled 96-well plates (GFP-multiTrap), allowing highly parallel RNA-binding measurements in a single experiment. Therefore, this method creates the possibility to conduct in vivo high-throughput RNA-binding assays. We believe that this fast and simple radioactivity-free method will find many useful applications in RNA biology.

The RNA-Binding Protein YBX3 Controls Amino Acid Levels by Regulating SLC mRNA Abundance.

Sufficient amino acid supplies are critical for protein synthesis and, thus, cell growth and proliferation. Specialized transporters mediate amino acid exchange across membranes and their regulation is critical for amino acid homeostasis. Here, we report that the DNA- and RNA-binding protein YBX3 regulates the expression of amino acid transporters. To investigate the functions of YBX3, we integrated proteomic and transcriptomic data from cells depleted of YBX3 with analyses of YBX3 RNA binding sites to identify RNAs directly regulated by YBX3. The data implicate YBX3 as a RNA-binding protein that regulates distinct sets of mRNAs by discrete mechanisms, including mRNA abundance. Among direct YBX3 targets, two solute carrier (SLC) amino acid transporters (SLC7A5 and SLC3A2) were identified. We show that YBX3 stabilizes these SLC mRNAs and that YBX3 depletion diminishes the expression of SLC7A5/SLC3A2, which specifically reduces SLC7A5/SLC3A2 amino acid substrates. Thus, YBX3 emerges as a key regulator of amino acid levels.

TRP-2 specifically decreases WM35 cell sensitivity to oxidative stress.

TRP-2 (dopachrome tautomerase) is a melanogenic enzyme whose expression was recently reported to modulate melanocyte response to different cytotoxic events. Here we studied a possible role of TRP-2 in the oxidative stress response in the amelanotic WM35 melanoma cell line. Cell viability assays showed that TRP-2 overexpression in WM35 cells reduced their sensitivity to oxidative stress. Comet assays linked TRP-2 expression to DNA damage protection, and high-performance liquid chromotography-tandem mass spectrometry experiments showed an increase in intracellular glutathione in TRP-2-overexpressing cells. These effects were specifically reversed when TRP-2 was silenced by RNA interference. Nevertheless, these properties appeared to depend on a particular cell environment because expression of TRP-2 failed to rescue HEK epithelial cells exposed to similar treatments.

Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap.

This protocol is an extension to: Nat. Protoc. 8, 491-500 (2013); doi:10.1038/nprot.2013.020; published online 14 February 2013RBDmap is a method for identifying, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native interactions with RNA. In brief, cells are irradiated with UV light to induce protein-RNA cross-links. Following stringent denaturing washes, the resulting covalently linked protein-RNA complexes are purified with oligo(dT) magnetic beads. After elution, RBPs are subjected to partial proteolysis, in which the protein regions still bound to the RNA and those released to the supernatant are separated by a second oligo(dT) selection. After sample preparation and mass-spectrometric analysis, peptide intensity ratios between the RNA-bound and released fractions are used to determine the RNA-binding regions. As a Protocol Extension, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol (for the RNA interactome capture method) describes how to identify the active RBPs in cultured cells, whereas this Protocol Extension also enables the identification of the RNA-binding domains of RBPs. The experimental workflow takes 1 week plus 2 additional weeks for proteomics and data analysis. Notably, RBDmap presents numerous advantages over classic methods for determining RNA-binding domains: it produces proteome-wide, high-resolution maps of the protein regions contacting the RNA in a physiological context and can be adapted to different biological systems and conditions. Because RBDmap relies on the isolation of polyadenylated RNA via oligo(dT), it will not provide RNA-binding information on proteins interacting exclusively with nonpolyadenylated transcripts. Applied to HeLa cells, RBDmap uncovered 1,174 RNA-binding sites in 529 proteins, many of which were previously unknown.

Exon Junction Complexes Show a Distributional Bias toward Alternatively Spliced mRNAs and against mRNAs Coding for Ribosomal Proteins.

The exon junction complex (EJC) connects spliced mRNAs to posttranscriptional processes including RNA localization, transport, and regulated degradation. Here, we provide a comprehensive analysis of bona fide EJC binding sites across the transcriptome including all four RNA binding EJC components eIF4A3, BTZ, UPF3B, and RNPS1. Integration of these data sets permits definition of high-confidence EJC deposition sites as well as assessment of whether EJC heterogeneity drives alternative nonsense-mediated mRNA decay pathways. Notably, BTZ (MLN51 or CASC3) emerges as the EJC subunit that is almost exclusively bound to sites 20-24 nucleotides upstream of exon-exon junctions, hence defining EJC positions. By contrast, eIF4A3, UPF3B, and RNPS1 display additional RNA binding sites suggesting accompanying non-EJC functions. Finally, our data show that EJCs are largely distributed across spliced RNAs in an orthodox fashion, with two notable exceptions: an EJC deposition bias in favor of alternatively spliced transcripts and against the mRNAs that encode ribosomal proteins.

Improved binding site assignment by high-resolution mapping of RNA-protein interactions using iCLIP.

Individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) allows the determination of crosslinking sites of RNA-binding proteins (RBPs) on RNAs. iCLIP is based on ultraviolet light crosslinking of RBPs to RNA, reverse transcription and high-throughput sequencing of fragments terminating at the site of crosslinking. As a result, start sites of iCLIP fragments are expected to cluster with a narrow distribution, typically representing the site of direct interaction between the RBP and the RNA. Here we show that for several RBPs (eIF4A3, PTB, SRSF3, SRSF4 and hnRNP L), the start sites of iCLIP fragments show a fragment length-dependent broader distribution that can be shifted to positions upstream of the known RNA-binding site. We developed an analysis tool that identifies these shifts and can improve the positioning of RBP binding sites.

The RNA-binding proteomes from yeast to man harbour conserved enigmRBPs.

RNA-binding proteins (RBPs) exert a broad range of biological functions. To explore the scope of RBPs across eukaryotic evolution, we determined the in vivo RBP repertoire of the yeast Saccharomyces cerevisiae and identified 678 RBPs from yeast and additionally 729 RBPs from human hepatocytic HuH-7 cells. Combined analyses of these and recently published data sets define the core RBP repertoire conserved from yeast to man. Conserved RBPs harbour defined repetitive motifs within disordered regions, which display striking evolutionary expansion. Only 60% of yeast and 73% of the human RBPs have functions assigned to RNA biology or structural motifs known to convey RNA binding, and many intensively studied proteins surprisingly emerge as RBPs (termed 'enigmRBPs'), including almost all glycolytic enzymes, pointing to emerging connections between gene regulation and metabolism. Analyses of the mitochondrial hydroxysteroid dehydrogenase (HSD17B10) uncover the RNA-binding specificity of an enigmRBP.

Cab45 is required for Ca(2+)-dependent secretory cargo sorting at the trans-Golgi network.

Ca(2+) import into the lumen of the trans-Golgi network (TGN) by the secretory pathway calcium ATPase1 (SPCA1) is required for the sorting of secretory cargo. How is Ca(2+) retained in the lumen of the Golgi, and what is its role in cargo sorting? We show here that a soluble, lumenal Golgi resident protein, Cab45, is required for SPCA1-dependent Ca(2+) import into the TGN; it binds secretory cargo in a Ca(2+)-dependent reaction and is required for its sorting at the TGN.

ADF/cofilin regulates secretory cargo sorting at the TGN via the Ca2+ ATPase SPCA1.

Actin-severing proteins ADF/cofilin are required for the sorting of secretory cargo at the trans-Golgi network (TGN) in mammalian cells. How do these cytoplasmic proteins interact with the cargoes in the lumen of the TGN? Put simply, how are these two sets of proteins connected across the TGN membrane? Mass spectrometry of cofilin1 immunoprecipitated from HeLa cells revealed the presence of actin and the Ca(2+) ATPase SPCA1. Moreover, cofilin1 was localized to the TGN and bound to SPCA1 via dynamic actin. SPCA1 knockdown, like ADF/cofilin1 knockdown, inhibited Ca(2+) uptake into the TGN and caused missorting of secretory cargo. These defects were rescued by the overexpression of the TGN-localized SPCA1. We propose that ADF/cofilin-dependent severing of actin filaments exposes and promotes the activation of SPCA1, which pumps Ca(2+) into the lumen of the TGN for the sorting of the class of secretory cargo that binds Ca(2+).

Actin remodeling by ADF/cofilin is required for cargo sorting at the trans-Golgi network.

Knockdown of the actin-severing protein actin-depolymerizing factor (ADF)/cofilin inhibited export of an exogenously expressed soluble secretory protein from Golgi membranes in Drosophila melanogaster and mammalian tissue culture cells. A stable isotope labeling by amino acids in cell culture mass spectrometry-based protein profiling revealed that a large number of endogenous secretory proteins in mammalian cells were not secreted upon ADF/cofilin knockdown. Although many secretory proteins were retained, a Golgi-resident protein and a lysosomal hydrolase were aberrantly secreted upon ADF/cofilin knockdown. Overall, our findings indicate that inactivation of ADF/cofilin perturbed the sorting of a subset of both soluble and integral membrane proteins at the trans-Golgi network (TGN). We suggest that ADF/cofilin-dependent actin trimming generates a sorting domain at the TGN, which filters secretory cargo for export, and that uncontrolled growth of this domain causes missorting of proteins. This type of actin-dependent compartmentalization and filtering of secretory cargo at the TGN by ADF/cofilin could explain sorting of proteins that are destined to the cell surface.

Trans-inactivation of receptor tyrosine kinases by novel angiotensin II AT2 receptor-interacting protein, ATIP.

Negative regulation of mitogenic pathways is a fundamental process that remains poorly characterized. The angiotensin II AT2 receptor is a rare example of a 7-transmembrane domain receptor that negatively cross-talks with receptor tyrosine kinases to inhibit cell growth. In the present study, we report the molecular cloning of a novel protein, ATIP1 (AT2-interacting protein), which interacts with the C-terminal tail of the AT2 receptor, but not with those of other receptors such as angiotensin AT1, bradykinin BK2, and adrenergic beta(2) receptor. ATIP1 defines a family of at least four members that possess the same domain of interaction with the AT2 receptor, contain a large coiled-coil region, and are able to dimerize. Ectopic expression of ATIP1 in eukaryotic cells leads to inhibition of insulin, basic fibroblast growth factor, and epidermal growth factor-induced ERK2 activation and DNA synthesis, and attenuates insulin receptor autophosphorylation, in the same way as the AT2 receptor. The inhibitory effect of ATIP1 requires expression, but not ligand activation, of the AT2 receptor and is further increased in the presence of Ang II, indicating that ATIP1 cooperates with AT2 to transinactivate receptor tyrosine kinases. Our findings therefore identify ATIP1 as a novel early component of growth inhibitory signaling cascade.