Proteins and RNA functionally and physically intersect in multiple biological processes, however, currently no universal method is available to purify protein-RNA complexes. Here, we introduce XRNAX, ...a method for the generic purification of protein-crosslinked RNA, and demonstrate its versatility to study the composition and dynamics of protein-RNA interactions by various transcriptomic and proteomic approaches. We show that XRNAX captures all RNA biotypes and use this to characterize the sub-proteomes that interact with coding and non-coding RNAs (ncRNAs) and to identify hundreds of protein-RNA interfaces. Exploiting the quantitative nature of XRNAX, we observe drastic remodeling of the RNA-bound proteome during arsenite-induced stress, distinct from autophagy-related changes in the total proteome. In addition, we combine XRNAX with crosslinking immunoprecipitation sequencing (CLIP-seq) to validate the interaction of ncRNA with lamin B1 and EXOSC2. Thus, XRNAX is a resourceful approach to study structural and compositional aspects of protein-RNA interactions to address fundamental questions in RNA-biology.
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•XRNAX purifies protein-crosslinked RNA of all biotypes from UV-crosslinked cells•Discovery of the WKF RNA-binding domain•Discovery of more than 700 proteins interacting with non-polyadenylated RNA•Profiling of stress-induced changes in RNA-binding proteomes
A general approach for characterizing cellular RNA-protein interactions allows examination of dynamic changes to the RNA-bound proteome.
Following the realization that eukaryotic RNA-binding proteomes are substantially larger than anticipated, we must now understand their detailed composition and dynamics. Methods such as RNA ...interactome capture (RIC) have begun to address this need. However, limitations of RIC have been reported. Here we describe enhanced RNA interactome capture (eRIC), a method based on the use of an LNA-modified capture probe, which yields numerous advantages including greater specificity and increased signal-to-noise ratios compared to existing methods. In Jurkat cells, eRIC reduces the rRNA and DNA contamination by >10-fold compared to RIC and increases the detection of RNA-binding proteins. Due to its low background, eRIC also empowers comparative analyses of changes of RNA-bound proteomes missed by RIC. For example, in cells treated with dimethyloxalylglycine, which inhibits RNA demethylases, eRIC identifies m6A-responsive RNA-binding proteins that escape RIC. eRIC will facilitate the unbiased characterization of RBP dynamics in response to biological and pharmacological cues.
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.
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•The selective human autophagy receptor p62/sequestosome-1 is an RNA-binding protein•p62 engages the small non-coding vault RNA1-1 as a major interacting RNA•Vault RNA1-1 riboregulates p62-dependent autophagy and aggregate clearance•Mechanistically, vault RNA1-1 interferes with p62 multimerization
A biological function of vault RNAs is to directly modulate the oligomerization state of p62, thereby controlling autophagy.
System-wide approaches have unveiled an unexpected breadth of the RNA-bound proteomes of cultured cells. Corresponding information regarding RNA-binding proteins (RBPs) of mammalian organs is still ...missing, largely due to technical challenges. Here, we describe ex vivo enhanced RNA interactome capture (eRIC) to characterize the RNA-bound proteomes of three different mouse organs. The resulting organ atlases encompass more than 1300 RBPs active in brain, kidney or liver. Nearly a quarter (291) of these had formerly not been identified in cultured cells, with more than 100 being metabolic enzymes. Remarkably, RBP activity differs between organs independent of RBP abundance, suggesting organ-specific levels of control. Similarly, we identify systematic differences in RNA binding between animal organs and cultured cells. The pervasive RNA binding of enzymes of intermediary metabolism in organs points to tightly knit connections between gene expression and metabolism, and displays a particular enrichment for enzymes that use nucleotide cofactors. We describe a generically applicable refinement of the eRIC technology and provide an instructive resource of RBPs active in intact mammalian organs, including the brain.
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.
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.
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•iCLIP analyses of EJC components provide a comprehensive map of bona fide EJCs•EJC proteins, in particular BTZ, are largely restricted to canonical deposition sites•EJCs are enriched on alternatively spliced mRNAs•EJCs are underrepresented on mRNAs encoding ribosomal proteins
Exon junction complexes govern multiple critical decisions in posttranscriptional gene regulation. Using all four RNA binding subunits of the complex, Hauer et al. provide a comprehensive map of bona fide EJCs across a mammalian transcriptome and show enrichment on alternatively spliced mRNAs and underrepresentation on RNAs encoding ribosomal proteins.
Modern omics technologies allow us to obtain global information on different types of biological networks. However, integrating these different types of analyses into a coherent framework for a ...comprehensive biological interpretation remains challenging. Here, we present a conceptual framework that integrates protein interaction, phosphoproteomics, and transcriptomics data. Applying this method to analyze HRAS signaling from different subcellular compartments shows that spatially defined networks contribute specific functions to HRAS signaling. Changes in HRAS protein interactions at different sites lead to different kinase activation patterns that differentially regulate gene transcription. HRAS-mediated signaling is the strongest from the cell membrane, but it regulates the largest number of genes from the endoplasmic reticulum. The integrated networks provide a topologically and functionally resolved view of HRAS signaling. They reveal distinct HRAS functions including the control of cell migration from the endoplasmic reticulum and TP53-dependent cell survival when signaling from the Golgi apparatus.
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•MiNETi (Mixed Network Integration) enables the integration of multi-omics datasets•MiNETi provides an integrated view of HRAS signaling from different subcellular sites•HRAS controls its interactome, phosphoproteome, and transcriptome site specifically•HRAS regulates cell migration and p53-mediated apoptosis from endomembranes
Santra et al. develop MiNETi (Mixed Network Integration) to integrate multi-omics data. Applying MiNETi to analyze the interactome, phosphoproteome, and transcriptome regulated by HRAS signaling from different subcellular compartments shows that HRAS controls phosphorylation-dependent signaling mainly from the cell membrane but regulates a large number of genes from endomembranes.
The aim of this paper is to assess opportunities the Clean Energy Package provides for Plus Energy Buildings (PEBs) and Plus Energy Districts (PEDs) regarding their economic optimization and market ...integration, possibly leading to new use cases and revenue streams. At the same time, insights into regulatory limitations at the national level in transposing the set of EU Clean Energy Package provisions are shown. The paper illustrates that the concepts of PEBs and PEDs are in principle compatible with the EU energy community concepts, as they relate to technical characteristics while energy communities provide a legal and regulatory framework for the organization and governance of a community, at the same time providing new regulatory space for specific activities and market integration. To realize new use cases, innovative ICT approaches are needed for a range of actors actively involved in creating and operating energy communities as presented in the paper. The paper discusses a range of different options to realize PEBs and PEDs as energy communities based on the H2020 EXCESS project. It concludes, however, that currently the transposition of the Clean Energy Package by the EU Member States is incomplete and limiting and as a consequence, in the short term, the full potential of PEBs and PEDs cannot be exploited.
A-Raf kinase can inhibit apoptosis by binding to the proapoptotic mammalian sterile 20-like kinase (MST2). This function relies on expression of hnRNP H, which ensures the correct splicing of a-raf ...mRNA needed to produce full-length A-Raf protein. Here, we showed that expression of hnRNP H and production of full-length A-Raf is positively controlled by c-Myc. Low c-Myc reduces hnRNP H expression and switches a-raf splicing to produce A-Raf(short), a truncated protein. Importantly, A-Raf(short) fails to regulate MST2 but retains the Ras-binding domain such that it functions as a dominant negative mutant suppressing Ras activation and transformation. Human colon and head and neck cancers exhibit high hnRNP H and high c-Myc levels resulting in enhanced A-Raf expression and reduced expression of A-Raf(short). Conversely, in normal cells and tissues in which c-Myc and hnRNP H are low, A-Raf(short) suppresses extracellular signal regulated kinase activation such that it may act as a safeguard against oncogenic transformation. Our findings offered a new paradigm to understand how c-Myc coordinates diverse cell functions by directly affecting alternate splicing of key signaling components.
A brave new world of RNA-binding proteins Hentze, Matthias W; Castello, Alfredo; Schwarzl, Thomas ...
Nature reviews. Molecular cell biology,
05/2018, Letnik:
19, Številka:
5
Journal Article
Recenzirano
Odprti dostop
RNA-binding proteins (RBPs) are typically thought of as proteins that bind RNA through one or multiple globular RNA-binding domains (RBDs) and change the fate or function of the bound RNAs. Several ...hundred such RBPs have been discovered and investigated over the years. Recent proteome-wide studies have more than doubled the number of proteins implicated in RNA binding and uncovered hundreds of additional RBPs lacking conventional RBDs. In this Review, we discuss these new RBPs and the emerging understanding of their unexpected modes of RNA binding, which can be mediated by intrinsically disordered regions, protein-protein interaction interfaces and enzymatic cores, among others. We also discuss the RNA targets and molecular and cellular functions of the new RBPs, as well as the possibility that some RBPs may be regulated by RNA rather than regulate RNA.
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