The Human RNA-Binding Proteome and Its Dynamics during Translational Arrest.

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.

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.

A brave new world of RNA-binding proteins.

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.

Riboregulation of Enolase 1 activity controls glycolysis and embryonic stem cell differentiation.

Differentiating stem cells must coordinate their metabolism and fate trajectories. Here, we report that the catalytic activity of the glycolytic enzyme Enolase 1 (ENO1) is directly regulated by RNAs leading to metabolic rewiring in mouse embryonic stem cells (mESCs). We identify RNA ligands that specifically inhibit ENO1's enzymatic activity in vitro and diminish glycolysis in cultured human cells and mESCs. Pharmacological inhibition or RNAi-mediated depletion of the protein deacetylase SIRT2 increases ENO1's acetylation and enhances its RNA binding. Similarly, induction of mESC differentiation leads to increased ENO1 acetylation, enhanced RNA binding, and inhibition of glycolysis. Stem cells expressing mutant forms of ENO1 that escape or hyper-activate this regulation display impaired germ layer differentiation. Our findings uncover acetylation-driven riboregulation of ENO1 as a physiological mechanism of glycolytic control and of the regulation of stem cell differentiation. Riboregulation may represent a more widespread principle of biological control.

RNA-binding proteins in human genetic disease.

RNA-binding proteins (RBPs) are critical effectors of gene expression, and as such their malfunction underlies the origin of many diseases. RBPs can recognize hundreds of transcripts and form extensive regulatory networks that help to maintain cell homeostasis. System-wide unbiased identification of RBPs has increased the number of recognized RBPs into the four-digit range and revealed new paradigms: from the prevalence of structurally disordered RNA-binding regions with roles in the formation of membraneless organelles to unsuspected and potentially pervasive connections between intermediary metabolism and RNA regulation. Together with an increasingly detailed understanding of molecular mechanisms of RBP function, these insights are facilitating the development of new therapies to treat malignancies. Here, we provide an overview of RBPs involved in human genetic disorders, both Mendelian and somatic, and discuss emerging aspects in the field with emphasis on molecular mechanisms of disease and therapeutic interventions.

Cytosolic RNA binding of the mitochondrial TCA cycle enzyme malate dehydrogenase.

Several enzymes of intermediary metabolism have been identified to bind RNA in cells, with potential consequences for the bound RNAs and/or the enzyme. In this study, we investigate the RNA-binding activity of the mitochondrial enzyme malate dehydrogenase 2 (MDH2), which functions in the tricarboxylic acid (TCA) cycle and the malate-aspartate shuttle. We confirmed in cellulo RNA binding of MDH2 using orthogonal biochemical assays and performed enhanced cross-linking and immunoprecipitation (eCLIP) to identify the cellular RNAs associated with endogenous MDH2. Surprisingly, MDH2 preferentially binds cytosolic over mitochondrial RNAs, although the latter are abundant in the milieu of the mature protein. Subcellular fractionation followed by RNA-binding assays revealed that MDH2-RNA interactions occur predominantly outside of mitochondria. We also found that a cytosolically retained N-terminal deletion mutant of MDH2 is competent to bind RNA, indicating that mitochondrial targeting is dispensable for MDH2-RNA interactions. MDH2 RNA binding increased when cellular NAD+ levels (MDH2's cofactor) were pharmacologically diminished, suggesting that the metabolic state of cells affects RNA binding. Taken together, our data implicate an as yet unidentified function of MDH2-binding RNA in the cytosol.

Improved discovery of RNA-binding protein binding sites in eCLIP data using DEWSeq.

Enhanced crosslinking and immunoprecipitation (eCLIP) sequencing is a method for transcriptome-wide detection of binding sites of RNA-binding proteins (RBPs). However, identified crosslink sites can deviate from experimentally established functional elements of even well-studied RBPs. Current peak-calling strategies result in low replication and high false positive rates. Here, we present the R/Bioconductor package DEWSeq that makes use of replicate information and size-matched input controls. We benchmarked DEWSeq on 107 RBPs for which both eCLIP data and RNA sequence motifs are available and were able to more than double the number of motif-containing binding regions relative to standard eCLIP processing. The improvement not only relates to the number of binding sites (3.1-fold with known motifs for RBFOX2), but also their subcellular localization (1.9-fold of mitochondrial genes for FASTKD2) and structural targets (2.2-fold increase of stem-loop regions for SLBP. On several orthogonal CLIP-seq datasets, DEWSeq recovers a larger number of motif-containing binding sites (3.3-fold). DEWSeq is a well-documented R/Bioconductor package, scalable to adequate numbers of replicates, and tends to substantially increase the proportion and total number of RBP binding sites containing biologically relevant features.

Small noncoding RNA interactome capture reveals pervasive, carbon source-dependent tRNA engagement of yeast glycolytic enzymes.

Small noncoding RNAs fulfill key functions in cellular and organismal biology, typically working in concert with RNA-binding proteins (RBPs). While proteome-wide methodologies have enormously expanded the repertoire of known RBPs, these methods do not distinguish RBPs binding to small noncoding RNAs from the rest. To specifically identify this relevant subclass of RBPs, we developed small noncoding RNA interactome capture (snRIC2C) based on the differential RNA-binding capacity of silica matrices (2C). We define the S. cerevisiae proteome of nearly 300 proteins that specifically binds to RNAs smaller than 200 nt in length (snRBPs), identifying informative distinctions from the total RNA-binding proteome determined in parallel. Strikingly, the snRBPs include most glycolytic enzymes from yeast. With further methodological developments using silica matrices, 12 tRNAs were identified as specific binders of the glycolytic enzyme GAPDH. We show that tRNA engagement of GAPDH is carbon source-dependent and regulated by the RNA polymerase III repressor Maf1, suggesting a regulatory interaction between glycolysis and RNA polymerase III activity. We conclude that snRIC2C and other 2C-derived methods greatly facilitate the study of RBPs, revealing previously unrecognized interactions.

htseq-clip: a toolset for the preprocessing of eCLIP/iCLIP datasets.

SUMMARY: Transcriptome-wide detection of binding sites of RNA-binding proteins is achieved using Individual-nucleotide crosslinking and immunoprecipitation (iCLIP) and its derivative enhanced CLIP (eCLIP) sequencing methods. Here, we introduce htseq-clip, a python package developed for preprocessing, extracting and summarizing crosslink site counts from i/eCLIP experimental data. The package delivers crosslink site count matrices along with other metrics, which can be directly used for filtering and downstream analyses such as the identification of differential binding sites. AVAILABILITY AND IMPLEMENTATION: The Python package htseq-clip is available via pypi (python package index), bioconda and the Galaxy Tool Shed under the open source MIT License. The code is hosted at https://github.com/EMBL-Hentze-group/htseq-clip and documentation is available under https://htseq-clip.readthedocs.io/en/latest.

Translational control of Ybx1 expression regulates cardiac function in response to pressure overload in vivo.

RNA-protein interactions are central to cardiac function, but how activity of individual RNA-binding protein is regulated through signaling cascades in cardiomyocytes during heart failure development is largely unknown. The mechanistic target of rapamycin kinase is a central signaling hub that controls mRNA translation in cardiomyocytes; however, a direct link between mTOR signaling and RNA-binding proteins in the heart has not been established. Integrative transcriptome and translatome analysis revealed mTOR dependent translational upregulation of the RNA binding protein Ybx1 during early pathological remodeling independent of mRNA levels. Ybx1 is necessary for pathological cardiomyocyte growth by regulating protein synthesis. To identify the molecular mechanisms how Ybx1 regulates cellular growth and protein synthesis, we identified mRNAs bound to Ybx1. We discovered that eucaryotic elongation factor 2 (Eef2) mRNA is bound to Ybx1, and its translation is upregulated during cardiac hypertrophy dependent on Ybx1 expression. Eef2 itself is sufficient to drive pathological growth by increasing global protein translation. Finally, Ybx1 depletion in vivo preserved heart function during pathological cardiac hypertrophy. Thus, activation of mTORC1 links pathological signaling cascades to altered gene expression regulation by activation of Ybx1 which in turn promotes translation through increased expression of Eef2.

Plasticity of nuclear and cytoplasmic stress responses of RNA-binding proteins.

Cellular stress causes multifaceted reactions to trigger adaptive responses to environmental cues at all levels of the gene expression pathway. RNA-binding proteins (RBP) are key contributors to stress-induced regulation of RNA fate and function. Here, we uncover the plasticity of the RNA interactome in stressed cells, differentiating between responses in the nucleus and in the cytoplasm. We applied enhanced RNA interactome capture (eRIC) analysis preceded by nucleo-cytoplasmic fractionation following arsenite-induced oxidative stress. The data reveal unexpectedly compartmentalized RNA interactomes and their responses to stress, including differential responses of RBPs in the nucleus versus the cytoplasm, which would have been missed by whole cell analyses.

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.

p19-INK4d inhibits neuroblastoma cell growth, induces differentiation and is hypermethylated and downregulated in MYCN-amplified neuroblastomas.

Uncontrolled cell cycle entry, resulting from deregulated CDK-RB1-E2F pathway activity, is a crucial determinant of neuroblastoma cell malignancy. Here we identify neuroblastoma-suppressive functions of the p19-INK4d CDK inhibitor and uncover mechanisms of its repression in high-risk neuroblastomas. Reduced p19-INK4d expression was associated with poor event-free and overall survival and neuroblastoma risk factors including amplified MYCN in a set of 478 primary neuroblastomas. High MYCN expression repressed p19-INK4d mRNA and protein levels in different neuroblastoma cell models with conditional MYCN expression. MassARRAY and 450K methylation analyses of 105 primary neuroblastomas uncovered a differentially methylated region within p19-INK4d. Hypermethylation of this region was associated with reduced p19-INK4d expression. In accordance, p19-INK4d expression was activated upon treatment with the demethylating agent, 2'-deoxy-5-azacytidine, in neuroblastoma cell lines. Ectopic p19-INK4d expression decreased viability, clonogenicity and the capacity for anchorage-independent growth of neuroblastoma cells, and shifted the cell cycle towards the G1/0 phase. p19-INK4d also induced neurite-like processes and markers of neuronal differentiation. Moreover, neuroblastoma cell differentiation, induced by all-trans retinoic acid or NGF-NTRK1-signaling, activated p19-INK4d expression. Our findings pinpoint p19-INK4d as a neuroblastoma suppressor and provide evidence for MYCN-mediated repression and for epigenetic silencing of p19-INK4d by DNA hypermethylation in high-risk neuroblastomas.

Prolyl hydroxylase-1 regulates hepatocyte apoptosis in an NF-κB-dependent manner.

Hepatocyte death is an important contributing factor in a number of diseases of the liver. PHD1 confers hypoxic sensitivity upon transcription factors including the hypoxia inducible factor (HIF) and nuclear factor-kappaB (NF-κB). Reduced PHD1 activity is linked to decreased apoptosis. Here, we investigated the underlying mechanism(s) in hepatocytes. Basal NF-κB activity was elevated in PHD1(-/-) hepatocytes compared to wild type controls. ChIP-seq analysis confirmed enhanced binding of NF-κB to chromatin in regions proximal to the promoters of genes involved in the regulation of apoptosis. Inhibition of NF-κB (but not knock-out of HIF-1 or HIF-2) reversed the anti-apoptotic effects of pharmacologic hydroxylase inhibition. We hypothesize that PHD1 inhibition leads to altered expression of NF-κB-dependent genes resulting in reduced apoptosis. This study provides new information relating to the possible mechanism of therapeutic action of hydroxylase inhibitors that has been reported in pre-clinical models of intestinal and hepatic disease.

The RNA-binding protein landscapes differ between mammalian organs and cultured cells.

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.

Transcriptional profiling of early differentiation of primary human mesenchymal stem cells into chondrocytes.

Articular cartilage has only very limited regenerative capacities in humans. Tissue engineering techniques for cartilage damage repair are limited in the production of hyaline cartilage. Mesenchymal stem/stromal cells (MSCs) are multipotent stem cells and can be differentiated into mature cartilage cells, chondrocytes, which could be used for repairing damaged cartilage. Chondrogenesis is a highly complex, relatively inefficient process lasting over 3 weeks in vitro. Methods: In order to better understand chondrogenic differentiation, especially the commitment phase, we have performed transcriptional profiling of MSC differentiation into chondrocytes from early timepoints starting 15 minutes after induction to 16 hours and fully differentiated chondrocytes at 21 days in triplicates.

GeneSigDB--a curated database of gene expression signatures.

The primary objective of most gene expression studies is the identification of one or more gene signatures; lists of genes whose transcriptional levels are uniquely associated with a specific biological phenotype. Whilst thousands of experimentally derived gene signatures are published, their potential value to the community is limited by their computational inaccessibility. Gene signatures are embedded in published article figures, tables or in supplementary materials, and are frequently presented using non-standard gene or probeset nomenclature. We present GeneSigDB (http://compbio.dfci.harvard.edu/genesigdb) a manually curated database of gene expression signatures. GeneSigDB release 1.0 focuses on cancer and stem cells gene signatures and was constructed from more than 850 publications from which we manually transcribed 575 gene signatures. Most gene signatures (n = 560) were successfully mapped to the genome to extract standardized lists of EnsEMBL gene identifiers. GeneSigDB provides the original gene signature, the standardized gene list and a fully traceable gene mapping history for each gene from the original transcribed data table through to the standardized list of genes. The GeneSigDB web portal is easy to search, allows users to compare their own gene list to those in the database, and download gene signatures in most common gene identifier formats.

A Pipeline for Analyzing eCLIP and iCLIP Data with Htseq-clip and DEWSeq.

Individual-nucleotide crosslinking and immunoprecipitation (iCLIP) sequencing and its derivative enhanced CLIP (eCLIP) sequencing are methods for the transcriptome-wide detection of binding sites of RNA-binding proteins (RBPs). This chapter provides a stepwise tutorial for analyzing iCLIP and eCLIP data with replicates and size-matched input (SMI) controls after read alignment using our open-source tools htseq-clip and DEWSeq. This includes the preparation of gene annotation, extraction, and preprocessing of truncation sites and the detection of significantly enriched binding sites using a sliding window based approach suitable for different binding modes of RBPs.

Identification of dynamic RNA-binding proteins uncovers a Cpeb4-controlled regulatory cascade during pathological cell growth of cardiomyocytes.

RNA-binding proteins (RBPs) control critical aspects of cardiomyocyte function, but the repertoire of active RBPs in cardiomyocytes during the growth response is largely unknown. We define RBPs in healthy and diseased cardiomyocytes at a system-wide level by RNA interactome capture. This identifies 67 cardiomyocyte-specific RBPs, including several contractile proteins. Furthermore, we identify the cytoplasmic polyadenylation element-binding protein 4 (Cpeb4) as a dynamic RBP, regulating cardiac growth both in vitro and in vivo. We identify mRNAs bound to and regulated by Cpeb4 in cardiomyocytes. Cpeb4 regulates cardiac remodeling by differential expression of transcription factors. Among Cpeb4 target mRNAs, two zinc finger transcription factors (Zeb1 and Zbtb20) are discovered. We show that Cpeb4 regulates the expression of these mRNAs and that Cpeb4 depletion increases their expression. Thus, Cpeb4 emerges as a critical regulator of cardiomyocyte function by differential binding to specific mRNAs in response to pathological growth stimulation.

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.