Condensation of Ded1p Promotes a Translational Switch from Housekeeping to Stress Protein Production.

Cells sense elevated temperatures and mount an adaptive heat shock response that involves changes in gene expression, but the underlying mechanisms, particularly on the level of translation, remain unknown. Here we report that, in budding yeast, the essential translation initiation factor Ded1p undergoes heat-induced phase separation into gel-like condensates. Using ribosome profiling and an in vitro translation assay, we reveal that condensate formation inactivates Ded1p and represses translation of housekeeping mRNAs while promoting translation of stress mRNAs. Testing a variant of Ded1p with altered phase behavior as well as Ded1p homologs from diverse species, we demonstrate that Ded1p condensation is adaptive and fine-tuned to the maximum growth temperature of the respective organism. We conclude that Ded1p condensation is an integral part of an extended heat shock response that selectively represses translation of housekeeping mRNAs to promote survival under conditions of severe heat stress.

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 Red Carpet for Iron Metabolism.

200 billion red blood cells (RBCs) are produced every day, requiring more than 2 × 1015 iron atoms every second to maintain adequate erythropoiesis. These numbers translate into 20 mL of blood being produced each day, containing 6 g of hemoglobin and 20 mg of iron. These impressive numbers illustrate why the making and breaking of RBCs is at the heart of iron physiology, providing an ideal context to discuss recent progress in understanding the systemic and cellular mechanisms that underlie the regulation of iron homeostasis and its disorders.

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.

Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.

RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and comprehensive approach, termed "interactome capture," to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings.

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.

Translational control via protein-regulated upstream open reading frames.

Analysis of the regulation of msl-2 mRNA by Sex lethal (SXL), which is critical for dosage compensation in Drosophila, has uncovered a mode of translational control based on common 5' untranslated region elements, upstream open reading frames (uORFs), and interaction sites for RNA-binding proteins. We show that SXL binding downstream of a short uORF imposes a strong negative effect on major reading frame translation. The underlying mechanism involves increasing initiation of scanning ribosomes at the uORF and augmenting its impediment to downstream translation. Our analyses reveal that SXL exerts its effect controlling initiation, not elongation or termination, at the uORF. Probing the generality of the underlying mechanism, we show that the regulatory module that we define experimentally functions in a heterologous context, and we identify natural Drosophila mRNAs that are regulated via this module. We propose that protein-regulated uORFs constitute a systematic principle for the regulation of protein synthesis.

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.

The RBPome of influenza A virus NP-mRNA reveals a role for TDP-43 in viral replication.

Genome-wide approaches have significantly advanced our knowledge of the repertoire of RNA-binding proteins (RBPs) that associate with cellular polyadenylated mRNAs within eukaryotic cells. Recent studies focusing on the RBP interactomes of viral mRNAs, notably SARS-Cov-2, have revealed both similarities and differences between the RBP profiles of viral and cellular mRNAs. However, the RBPome of influenza virus mRNAs remains unexplored. Herein, we identify RBPs that associate with the viral mRNA encoding the nucleoprotein (NP) of an influenza A virus. Focusing on TDP-43, we show that it binds several influenza mRNAs beyond the NP-mRNA, and that its depletion results in lower levels of viral mRNAs and proteins within infected cells, and a decreased yield of infectious viral particles. We provide evidence that the viral polymerase recruits TDP-43 onto viral mRNAs through a direct interaction with the disordered C-terminal domain of TDP-43. Notably, other RBPs found to be associated with influenza virus mRNAs also interact with the viral polymerase, which points to a role of the polymerase in orchestrating the assembly of viral messenger ribonucleoproteins.

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.

Two to tango: regulation of Mammalian iron metabolism.

Disruptions in iron homeostasis from both iron deficiency and overload account for some of the most common human diseases. Iron metabolism is balanced by two regulatory systems, one that functions systemically and relies on the hormone hepcidin and the iron exporter ferroportin, and another that predominantly controls cellular iron metabolism through iron-regulatory proteins that bind iron-responsive elements in regulated messenger RNAs. We describe how the two distinct systems function and how they "tango" together in a coordinated manner. We also highlight some of the current questions in mammalian iron metabolism and discuss therapeutic opportunities arising from a better understanding of the underlying biological principles.

Vault RNA1-1 riboregulates the autophagic function of p62 by binding to lysine 7 and arginine 21, both of which are critical for p62 oligomerization.

Cellular processes can be regulated at multiple levels, including transcriptional, post-transcriptional, and post-translational mechanisms. We have recently shown that the small, noncoding vault RNA1-1 negatively riboregulates p62 oligomerization in selective autophagy through direct interaction with the autophagic receptor. This function is highly specific for this Pol III transcript, but the determinants of this specificity and a mechanistic explanation of how vault RNA1-1 inhibits p62 oligomerization are lacking. Here, we combine biochemical and functional experiments to answer these questions. We show that the PB1 domain and adjacent linker region of p62 (aa 1-122) are necessary and sufficient for specific vault RNA1-1 binding, and we identify lysine 7 and arginine 21 as key hinges for p62 riboregulation. Chemical structure probing of vault RNA1-1 further reveals a central flexible loop within vault RNA1-1 that is required for the specific interaction with p62. Overall, our data provide molecular insight into how a small RNA riboregulates protein-protein interactions critical to the activation of specific autophagy.

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.

RNA-Binding Proteins Regulate Post-Transcriptional Responses to TGF-ß to Coordinate Function and Mesenchymal Activation of Murine Endothelial Cells.

BACKGROUND: Endothelial cells (ECs) are primed to respond to various signaling cues. For example, TGF (transforming growth factor)-ß has major effects on EC function and phenotype by driving ECs towards a more mesenchymal state (ie, triggering endothelial to mesenchymal activation), a dynamic process associated with cardiovascular diseases. Although transcriptional regulation triggered by TGF-ß in ECs is well characterized, post-transcriptional regulatory mechanisms induced by TGF-ß remain largely unknown. METHODS: Using RNA interactome capture, we identified global TGF-ß driven changes in RNA-binding proteins in ECs. We investigated specific changes in the RNA-binding patterns of hnRNP H1 (heterogeneous nuclear ribonucleoprotein H1) and Csde1 (cold shock domain containing E1) using RNA immunoprecipitation and overlapped this with RNA-sequencing data after knockdown of either protein for functional insight. Using a modified proximity ligation assay, we visualized the specific interactions between hnRNP H1 and Csde1 and target RNAs in situ both in vitro and in mouse heart sections. RESULTS: Characterization of TGF-ß-regulated RBPs (RNA-binding proteins) revealed hnRNP H1 and Csde1 as key regulators of the cellular response to TGF-ß at the post-transcriptional level, with loss of either protein-promoting mesenchymal activation in ECs. We found that TGF-ß drives an increase in binding of hnRNP H1 to its target RNAs, offsetting mesenchymal activation, but a decrease in Csde1 RNA-binding, facilitating this process. Both, hnRNP H1 and Csde1, dynamically bind and regulate specific subsets of mRNAs related to mesenchymal activation and endothelial function. CONCLUSIONS: Together, we show that RBPs play a key role in the endothelial response to TGF-ß stimulation at the post-transcriptional level and that the RBPs hnRNP H1 and Csde1 serve to maintain EC function and counteract mesenchymal activation. We propose that TGF-ß profoundly modifies RNA-protein interaction entailing feedback and feed-forward control at the post-transcriptional level, to fine-tune mesenchymal activation in ECs.

Disassembly of exon junction complexes by PYM.

Exon junction complexes (EJCs) are deposited onto mRNAs during splicing, serve as positional landmarks for the intron exon structure of genes, and direct posttranscriptional processes in the cytoplasm. EJC removal and recycling by translation are ill understood and have been attributed to ribosomal passage. This work identifies the ribosome-associated protein PYM as an EJC disassembly factor and defines its mechanism of function. Whereas EJC assembly intermediates are resistant to PYM, fully assembled EJCs are dissociated from spliced mRNAs by PYM. This disassembly involves PYM binding to the EJC proteins MAGOH-Y14. PYM overexpression in cells disrupts EJC association with spliced mRNA and inhibits nonsense-mediated mRNA decay. In cells depleted of PYM, EJCs accumulate on spliced mRNAs and EJC protein recycling is impaired. Hence, PYM is an EJC disassembly factor that acts both in vitro and in living cells, and that antagonizes important EJC functions.

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.

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.