Inhibition of YTHDF2 triggers proteotoxic cell death in MYC-driven breast cancer.

RNA-binding proteins (RBPs) are critical regulators of post-transcriptional gene expression, and aberrant RBP-RNA interactions can promote cancer progression. Here, we interrogate the function of RBPs in cancer using pooled CRISPR-Cas9 screening and identify 57 RBP candidates with distinct roles in supporting MYC-driven oncogenic pathways. We find that disrupting YTHDF2-dependent mRNA degradation triggers apoptosis in triple-negative breast cancer (TNBC) cells and tumors. eCLIP and m6A sequencing reveal that YTHDF2 interacts with mRNAs encoding proteins in the MAPK pathway that, when stabilized, induce epithelial-to-mesenchymal transition and increase global translation rates. scRibo-STAMP profiling of translating mRNAs reveals unique alterations in the translatome of single cells within YTHDF2-depleted solid tumors, which selectively contribute to endoplasmic reticulum stress-induced apoptosis in TNBC cells. Thus, our work highlights the therapeutic potential of RBPs by uncovering a critical role for YTHDF2 in counteracting the global increase of mRNA synthesis in MYC-driven breast cancers.

Zmat3 Is a Key Splicing Regulator in the p53 Tumor Suppression Program.

Although TP53 is the most commonly mutated gene in human cancers, the p53-dependent transcriptional programs mediating tumor suppression remain incompletely understood. Here, to uncover critical components downstream of p53 in tumor suppression, we perform unbiased RNAi and CRISPR-Cas9-based genetic screens in vivo. These screens converge upon the p53-inducible gene Zmat3, encoding an RNA-binding protein, and we demonstrate that ZMAT3 is an important tumor suppressor downstream of p53 in mouse KrasG12D-driven lung and liver cancers and human carcinomas. Integrative analysis of the ZMAT3 RNA-binding landscape and transcriptomic profiling reveals that ZMAT3 directly modulates exon inclusion in transcripts encoding proteins of diverse functions, including the p53 inhibitors MDM4 and MDM2, splicing regulators, and components of varied cellular processes. Interestingly, these exons are enriched in NMD signals, and, accordingly, ZMAT3 broadly affects target transcript stability. Collectively, these studies reveal ZMAT3 as a novel RNA-splicing and homeostasis regulator and a key component of p53-mediated tumor suppression.

Long-read Ribo-STAMP simultaneously measures transcription and translation with isoform resolution.

Transcription and translation are intertwined processes where mRNA isoforms are crucial intermediaries. However, methodological limitations in analyzing translation at the mRNA isoform level have left gaps in our understanding of critical biological processes. To address these gaps, we developed an integrated computational and experimental framework called long-read Ribo-STAMP (LR-Ribo-STAMP) that capitalizes on advancements in long-read sequencing and RNA-base editing-mediated technologies to simultaneously profile translation and transcription at both gene and mRNA isoform levels. We also developed the EditsC metric to quantify editing and leverage the single-molecule, full-length transcript information provided by long-read sequencing. Here, we report concordance between gene-level translation profiles obtained with long-read and short-read Ribo-STAMP. We show that LR-Ribo-STAMP successfully profiles translation of mRNA isoforms and links regulatory features, such as upstream open reading frames (uORFs), to translation measurements. We apply LR-Ribo-STAMP to discovering translational differences at both gene and isoform levels in a triple-negative breast cancer cell line under normoxia and hypoxia and find that LR-Ribo-STAMP effectively delineates orthogonal transcriptional and translation shifts between conditions. We also discover regulatory elements that distinguish translational differences at the isoform level. We highlight GRK6, where hypoxia is observed to increase expression and translation of a shorter mRNA isoform, giving rise to a truncated protein without the AGC Kinase domain. Overall, LR-Ribo-STAMP is an important advance in our repertoire of methods that measure mRNA translation with isoform sensitivity.

Multiplexed transcriptome discovery of RNA-binding protein binding sites by antibody-barcode eCLIP.

Ultraviolet crosslinking and immunoprecipitation (CLIP) methodologies enable the identification of RNA binding sites of RNA-binding proteins (RBPs). Despite improvements in the library preparation of RNA fragments, the enhanced CLIP (eCLIP) protocol requires 4 days of hands-on time and lacks the ability to process several RBPs in parallel. We present a new method termed antibody-barcode eCLIP that utilizes DNA-barcoded antibodies and proximity ligation of the DNA oligonucleotides to RBP-protected RNA fragments to interrogate several RBPs simultaneously. We observe performance comparable with that of eCLIP with the advantage of dramatically increased scaling while maintaining the same material requirement of a single eCLIP experiment.

A large-scale binding and functional map of human RNA-binding proteins.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection.

Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

Seryl-tRNA synthetase promotes translational readthrough by mRNA binding and involvement of the selenocysteine incorporation machinery.

Translational readthrough of UGA stop codons by selenocysteine-specific tRNA (tRNASec) enables the synthesis of selenoproteins. Seryl-tRNA synthetase (SerRS) charges tRNASec with serine, which is modified into selenocysteine and delivered to the ribosome by a designated elongation factor (eEFSec in eukaryotes). Here we found that components of the human selenocysteine incorporation machinery (SerRS, tRNASec, and eEFSec) also increased translational readthrough of non-selenocysteine genes, including VEGFA, to create C-terminally extended isoforms. SerRS recognizes target mRNAs through a stem-loop structure that resembles the variable loop of its cognate tRNAs. This function of SerRS depends on both its enzymatic activity and a vertebrate-specific domain. Through eCLIP-seq, we identified additional SerRS-interacting mRNAs as potential readthrough genes. Moreover, SerRS overexpression was sufficient to reverse premature termination caused by a pathogenic nonsense mutation. Our findings expand the repertoire of selenoprotein biosynthesis machinery and suggest an avenue for therapeutic targeting of nonsense mutations using endogenous factors.

Pyruvate Kinase M (PKM) binds ribosomes in a poly-ADP ribosylation dependent manner to induce translational stalling.

In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we asked whether there are factors that regulate compartment specific mRNA translation in human cells. Using a proteomic survey of spatially regulated polysome interacting proteins, we identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic (i.e. ER-excluded) polysome interactor and investigated how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels-providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode lysine- and glutamate-enriched tracts. Using ribosome footprint protection sequencing, we found that PKM binding to ribosomes causes translational stalling near lysine and glutamate encoding sequences. Lastly, we observed that PKM recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation)-and may depend on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, our study uncovers a novel role for PKM in post-transcriptional gene regulation, linking cellular metabolism and mRNA translation.

Robust single-cell discovery of RNA targets of RNA-binding proteins and ribosomes.

RNA-binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC-Mediated Profiling), which efficiently detects RBP-RNA interactions. STAMP does not rely on ultraviolet cross-linking or immunoprecipitation and, when coupled with single-cell capture, can identify RBP-specific and cell-type-specific RNA-protein interactions for multiple RBPs and cell types in single, pooled experiments. Pairing STAMP with long-read sequencing yields RBP target sites in an isoform-specific manner. Finally, Ribo-STAMP leverages small ribosomal subunits to measure transcriptome-wide ribosome association in single cells. STAMP enables the study of RBP-RNA interactomes and translational landscapes with unprecedented cellular resolution.

RNA binding protein DDX5 directs tuft cell specification and function to regulate microbial repertoire and disease susceptibility in the intestine.

OBJECTIVE: Tuft cells residing in the intestinal epithelium have diverse functions. In the small intestine, they provide protection against inflammation, combat against helminth and protist infections, and serve as entry portals for enteroviruses. In the colon, they had been implicated in tumourigenesis. Commitment of intestinal progenitor cells to the tuft cell lineage requires Rho GTPase Cell Division Cycle 42 (CDC42), a Rho GTPase that acts downstream of the epidermal growth factor receptor and wingless-related integration site signalling cascades, and the master transcription factor POU class 2 homeobox 3 (POU2F3). This study investigates how this pathway is regulated by the DEAD box containing RNA binding protein DDX5 in vivo. DESIGN: We assessed the role of DDX5 in tuft cell specification and function in control and epithelial cell-specific Ddx5 knockout mice (DDX5ΔIEC) using transcriptomic approaches. RESULTS: DDX5ΔIEC mice harboured a loss of intestinal tuft cell populations, modified microbial repertoire, and altered susceptibilities to ileal inflammation and colonic tumourigenesis. Mechanistically, DDX5 promotes CDC42 protein synthesis through a post-transcriptional mechanism to license tuft cell specification. Importantly, the DDX5-CDC42 axis is parallel but distinct from the known interleukin-13 circuit implicated in tuft cell hyperplasia, and both pathways augment Pou2f3 expression in secretory lineage progenitors. In mature tuft cells, DDX5 not only promotes integrin signalling and microbial responses, it also represses gene programmes involved in membrane transport and lipid metabolism. CONCLUSION: RNA binding protein DDX5 directs tuft cell specification and function to regulate microbial repertoire and disease susceptibility in the intestine.

The Thermus thermophilus DEAD-box protein Hera is a general RNA binding protein and plays a key role in tRNA metabolism.

RNA helicases catalyze the ATP-dependent destabilization of RNA duplexes. DEAD-box helicases share a helicase core that mediates ATP binding and hydrolysis, RNA binding and unwinding. Most members of this family contain domains flanking the core that can confer RNA substrate specificity and guide the helicase to a specific RNA. However, the in vivo RNA substrates of most helicases are currently not defined. The DEAD-box helicase Hera from Thermus thermophilus contains a helicase core, followed by a dimerization domain and an RNA binding domain that folds into an RNA recognition motif (RRM). The RRM mediates high affinity binding to an RNA hairpin, and an adjacent duplex is then unwound by the helicase core. Hera is a cold-shock protein, and has been suggested to act as an RNA chaperone under cold-shock conditions. Using crosslinking immunoprecipitation of Hera/RNA complexes and sequencing, we show that Hera binds to a large fraction of T. thermophilus RNAs under normal-growth and cold-shock conditions without a strong sequence preference, in agreement with a structure-specific recognition of RNAs and a general function in RNA metabolism. Under cold-shock conditions, Hera is recruited to RNAs with high propensities to form stable secondary structures. We show that selected RNAs identified, including a set of tRNAs, bind to Hera in vitro, and activate the Hera helicase core. Gene ontology analysis reveals an enrichment of genes related to translation, including mRNAs of ribosomal proteins, tRNAs, tRNA ligases, and tRNA-modifying enzymes, consistent with a key role of Hera in ribosome and tRNA metabolism.

RBP-Maps enables robust generation of splicing regulatory maps.

Alternative splicing of pre-messenger RNA transcripts enables the generation of multiple protein isoforms from the same gene locus, providing a major source of protein diversity in mammalian genomes. RNA binding proteins (RBPs) bind to RNA to control splice site choice and define which exons are included in the resulting mature RNA transcript. However, depending on where the RBPs bind relative to splice sites, they can activate or repress splice site usage. To explore this position-specific regulation, in vivo binding sites identified by methods such as cross-linking and immunoprecipitation (CLIP) are integrated with alternative splicing events identified by RNA-seq or microarray. Merging these data sets enables the generation of a "splicing map," where CLIP signal relative to a merged meta-exon provides a simple summary of the position-specific effect of binding on splicing regulation. Here, we provide RBP-Maps, a software tool to simplify generation of these maps and enable researchers to rapidly query regulatory patterns of an RBP of interest. Further, we discuss various alternative approaches to generate such splicing maps, focusing on how decisions in construction (such as the use of peak versus read density, or whole-reads versus only single-nucleotide candidate crosslink positions) can affect the interpretation of these maps using example eCLIP data from the 150 RBPs profiled by the ENCODE consortium.

Transcriptome regulation by PARP13 in basal and antiviral states in human cells.

The RNA-binding protein PARP13 is a primary factor in the innate antiviral response, which suppresses translation and drives decay of bound viral and host RNA. PARP13 interacts with many proteins encoded by interferon-stimulated genes (ISG) to activate antiviral pathways including co-translational addition of ISG15, or ISGylation. We performed enhanced crosslinking immunoprecipitation (eCLIP) and RNA-seq in human cells to investigate PARP13's role in transcriptome regulation for both basal and antiviral states. We find that the antiviral response shifts PARP13 target localization, but not its binding preferences, and that PARP13 supports the expression of ISGylation-related genes, including PARP13's cofactor, TRIM25. PARP13 associates with TRIM25 via RNA-protein interactions, and we elucidate a transcriptome-wide periodicity of PARP13 binding around TRIM25. Taken together, our study implicates PARP13 in creating and maintaining a cellular environment poised for an antiviral response through limiting PARP13 translation, regulating access to distinct mRNA pools, and elevating ISGylation machinery expression.

Protocol to process crosslinking and immunoprecipitation data into annotated binding sites.

Here, we present a protocol for using Skipper, a pipeline designed to process crosslinking and immunoprecipitation (CLIP) data into annotated binding sites. We describe steps for partitioning annotated transcript regions and fitting data to a beta-binomial model to call windows of enriched binding. From raw CLIP data, we detail how users can map reproducible RNA-binding sites to call enriched windows and perform downstream analysis. This protocol supports optional customizations for different use cases. For complete details on the use and execution of this protocol, please refer to Boyle et al.1.

Epistatic interactions between NMD and TRP53 control progenitor cell maintenance and brain size.

Mutations in human nonsense-mediated mRNA decay (NMD) factors are enriched in neurodevelopmental disorders. We show that deletion of key NMD factor Upf2 in mouse embryonic neural progenitor cells causes perinatal microcephaly but deletion in immature neurons does not, indicating NMD's critical roles in progenitors. Upf2 knockout (KO) prolongs the cell cycle of radial glia progenitor cells, promotes their transition into intermediate progenitors, and leads to reduced upper-layer neurons. CRISPRi screening identified Trp53 knockdown rescuing Upf2KO progenitors without globally reversing NMD inhibition, implying marginal contributions of most NMD targets to the cell cycle defect. Integrated functional genomics shows that NMD degrades selective TRP53 downstream targets, including Cdkn1a, which, without NMD suppression, slow the cell cycle. Trp53KO restores the progenitor cell pool and rescues the microcephaly of Upf2KO mice. Therefore, one physiological role of NMD in the developing brain is to degrade selective TRP53 targets to control progenitor cell cycle and brain size.

RNA binding protein DDX5 restricts RORγt+ Treg suppressor function to promote intestine inflammation.

Retinoid-related orphan receptor (RAR) gamma (RORγt)-expressing regulatory T cells (RORγt+ Tregs) play pivotal roles in preventing T cell hyperactivation and maintaining tissue homeostasis, in part by secreting the anti-inflammation cytokine interleukin-10 (IL-10). Here, we report that hypoxia-induced factor 1α (HIF1α) is the master transcription factor for Il10 in RORγt+ Tregs. This critical anti-inflammatory pathway is negatively regulated by an RNA binding protein DEAD box helicase 5 (DDX5). As a transcriptional corepressor, DDX5 restricts the expression of HIF1α and its downstream target gene Il10 in RORγt+ Tregs. T cell-specific Ddx5 knockout (DDX5ΔT) mice have augmented RORγt+ Treg suppressor activities and are better protected from intestinal inflammation. Genetic ablation or pharmacologic inhibition of HIF1α restores enteropathy susceptibility in DDX5ΔT mice. The DDX5-HIF1α-IL-10 pathway is conserved in mice and humans. These findings reveal potential therapeutic targets for intestinal inflammatory diseases.

Splicing factor SRSF1 deficiency in the liver triggers NASH-like pathology and cell death.

Regulation of RNA processing contributes profoundly to tissue development and physiology. Here, we report that serine-arginine-rich splicing factor 1 (SRSF1) is essential for hepatocyte function and survival. Although SRSF1 is mainly known for its many roles in mRNA metabolism, it is also crucial for maintaining genome stability. We show that acute liver damage in the setting of targeted SRSF1 deletion in mice is associated with the excessive formation of deleterious RNA-DNA hybrids (R-loops), which induce DNA damage. Combining hepatocyte-specific transcriptome, proteome, and RNA binding analyses, we demonstrate that widespread genotoxic stress following SRSF1 depletion results in global inhibition of mRNA transcription and protein synthesis, leading to impaired metabolism and trafficking of lipids. Lipid accumulation in SRSF1-deficient hepatocytes is followed by necroptotic cell death, inflammation, and fibrosis, resulting in NASH-like liver pathology. Importantly, SRSF1-depleted human liver cancer cells recapitulate this pathogenesis, illustrating a conserved and fundamental role for SRSF1 in preserving genome integrity and tissue homeostasis. Thus, our study uncovers how the accumulation of detrimental R-loops impedes hepatocellular gene expression, triggering metabolic derangements and liver damage.

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection.

Large-genome bacteriophages (jumbo phages) of the Chimalliviridae family assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and CRISPR/Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d halts infections at an early stage. Taken together, our data suggest that the conserved ChmC protein acts as a chaperone for phage mRNAs, potentially stabilizing these mRNAs and driving their translocation through the nuclear shell to promote translation and infection progression.

In Vivo Screening Unveils Pervasive RNA-Binding Protein Dependencies in Leukemic Stem Cells and Identifies ELAVL1 as a Therapeutic Target.

Acute myeloid leukemia (AML) is fueled by leukemic stem cells (LSC) whose determinants are challenging to discern from hematopoietic stem cells (HSC) or uncover by approaches focused on general cell properties. We have identified a set of RNA-binding proteins (RBP) selectively enriched in human AML LSCs. Using an in vivo two-step CRISPR-Cas9 screen to assay stem cell functionality, we found 32 RBPs essential for LSCs in MLL-AF9;NrasG12D AML. Loss-of-function approaches targeting key hit RBP ELAVL1 compromised LSC-driven in vivo leukemic reconstitution, and selectively depleted primitive malignant versus healthy cells. Integrative multiomics revealed differentiation, splicing, and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with mitochondrial import protein, TOMM34, being a direct ELAVL1-stabilized target whose repression impairs AML propagation. Altogether, using a stem cell-adapted in vivo CRISPR screen, this work demonstrates pervasive reliance on RBPs as regulators of LSCs and highlights their potential as therapeutic targets in AML. SIGNIFICANCE: LSC-targeted therapies remain a significant unmet need in AML. We developed a stem-cell-adapted in vivo CRISPR screen to identify key LSC drivers. We uncover widespread RNA-binding protein dependencies in LSCs, including ELAVL1, which we identify as a novel therapeutic vulnerability through its regulation of mitochondrial metabolism. This article is highlighted in the In This Issue feature, p. 171.