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Many cancers are defined by gene fusions that frequently encode oncogenic transcription factors (TFs), such as EWSR1::FLI1 in Ewing sarcoma (EwS). Here, we report that independently to its canonical roles in transcription, EWSR1::FLI1 also functions as an mRNA decay factor, reshaping mRNA stability in EwS. This function participates in EWSR1::FLI1 tumorigenicity and involves interactions of EWSR1::FLI1 with the CCR4-NOT deadenylation complex via its EWSR1-derived low-complexity domain and with the RNA-binding protein HuR/ELAVL1 via its FLI1-derived region. Strikingly, we find that EWSR1::FLI1-mediated mRNA decay antagonizes the normal mRNA protective function of HuR and renders EwS cells highly sensitive to HuR inhibition. Our findings uncover a post-transcriptional function of EWSR1::FLI1 and suggest that targeting mRNA stability mechanisms may offer therapeutic opportunities for EwS. The EWSR1::FLI1 fusion protein is the oncogenic driver of Ewing sarcoma (EwS). Here, the authors find that EWSR1::FLI1 plays a non-canonical role in mRNA decay via interactions with the CCR4-NOT deadenylation complex and the RNA-binding protein HuR. This role uncovers a new therapeutic vulnerability of EwS to HuR inhibition.

Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.Leidal et al. show that the LC3-conjugation pathway, which is part of the autophagy machinery, controls extracellular vesicle cargo loading and secretion of RNA-binding proteins.

Background Gliomas are the most common malignant primary brain tumours with a highly immunosuppressive tumour microenvironment (TME) and poor prognosis. Circular RNAs (circRNA), a newly found type of endogenous noncoding RNA, characterized by high stability, abundance, conservation, have been shown to play an important role in the pathophysiological processes and TME remodelling of various tumours. Methods CircRNA sequencing analysis was performed to explore circRNA expression profiles in normal and glioma tissues. The biological function of a novel circRNA, namely, circNEIL3, in glioma development was confirmed both in vitro and in vivo. Mechanistically, RNA pull-down, mass spectrum, RNA immunoprecipitation (RIP), luciferase reporter, and co-immunoprecipitation assays were conducted. Results We identified circNEIL3, which could be cyclized by EWS RNA-binding protein 1(EWSR1), to be upregulated in glioma tissues and to correlate positively with glioma malignant progression. Functionally, we confirmed that circNEIL3 promotes tumorigenesis and carcinogenic progression of glioma in vitro and in vivo. Mechanistically, circNEIL3 stabilizes IGF2BP3 (insulin-like growth factor 2 mRNA binding protein 3) protein, a known oncogenic protein, by preventing HECTD4-mediated ubiquitination. Moreover, circNEIL3 overexpression glioma cells drives macrophage infiltration into the tumour microenvironment (TME). Finally, circNEIL3 is packaged into exosomes by hnRNPA2B1 and transmitted to infiltrated tumour associated macrophages (TAMs), enabling them to acquire immunosuppressive properties by stabilizing IGF2BP3 and in turn promoting glioma progression. Conclusions This work reveals that circNEIL3 plays a nonnegligible multifaceted role in promoting gliomagenesis, malignant progression and macrophage tumour-promoting phenotypes polarization, highlighting that circNEIL3 is a potential prognostic biomarker and therapeutic target in glioma.

Protein domains without the usual distribution of amino acids, called low complexity (LC) domains, can be prone to self-assembly into amyloid-like fibrils. Self-assembly of LC domains that are nearly devoid of hydrophobic residues, such as the 214-residue LC domain of the RNA-binding protein FUS, is particularly intriguing from the biophysical perspective and is biomedically relevant due to its occurrence within neurons in amyotrophic lateral sclerosis, frontotemporal dementia, and other neurodegenerative diseases. We report a high-resolution molecular structural model for fibrils formed by the C-terminal half of the FUS LC domain (FUS-LC-C, residues 111-214), based on a density map with 2.62 Å resolution from cryo-electron microscopy (cryo-EM). In the FUS-LC-C fibril core, residues 112-150 adopt U-shaped conformations and form two subunits with in-register, parallel cross-β structures, arranged with quasi-2 1 symmetry. All-atom molecular dynamics simulations indicate that the FUS-LC-C fibril core is stabilized by a plethora of hydrogen bonds involving sidechains of Gln, Asn, Ser, and Tyr residues, both along and transverse to the fibril growth direction, including diverse sidechain-to-backbone, sidechain-to-sidechain, and sidechain-to-water interactions. Nuclear magnetic resonance measurements additionally show that portions of disordered residues 151-214 remain highly dynamic in FUS-LC-C fibrils and that fibrils formed by the N-terminal half of the FUS LC domain (FUS-LC-N, residues 2-108) have the same core structure as fibrils formed by the full-length LC domain. These results contribute to our understanding of the molecular structural basis for amyloid formation by FUS and by LC domains in general. The low-complexity (LC) domain mediates liquid-liquid phase separation and fibril formation of the RNA-binding protein FUS (FUsed in Sarcoma). Here, the authors combine cryo-EM, solid-state NMR measurements and MD simulations to structurally characterise the fibrils formed by the C-terminal half of the FUS LC domain and discuss stabilizing interactions within the fibril core.

Protein phase separation drives the assembly of membraneless organelles, but little is known about how these membraneless organelles are maintained in a metastable liquid- or gel-like phase rather than proceeding to solid aggregation. Here, we find that human small heat-shock protein 27 (Hsp27), a canonical chaperone that localizes to stress granules (SGs), prevents FUS from undergoing liquid−liquid phase separation (LLPS) via weak interactions with the FUS low complexity (LC) domain. Remarkably, stress-induced phosphorylation of Hsp27 alters its activity, leading Hsp27 to partition with FUS LC to preserve the liquid phase against amyloid fibril formation. NMR spectroscopy demonstrates that Hsp27 uses distinct structural mechanisms for both functions. Our work reveals a fine-tuned regulation of Hsp27 for chaperoning FUS into either a polydispersed state or a LLPS state and suggests an essential role for Hsp27 in stabilizing the dynamic phase of stress granules.The chaperone Hsp27 prevents FUS from undergoing liquid–liquid phase separation until stress-induced phosphorylation causes Hsp27 to partition with FUS to preserve the liquid phase against amyloid fibril formation.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by preferential motor neuron death. Approximately 15% of ALS cases are familial, and mutations in the fused in sarcoma (FUS) gene contribute to a subset of familial ALS cases. FUS is a multifunctional protein participating in many RNA metabolism pathways. ALS-linked mutations cause a liquid–liquid phase separation of FUS protein in vitro, inducing the formation of cytoplasmic granules and inclusions. However, it remains elusive what other proteins are sequestered into the inclusions and how such a process leads to neuronal dysfunction and degeneration. In this study, we developed a protocol to isolate the dynamic mutant FUS-positive cytoplasmic granules. Proteomic identification of the protein composition and subsequent pathway analysis led us to hypothesize that mutant FUS can interfere with protein translation. We demonstrated that the ALS mutations in FUS indeed suppressed protein translation in N2a cells expressing mutant FUS and fibroblast cells derived from FUS ALS cases. In addition, the nonsense-mediated decay (NMD) pathway, which is closely related to protein translation, was altered by mutant FUS. Specifically, NMD-promoting factors UPF1 and UPF3b increased, whereas a negative NMD regulator, UPF3a, decreased, leading to the disruption of NMD autoregulation and the hyperactivation of NMD. Alterations in NMD factors and elevated activity were also observed in the fibroblast cells of FUS ALS cases. We conclude that mutant FUS suppresses protein biosynthesis and disrupts NMD regulation, both of which likely contribute to motor neuron death.

Quantitative measurements of biomolecule associations are central to biological understanding and are needed to build and test predictive and mechanistic models. Given the advances in high-throughput technologies and the projected increase in the availability of binding data, we found it especially timely to evaluate the current standards for performing and reporting binding measurements. A review of 100 studies revealed that in most cases essential controls for establishing the appropriate incubation time and concentration regime were not documented, making it impossible to determine measurement reliability. Moreover, several reported affinities could be concluded to be incorrect, thereby impacting biological interpretations. Given these challenges, we provide a framework for a broad range of researchers to evaluate, teach about, perform, and clearly document high-quality equilibrium binding measurements. We apply this framework and explain underlying fundamental concepts through experimental examples with the RNA-binding protein Puf4.

Background Recent work has identified and mapped a range of posttranscriptional modifications in mRNA, including methylation of the N6 and N1 positions in adenine, pseudouridylation, and methylation of carbon 5 in cytosine (m5C). However, knowledge about the prevalence and transcriptome-wide distribution of m5C is still extremely limited; thus, studies in different cell types, tissues, and organisms are needed to gain insight into possible functions of this modification and implications for other regulatory processes. Results We have carried out an unbiased global analysis of m5C in total and nuclear poly(A) RNA of mouse embryonic stem cells and murine brain. We show that there are intriguing differences in these samples and cell compartments with respect to the degree of methylation, functional classification of methylated transcripts, and position bias within the transcript. Specifically, we observe a pronounced accumulation of m5C sites in the vicinity of the translational start codon, depletion in coding sequences, and mixed patterns of enrichment in the 3′ UTR. Degree and pattern of methylation distinguish transcripts modified in both embryonic stem cells and brain from those methylated in either one of the samples. We also analyze potential correlations between m5C and micro RNA target sites, binding sites of RNA binding proteins, and N 6-methyladenosine. Conclusion Our study presents the first comprehensive picture of cytosine methylation in the epitranscriptome of pluripotent and differentiated stages in the mouse. These data provide an invaluable resource for future studies of function and biological significance of m5C in mRNA in mammals.

Background Circular RNAs (circRNAs) are stable, often highly expressed RNA transcripts with potential to modulate other regulatory RNAs. A few circRNAs have been shown to bind RNA-binding proteins (RBPs); however, little is known about the prevalence and distribution of these interactions in different biological contexts. Methods We conduct an extensive screen of circRNA-RBP interactions in the ENCODE cell lines HepG2 and K562. We profile circRNAs in deep-sequenced total RNA samples and analyze circRNA-RBP interactions using a large set of eCLIP data with binding sites of 150 RBPs. We validate interactions for select circRNAs and RBPs by performing RNA immunoprecipitation and functionally characterize our most interesting candidates by conducting knockdown studies followed by RNA-Seq. Results We generate a comprehensive catalog of circRNA-RBP interactions in HepG2 and K562 cells. We show that KHSRP binding sites are enriched in flanking introns of circRNAs and that KHSRP depletion affects circRNA biogenesis. We identify circRNAs that are highly covered by RBP binding sites and experimentally validate individual circRNA-RBP interactions. We show that circCDYL, a highly expressed circRNA with clinical and functional implications in bladder cancer, is almost completely covered with GRWD1 binding sites in HepG2 cells, and that circCDYL depletion counteracts the effect of GRWD1 depletion. Furthermore, we confirm interactions between circCDYL and RBPs in bladder cancer cells and demonstrate that circCDYL depletion affects hallmarks of cancer and perturbs the expression of key cancer genes, e.g., TP53 . Finally, we show that elevated levels of circCDYL are associated with overall survival of bladder cancer patients. Conclusions Our study demonstrates transcriptome-wide and cell-type-specific circRNA-RBP interactions that could play important regulatory roles in tumorigenesis.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome 1 . 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 combination of five assays is used to produce a catalogue of RNA elements to which RNA-binding proteins bind in human cells.