Coilin protein scaffolds Cajal bodies (CBs)-subnuclear compartments enriched in small nuclear RNAs (snRNAs)-and promotes efficient spliceosomal snRNP assembly. The molecular function of coilin, which is intrinsically disordered with no defined motifs, is poorly understood. We use UV crosslinking and immunoprecipitation (iCLIP) to determine whether mammalian coilin binds RNA in vivo and to identify targets. Robust detection of snRNA transcripts correlated with coilin ChIP-seq peaks on snRNA genes, indicating that coilin binding to nascent snRNAs is a site-specific CB nucleator. Surprisingly, several hundred small nucleolar RNAs (snoRNAs) were identified as coilin interactors, including numerous unannotated mouse and human snoRNAs. We show that all classes of snoRNAs concentrate in CBs. Moreover, snoRNAs lacking specific CB retention signals traffic through CBs en route to nucleoli, consistent with the role of CBs in small RNP assembly. Thus, coilin couples snRNA and snoRNA biogenesis, making CBs the cellular hub of small ncRNA metabolism.

AAUAAA is the most highly conserved motif in eukaryotic mRNA polyadenylation sites and, in mammals, is specifically recognized by the multisubunit CPSF (cleavage and polyadenylation specificity factor) complex. Despite its critical functions in mRNA 3' end formation, the molecular basis for CPSF-AAUAAA interaction remains poorly defined. The CPSF subunit CPSF160 has been implicated in AAUAAA recognition, but direct evidence has been lacking. Using in vitro and in vivo assays, we unexpectedly found that CPSF subunits CPSF30 and Wdr33 directly contact AAUAAA. Importantly, the CPSF30-RNA interaction is essential for mRNA 3' processing and is primarily mediated by its zinc fingers 2 and 3, which are specifically targeted by the influenza protein NS1A to suppress host mRNA 3' processing. Our data suggest that AAUAAA recognition in mammalian mRNA 3' processing is more complex than previously thought and involves multiple protein-RNA interactions.

RNA-binding proteins (RBPs) regulate splicing according to position-dependent principles, which can be exploited for analysis of regulatory motifs. Here we present RNAmotifs, a method that evaluates the sequence around differentially regulated alternative exons to identify clusters of short and degenerate sequences, referred to as multivalent RNA motifs. We show that diverse RBPs share basic positional principles, but differ in their propensity to enhance or repress exon inclusion. We assess exons differentially spliced between brain and heart, identifying known and new regulatory motifs, and predict the expression pattern of RBPs that bind these motifs. RNAmotifs is available at https://bitbucket.org/rogrro/rna_motifs.

Heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C) is a core component of 40S ribonucleoprotein particles that bind pre-mRNAs and influence their processing, stability and export. Breast cancer tumor suppressors BRCA1, BRCA2 and PALB2 form a complex and play key roles in homologous recombination (HR), DNA double strand break (DSB) repair and cell cycle regulation following DNA damage.

The SR proteins comprise a family of essential, structurally related RNA binding proteins. The complexity of their RNA targets and specificity of RNA recognition in vivo is not well understood. Here we use iCLIP to globally analyze and compare the RNA binding properties of two SR proteins, SRSF3 and SRSF4, in murine cells.

RNA-binding proteins have emerged as causal agents of complex neurological diseases. Mice deficient for neuronal RNA-binding protein CELF4 have a complex neurological disorder with epilepsy as a prominent feature. Human CELF4 has recently been associated with clinical features similar to those seen in mutant mice. CELF4 is expressed primarily in excitatory neurons, including large pyramidal cells of the cerebral cortex and hippocampus, and it regulates excitatory but not inhibitory neurotransmission. We examined mechanisms underlying neuronal hyperexcitability in Celf4 mutants by identifying CELF4 target mRNAs and assessing their fate in the absence of CELF4 in view of their known functions. CELF4 binds to at least 15%-20% of the transcriptome, with striking specificity for the mRNA 3' untranslated region. CELF4 mRNA targets encode a variety of proteins, many of which are well established in neuron development and function. While the overall abundance of these mRNA targets is often dysregulated in Celf4 deficient mice, the actual expression changes are modest at the steady-state level. In contrast, by examining the transcriptome of polysome fractions and the mRNA distribution along the neuronal cell body-neuropil axis, we found that CELF4 is critical for maintaining mRNA stability and availability for translation. Among biological processes associated with CELF4 targets that accumulate in neuropil of mutants, regulation of synaptic plasticity and transmission are the most prominent. Together with a related study of the impact of CELF4 loss on sodium channel Na(v)1.6 function, we suggest that CELF4 deficiency leads to abnormal neuronal function by combining a specific effect on neuronal excitation with a general impairment of synaptic transmission. These results also expand our understanding of the vital roles RNA-binding proteins play in regulating and shaping the activity of neural circuits.

We established a modified iCLIP protocol, called 'read-through marking', which facilitates the detection of cDNAs that have not been truncated upon encountering the RNA-peptide complex during reverse transcription (read-through cDNAs). A large proportion of these cDNAs would be undesirable in an iCLIP library, as it could affect the resolution of the method. To this end, we added an oligonucleotide to the 5'-end of RNA fragments-a 5'-marker-to mark the read-through cDNAs. By applying this modified iCLIP protocol to PTBP1 and eIF4A3, we found that the start sites of read-through cDNAs are enriched in adenosines, while the remaining cDNAs have a markedly different sequence content at their starts, preferentially containing thymidines. This finding in turn indicates that most of the reads in our iCLIP libraries are a product of truncation with valuable information regarding the proteins' RNA-binding sites. Thus, cDNA start sites confidently identify a protein's RNA-crosslink sites and we can account for the impact of read-through cDNAs by commonly adding a 5'-marker.

Recursive splicing (RS) starts by defining an "RS-exon," which is then spliced to the preceding exon, thus creating a recursive 5' splice site (RS-5ss). Previous studies focused on cryptic RS-exons, and now we find that the exon junction complex (EJC) represses RS of hundreds of annotated, mainly constitutive RS-exons. The core EJC factors, and the peripheral factors PNN and RNPS1, maintain RS-exon inclusion by repressing spliceosomal assembly on RS-5ss. The EJC also blocks 5ss located near exon-exon junctions, thus repressing inclusion of cryptic microexons. The prevalence of annotated RS-exons is high in deuterostomes, while the cryptic RS-exons are more prevalent in Drosophila, where EJC appears less capable of repressing RS. Notably, incomplete repression of RS also contributes to physiological alternative splicing of several human RS-exons. Finally, haploinsufficiency of the EJC factor Magoh in mice is associated with skipping of RS-exons in the brain, with relevance to the microcephaly phenotype and human diseases.

Mutations causing amyotrophic lateral sclerosis (ALS) strongly implicate ubiquitously expressed regulators of RNA processing. To understand the molecular impact of ALS-causing mutations on neuronal development and disease, we analysed transcriptomes during in vitro differentiation of motor neurons (MNs) from human control and patient-specific VCP mutant induced-pluripotent stem cells (iPSCs). We identify increased intron retention (IR) as a dominant feature of the splicing programme during early neural differentiation. Importantly, IR occurs prematurely in VCP mutant cultures compared with control counterparts. These aberrant IR events are also seen in independent RNAseq data sets from SOD1- and FUS-mutant MNs. The most significant IR is seen in the SFPQ transcript. The SFPQ protein binds extensively to its retained intron, exhibits lower nuclear abundance in VCP mutant cultures and is lost from nuclei of MNs in mouse models and human sporadic ALS. Collectively, we demonstrate SFPQ IR and nuclear loss as molecular hallmarks of familial and sporadic ALS.

Methyl-5-uridine (m5U) is one the most abundant non-canonical bases present in cellular RNA, and in yeast is found at position U54 of tRNAs where modification is catalysed by the methyltransferase Trm2. Although the mammalian enzymes that catalyse m5U formation are yet to be identified via experimental evidence, based on sequence homology to Trm2, two candidates currently exist, TRMT2A and TRMT2B. Here we developed a genome-wide single-nucleotide resolution mapping method, Fluorouracil-Induced-Catalytic-Crosslinking-Sequencing (FICC-Seq), in order to identify the relevant enzymatic targets. We demonstrate that TRMT2A is responsible for the majority of m5U present in human RNA, and that it commonly targets U54 of cytosolic tRNAs. By comparison to current methods, we show that FICC-Seq is a particularly robust method for accurate and reliable detection of relevant enzymatic target sites. Our associated finding of extensive irreversible TRMT2A-tRNA crosslinking in vivo following 5-Fluorouracil exposure is also intriguing, as it suggests a tangible mechanism for a previously suspected RNA-dependent route of Fluorouracil-mediated cytotoxicity.

Nonsense-mediated mRNA decay (NMD) controls gene expression by eliminating mRNAs with premature or aberrant translation termination. Degradation of NMD substrates is initiated by the central NMD factor UPF1, which recruits the endonuclease SMG6 and the deadenylation-promoting SMG5/7 complex. The extent to which SMG5/7 and SMG6 contribute to the degradation of individual substrates and their regulation by UPF1 remains elusive. Here we map transcriptome-wide sites of SMG6-mediated endocleavage via 3' fragment capture and degradome sequencing. This reveals that endogenous transcripts can have NMD-eliciting features at various positions, including upstream open reading frames (uORFs), premature termination codons (PTCs), and long 3' UTRs. We find that NMD substrates with PTCs undergo constitutive SMG6-dependent endocleavage, rather than SMG7-dependent exonucleolytic decay. In contrast, the turnover of NMD substrates containing uORFs and long 3' UTRs involves both SMG6- and SMG7-dependent endo- and exonucleolytic decay, respectively. This suggests that the extent to which SMG6 and SMG7 degrade NMD substrates is determined by the mRNA architecture.

Exercise has a wide range of systemic effects. In animal models, repeated exertion reduces malignant tumor progression, and clinically, exercise can improve outcome for cancer patients. The etiology of the effects of exercise on tumor progression are unclear, as are the cellular actors involved. We show here that in mice, exercise-induced reduction in tumor growth is dependent on CD8+ T cells, and that metabolites produced in skeletal muscle and excreted into plasma at high levels during exertion in both mice and humans enhance the effector profile of CD8+ T-cells. We found that activated murine CD8+ T cells alter their central carbon metabolism in response to exertion in vivo, and that immune cells from trained mice are more potent antitumor effector cells when transferred into tumor-bearing untrained animals. These data demonstrate that CD8+ T cells are metabolically altered by exercise in a manner that acts to improve their antitumoral efficacy.

The TGFβ pathway has essential roles in embryonic development, organ homeostasis, tissue repair and disease. These diverse effects are mediated through the intracellular effectors SMAD2 and SMAD3 (hereafter SMAD2/3), whose canonical function is to control the activity of target genes by interacting with transcriptional regulators. Therefore, a complete description of the factors that interact with SMAD2/3 in a given cell type would have broad implications for many areas of cell biology. Here we describe the interactome of SMAD2/3 in human pluripotent stem cells. This analysis reveals that SMAD2/3 is involved in multiple molecular processes in addition to its role in transcription. In particular, we identify a functional interaction with the METTL3-METTL14-WTAP complex, which mediates the conversion of adenosine to N6-methyladenosine (m6A) on RNA. We show that SMAD2/3 promotes binding of the m6A methyltransferase complex to a subset of transcripts involved in early cell fate decisions. This mechanism destabilizes specific SMAD2/3 transcriptional targets, including the pluripotency factor gene NANOG, priming them for rapid downregulation upon differentiation to enable timely exit from pluripotency. Collectively, these findings reveal the mechanism by which extracellular signalling can induce rapid cellular responses through regulation of the epitranscriptome. These aspects of TGFβ signalling could have far-reaching implications in many other cell types and in diseases such as cancer.

Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These "binding-region condensates" are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution.

Here, we show that direct recruitment of U1A to target transcripts can increase gene expression. This is a new regulatory role, in addition to previous knowledge showing that U1A decreases the levels of U1A mRNA and other specific targets. In fact, genome-wide, U1A more often increases rather than represses gene expression and many U1A-upregulated transcripts are directly bound by U1A according to individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) studies. Interestingly, U1A-mediated positive regulation can be transferred to a heterologous system for biotechnological purposes. Finally, U1A-bound genes are enriched for those involved in cell cycle and adhesion. In agreement with this, higher U1A mRNA expression associates with lower disease-free survival and overall survival in many cancer types, and U1A mRNA levels positively correlate with those of some oncogenes involved in cell proliferation. Accordingly, U1A depletion leads to decreased expression of these genes and the migration-related gene CCN2/CTGF, which shows the strongest regulation by U1A. A decrease in U1A causes a strong drop in CCN2 expression and CTGF secretion and defects in the expression of CTGF EMT targets, cell migration, and proliferation. These results support U1A as a putative therapeutic target for cancer treatment. In addition, U1A-binding sequences should be considered in biotechnological applications.

Recent studies have revealed multiple mechanisms that can lead to heterogeneity in ribosomal composition. This heterogeneity can lead to preferential translation of specific panels of mRNAs, and is defined in large part by the ribosomal protein (RP) content, amongst other things. However, it is currently unknown to what extent ribosomal composition is heterogeneous across tissues, which is compounded by a lack of tools available to study it. Here we present dripARF, a method for detecting differential RP incorporation into the ribosome using Ribosome Profiling (Ribo-seq) data. We combine the 'waste' rRNA fragment data generated in Ribo-seq with the known 3D structure of the human ribosome to predict differences in the composition of ribosomes in the material being studied. We have validated this approach using publicly available data, and have revealed a potential role for eS25/RPS25 in development. Our results indicate that ribosome heterogeneity can be detected in Ribo-seq data, providing a new method to study this phenomenon. Furthermore, with dripARF, previously published Ribo-seq data provides a wealth of new information, allowing the identification of RPs of interest in many disease and normal contexts. dripARF is available as part of the ARF R package and can be accessed through https://github.com/fallerlab/ARF.

The structure of mRNA molecules plays an important role in its interactions with trans-acting factors, notably RNA binding proteins (RBPs), thus contributing to the functional consequences of this interplay. However, current transcriptome-wide experimental methods to chart these interactions are limited by their poor sensitivity. Here we extend the hiCLIP atlas of duplexes bound by Staufen1 (STAU1) ∼10-fold, through careful consideration of experimental assumptions, and the development of bespoke computational methods which we apply to existing data. We present Tosca, a Nextflow computational pipeline for the processing, analysis and visualisation of proximity ligation sequencing data generally. We use our extended duplex atlas to discover insights into the RNA selectivity of STAU1, revealing the importance of structural symmetry and duplex-span-dependent nucleotide composition. Furthermore, we identify heterogeneity in the relationship between transcripts with STAU1-bound 3' UTR duplexes and metabolism of the associated RNAs that we relate to RNA structure: transcripts with short-range proximal 3' UTR duplexes have high degradation rates, but those with long-range duplexes have low rates. Overall, our work enables the integrative analysis of proximity ligation data delivering insights into specific features and effects of RBP-RNA structure interactions.

Enhanced expression of the cold-shock protein RNA binding motif 3 (RBM3) is highly neuroprotective both in vitro and in vivo. Whilst upstream signalling pathways leading to RBM3 expression have been described, the precise molecular mechanism of RBM3 cold induction remains elusive. To identify temperature-dependent modulators of RBM3, we performed a genome-wide CRISPR-Cas9 knockout screen using RBM3-reporter human iPSC-derived neurons. We found that RBM3 mRNA and protein levels are robustly regulated by several splicing factors, with heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) being the strongest positive regulator. Splicing analysis revealed that moderate hypothermia significantly represses the inclusion of a poison exon, which, when retained, targets the mRNA for nonsense-mediated decay. Importantly, we show that HNRNPH1 mediates this cold-dependent exon skipping via its thermosensitive interaction with a G-rich motif within the poison exon. Our study provides novel mechanistic insights into the regulation of RBM3 and provides further targets for neuroprotective therapeutic strategies.

An intronic GGGGCC repeat expansion in C9orf72 is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense and antisense directions to generate distinct dipeptide repeat proteins, of which poly(GA), poly(GR), and poly(PR) have been implicated in contributing to neurodegeneration. Poly(PR) binding to RNA may contribute to toxicity, but analysis of poly(PR)-RNA binding on a transcriptome-wide scale has not yet been carried out. We therefore performed crosslinking and immunoprecipitation (CLIP) analysis in human cells to identify the RNA binding sites of poly(PR). We found that poly(PR) binds to nearly 600 RNAs, with the sequence GAAGA enriched at the binding sites. In vitro experiments showed that poly(GAAGA) RNA binds poly(PR) with higher affinity than control RNA and induces the phase separation of poly(PR) into condensates. These data indicate that poly(PR) preferentially binds to poly(GAAGA)-containing RNAs, which may have physiological consequences.

A system enabling the expression of therapeutic proteins specifically in diseased cells would be transformative, providing greatly increased safety and the possibility of pre-emptive treatment. Here we describe "TDP-REG", a precision medicine approach primarily for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which exploits the cryptic splicing events that occur in cells with TDP-43 loss-of-function (TDP-LOF) in order to drive expression specifically in diseased cells. In addition to modifying existing cryptic exons for this purpose, we develop a deep-learning-powered algorithm for generating customisable cryptic splicing events, which can be embedded within virtually any coding sequence. By placing part of a coding sequence within a novel cryptic exon, we tightly couple protein expression to TDP-LOF. Protein expression is activated by TDP-LOF in vitro and in vivo, including TDP-LOF induced by cytoplasmic TDP-43 aggregation. In addition to generating a variety of fluorescent and luminescent reporters, we use this system to perform TDP-LOF-dependent genomic prime editing to ablate the UNC13A cryptic donor splice site. Furthermore, we design a panel of tightly gated, autoregulating vectors encoding a TDP-43/Raver1 fusion protein, which rescue key pathological cryptic splicing events. In summary, we combine deep-learning and rational design to create sophisticated splicing sensors, resulting in a platform that provides far safer therapeutics for neurodegeneration, potentially even enabling preemptive treatment of at-risk individuals.