FUBP1 is a general splicing factor facilitating 3' splice site recognition and splicing of long introns.

Splicing of pre-mRNAs critically contributes to gene regulation and proteome expansion in eukaryotes, but our understanding of the recognition and pairing of splice sites during spliceosome assembly lacks detail. Here, we identify the multidomain RNA-binding protein FUBP1 as a key splicing factor that binds to a hitherto unknown cis-regulatory motif. By collecting NMR, structural, and in vivo interaction data, we demonstrate that FUBP1 stabilizes U2AF2 and SF1, key components at the 3' splice site, through multivalent binding interfaces located within its disordered regions. Transcriptional profiling and kinetic modeling reveal that FUBP1 is required for efficient splicing of long introns, which is impaired in cancer patients harboring FUBP1 mutations. Notably, FUBP1 interacts with numerous U1 snRNP-associated proteins, suggesting a unique role for FUBP1 in splice site bridging for long introns. We propose a compelling model for 3' splice site recognition of long introns, which represent 80% of all human introns.

RNA stability controlled by m6A methylation contributes to X-to-autosome dosage compensation in mammals.

In mammals, X-chromosomal genes are expressed from a single copy since males (XY) possess a single X chromosome, while females (XX) undergo X inactivation. To compensate for this reduction in dosage compared with two active copies of autosomes, it has been proposed that genes from the active X chromosome exhibit dosage compensation. However, the existence and mechanisms of X-to-autosome dosage compensation are still under debate. Here we show that X-chromosomal transcripts have fewer m6A modifications and are more stable than their autosomal counterparts. Acute depletion of m6A selectively stabilizes autosomal transcripts, resulting in perturbed dosage compensation in mouse embryonic stem cells. We propose that higher stability of X-chromosomal transcripts is directed by lower levels of m6A, indicating that mammalian dosage compensation is partly regulated by epitranscriptomic RNA modifications.

Depletion of the RNA-binding protein PURA triggers changes in posttranscriptional gene regulation and loss of P-bodies.

The RNA-binding protein PURA has been implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. PURA binds both DNA and RNA and has been associated with various cellular functions. Only little is known about its main cellular roles and the molecular pathways affected upon PURA depletion. Here, we show that PURA is predominantly located in the cytoplasm, where it binds to thousands of mRNAs. Many of these transcripts change abundance in response to PURA depletion. The encoded proteins suggest a role for PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity. Intriguingly, reduced PURA levels decrease the expression of the integral P-body components LSM14A and DDX6 and strongly affect P-body formation in human cells. Furthermore, PURA knockdown results in stabilization of P-body-enriched transcripts, whereas other mRNAs are not affected. Hence, reduced PURA levels, as reported in patients with PURA Syndrome, influence the formation and composition of this phase-separated RNA processing machinery. Our study proposes PURA Syndrome as a new model to study the tight connection between P-body-associated RNA regulation and neurodevelopmental disorders.

TERRA increases at short telomeres in yeast survivors and regulates survivor associated senescence (SAS).

Cancer cells achieve immortality by employing either homology-directed repair (HDR) or the telomerase enzyme to maintain telomeres. ALT (alternative lengthening of telomeres) refers to the subset of cancer cells that employ HDR. Many ALT features are conserved from yeast to human cells, with the yeast equivalent being referred to as survivors. The non-coding RNA TERRA, and its ability to form RNA-DNA hybrids, has been implicated in ALT/survivor maintenance by promoting HDR. It is not understood which telomeres in ALT/survivors engage in HDR, nor is it clear which telomeres upregulate TERRA. Using yeast survivors as a model for ALT, we demonstrate that HDR only occurs at telomeres when they become critically short. Moreover, TERRA levels steadily increase as telomeres shorten and decrease again following HDR-mediated recombination. We observe that survivors undergo cycles of senescence, in a similar manner to non-survivors following telomerase loss, which we refer to as survivor associated senescence (SAS). Similar to 'normal' senescence, we report that RNA-DNA hybrids slow the rate of SAS, likely through the elongation of critically short telomeres, however decreasing the rate of telomere shortening may contribute to this effect. In summary, TERRA RNA-DNA hybrids regulate telomere dysfunction-induced senescence before and after survivor formation.

High-throughput mutagenesis identifies mutations and RNA-binding proteins controlling CD19 splicing and CART-19 therapy resistance.

Following CART-19 immunotherapy for B-cell acute lymphoblastic leukaemia (B-ALL), many patients relapse due to loss of the cognate CD19 epitope. Since epitope loss can be caused by aberrant CD19 exon 2 processing, we herein investigate the regulatory code that controls CD19 splicing. We combine high-throughput mutagenesis with mathematical modelling to quantitatively disentangle the effects of all mutations in the region comprising CD19 exons 1-3. Thereupon, we identify ~200 single point mutations that alter CD19 splicing and thus could predispose B-ALL patients to developing CART-19 resistance. Furthermore, we report almost 100 previously unknown splice isoforms that emerge from cryptic splice sites and likely encode non-functional CD19 proteins. We further identify cis-regulatory elements and trans-acting RNA-binding proteins that control CD19 splicing (e.g., PTBP1 and SF3B4) and validate that loss of these factors leads to pervasive CD19 mis-splicing. Our dataset represents a comprehensive resource for identifying predictive biomarkers for CART-19 therapy.

Deep and accurate detection of m6A RNA modifications using miCLIP2 and m6Aboost machine learning.

N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic mRNAs and influences many aspects of RNA processing. miCLIP (m6A individual-nucleotide resolution UV crosslinking and immunoprecipitation) is an antibody-based approach to map m6A sites with single-nucleotide resolution. However, due to broad antibody reactivity, reliable identification of m6A sites from miCLIP data remains challenging. Here, we present miCLIP2 in combination with machine learning to significantly improve m6A detection. The optimized miCLIP2 results in high-complexity libraries from less input material. Importantly, we established a robust computational pipeline to tackle the inherent issue of false positives in antibody-based m6A detection. The analyses were calibrated with Mettl3 knockout cells to learn the characteristics of m6A deposition, including m6A sites outside of DRACH motifs. To make our results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m6A sites in miCLIP2 data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m6A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m6A identification.

Ythdf is a N6-methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila.

N6-methyladenosine (m6 A) regulates a variety of physiological processes through modulation of RNA metabolism. This modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m6 A alters fly behavior, albeit the underlying molecular mechanism and the role of m6 A during nervous system development have remained elusive. Here we find that impairment of the m6 A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m6 A reader in the nervous system, being required to limit axonal growth. Mechanistically, we show that the m6 A reader Ythdf directly interacts with Fmr1, the fly homolog of Fragile X mental retardation RNA binding protein (FMRP), to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6 A pathway controls development of the nervous system and modulates Fmr1 target transcript selection.

The RNA-binding profile of the splicing factor SRSF6 in immortalized human pancreatic ß-cells.

In pancreatic ß-cells, the expression of the splicing factor SRSF6 is regulated by GLIS3, a transcription factor encoded by a diabetes susceptibility gene. SRSF6 down-regulation promotes ß-cell demise through splicing dysregulation of central genes for ß-cells function and survival, but how RNAs are targeted by SRSF6 remains poorly understood. Here, we define the SRSF6 binding landscape in the human pancreatic ß-cell line EndoC-ßH1 by integrating individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) under basal conditions with RNA sequencing after SRSF6 knockdown. We detect thousands of SRSF6 bindings sites in coding sequences. Motif analyses suggest that SRSF6 specifically recognizes a purine-rich consensus motif consisting of GAA triplets and that the number of contiguous GAA triplets correlates with increasing binding site strength. The SRSF6 positioning determines the splicing fate. In line with its role in ß-cell function, we identify SRSF6 binding sites on regulated exons in several diabetes susceptibility genes. In a proof-of-principle, the splicing of the susceptibility gene LMO7 is modulated by antisense oligonucleotides. Our present study unveils the splicing regulatory landscape of SRSF6 in immortalized human pancreatic ß-cells.

Checkpoint adaptation in recombination-deficient cells drives aneuploidy and resistance to genotoxic agents.

Human cancers frequently harbour mutations in DNA repair genes, rendering the use of DNA damaging agents as an effective therapeutic intervention. As therapy-resistant cells often arise, it is important to better understand the molecular pathways that drive resistance in order to facilitate the eventual targeting of such processes. We employ recombination-defective diploid yeast as a model to demonstrate that, in response to genotoxic challenges, nearly all cells eventually undergo checkpoint adaptation, resulting in the generation of aneuploid cells with whole chromosome losses that have acquired resistance to the initial genotoxic challenge. We demonstrate that adaptation inhibition, either pharmacologically, or genetically, drastically reduces the occurrence of resistant cells. Additionally, the aneuploid phenotypes of the resistant cells can be specifically targeted to induce cytotoxicity. We provide evidence that TORC1 inhibition with rapamycin, in combination with DNA damaging agents, can prevent both checkpoint adaptation and the continued growth of aneuploid resistant cells.

Exon Definition Facilitates Reliable Control of Alternative Splicing in the RON Proto-Oncogene.

Alternative splicing is a key step in eukaryotic gene expression that allows for the production of multiple transcript and protein isoforms from the same gene. Even though splicing is perturbed in many diseases, we currently lack insights into regulatory mechanisms promoting its precision and efficiency. We analyze high-throughput mutagenesis data obtained for an alternatively spliced exon in the proto-oncogene RON and determine the functional units that control this splicing event. Using mathematical modeling of distinct splicing mechanisms, we show that alternative splicing is based in RON on a so-called "exon definition" mechanism. Here, the recognition of the adjacent exons by the spliceosome is required for removal of an intron. We use our model to analyze the differences between the exon and intron definition scenarios and find that exon definition prevents the accumulation of deleterious, partially spliced retention products during alternative splicing regulation. Furthermore, it modularizes splicing control, as multiple regulatory inputs are integrated into a common net input, irrespective of the location and nature of the corresponding cis-regulatory elements in the pre-messenger RNA. Our analysis suggests that exon definition promotes robust and reliable splicing outcomes in RON splicing.

An autoinhibitory intramolecular interaction proof-reads RNA recognition by the essential splicing factor U2AF2.

The recognition of cis-regulatory RNA motifs in human transcripts by RNA binding proteins (RBPs) is essential for gene regulation. The molecular features that determine RBP specificity are often poorly understood. Here, we combined NMR structural biology with high-throughput iCLIP approaches to identify a regulatory mechanism for U2AF2 RNA recognition. We found that the intrinsically disordered linker region connecting the two RNA recognition motif (RRM) domains of U2AF2 mediates autoinhibitory intramolecular interactions to reduce nonproductive binding to weak Py-tract RNAs. This proofreading favors binding of U2AF2 at stronger Py-tracts, as required to define 3' splice sites at early stages of spliceosome assembly. Mutations that impair the linker autoinhibition enhance the affinity for weak Py-tracts result in promiscuous binding of U2AF2 along mRNAs and impact on splicing fidelity. Our findings highlight an important role of intrinsically disordered linkers to modulate RNA interactions of multidomain RBPs.

Makorin 1 controls embryonic patterning by alleviating Bruno1-mediated repression of oskar translation.

Makorins are evolutionary conserved proteins that contain C3H-type zinc finger modules and a RING E3 ubiquitin ligase domain. In Drosophila, maternal Makorin 1 (Mkrn1) has been linked to embryonic patterning but the mechanism remained unsolved. Here, we show that Mkrn1 is essential for axis specification and pole plasm assembly by translational activation of oskar (osk). We demonstrate that Mkrn1 interacts with poly(A) binding protein (pAbp) and binds specifically to osk 3' UTR in a region adjacent to A-rich sequences. Using Drosophila S2R+ cultured cells we show that this binding site overlaps with a Bruno1 (Bru1) responsive element (BREs) that regulates osk translation. We observe increased association of the translational repressor Bru1 with osk mRNA upon depletion of Mkrn1, indicating that both proteins compete for osk binding. Consistently, reducing Bru1 dosage partially rescues viability and Osk protein level in ovaries from Mkrn1 females. We conclude that Mkrn1 controls embryonic patterning and germ cell formation by specifically activating osk translation, most likely by competing with Bru1 to bind to osk 3' UTR.

Improved library preparation with the new iCLIP2 protocol.

Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) is a state-of-the-art technology to map the RNA interaction sites of an RNA-binding protein (RBP) across the transcriptome. Here, we present the new iCLIP2 protocol that allows to obtain high-quality iCLIP libraries in a fast and efficient manner. The new protocol comprises separate adapter ligations, two cDNA amplification steps and bead-based size selection. The full procedure can be completed within four days. Our advances significantly increase the complexity of the iCLIP2 libraries, resulting in a more comprehensive representation of RBP binding sites. Overall, the methodological advances in iCLIP2 allow efficient library generation and thereby promote the versatile and flexible application of this important technology.

iCLIP data analysis: A complete pipeline from sequencing reads to RBP binding sites.

Precise knowledge on the binding sites of an RNA-binding protein (RBP) is key to understanding the complex post-transcriptional regulation of gene expression. This information can be obtained from individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) experiments. Here, we present a complete data analysis workflow to reliably detect RBP binding sites from iCLIP data. The workflow covers all steps from the initial quality control of the sequencing reads up to peak calling and quantification of RBP binding. For each tool, we explain the specific requirements for iCLIP data analysis and suggest optimised parameter settings.

The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation.

BACKGROUND: Cells have evolved quality control mechanisms to ensure protein homeostasis by detecting and degrading aberrant mRNAs and proteins. A common source of aberrant mRNAs is premature polyadenylation, which can result in non-functional protein products. Translating ribosomes that encounter poly(A) sequences are terminally stalled, followed by ribosome recycling and decay of the truncated nascent polypeptide via ribosome-associated quality control. RESULTS: Here, we demonstrate that the conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) promotes ribosome stalling at poly(A) sequences during ribosome-associated quality control. We show that MKRN1 directly binds to the cytoplasmic poly(A)-binding protein (PABPC1) and associates with polysomes. MKRN1 is positioned upstream of poly(A) tails in mRNAs in a PABPC1-dependent manner. Ubiquitin remnant profiling and in vitro ubiquitylation assays uncover PABPC1 and ribosomal protein RPS10 as direct ubiquitylation substrates of MKRN1. CONCLUSIONS: We propose that MKRN1 mediates the recognition of poly(A) tails to prevent the production of erroneous proteins from prematurely polyadenylated transcripts, thereby maintaining proteome integrity.

Pancreatic pericytes originate from the embryonic pancreatic mesenchyme.

The embryonic origin of pericytes is heterogeneous, both between and within organs. While pericytes of coelomic organs were proposed to differentiate from the mesothelium, a single-layer squamous epithelium, the embryonic origin of pancreatic pericytes has yet to be reported. Here, we show that adult pancreatic pericytes originate from the embryonic pancreatic mesenchyme. Our analysis indicates that pericytes of the adult mouse pancreas originate from cells expressing the transcription factor Nkx3.2. In the embryonic pancreas, Nkx3.2-expressing cells constitute the multilayered mesenchyme, which surrounds the pancreatic epithelium and supports multiple events in its development. Thus, we traced the fate of the pancreatic mesenchyme. Our analysis reveals that pancreatic mesenchymal cells acquire various pericyte characteristics, including gene expression, typical morphology, and periendothelial location, during embryogenesis. Importantly, we show that the vast majority of pancreatic mesenchymal cells differentiate into pericytes already at embryonic day 13.5 and progressively acquires a more mature pericyte phenotype during later stages of pancreas organogenesis. Thus, our study indicates the embryonic pancreatic mesenchyme as the primary origin to adult pancreatic pericytes. As pericytes of other coelomic organs were suggested to differentiate from the mesothelium, our findings point to a distinct origin of these cells in the pancreas. Thus, our study proposes a complex ontogeny of pericytes of coelomic organs.

The key protein of endosomal mRNP transport Rrm4 binds translational landmark sites of cargo mRNAs.

RNA-binding proteins (RBPs) determine spatiotemporal gene expression by mediating active transport and local translation of cargo mRNAs. Here, we cast a transcriptome-wide view on the transported mRNAs and cognate RBP binding sites during endosomal messenger ribonucleoprotein (mRNP) transport in Ustilago maydis Using individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP), we compare the key transport RBP Rrm4 and the newly identified endosomal mRNP component Grp1 that is crucial to coordinate hyphal growth. Both RBPs bind predominantly in the 3' untranslated region of thousands of shared cargo mRNAs, often in close proximity. Intriguingly, Rrm4 precisely binds at stop codons, which constitute landmark sites of translation, suggesting an intimate connection of mRNA transport and translation. Towards uncovering the code of recognition, we identify UAUG as specific binding motif of Rrm4 that is bound by its third RRM domain. Altogether, we provide first insights into the positional organisation of co-localising RBPs on individual cargo mRNAs.

Decoding a cancer-relevant splicing decision in the RON proto-oncogene using high-throughput mutagenesis.

Mutations causing aberrant splicing are frequently implicated in human diseases including cancer. Here, we establish a high-throughput screen of randomly mutated minigenes to decode the cis-regulatory landscape that determines alternative splicing of exon 11 in the proto-oncogene MST1R (RON). Mathematical modelling of splicing kinetics enables us to identify more than 1000 mutations affecting RON exon 11 skipping, which corresponds to the pathological isoform RON∆165. Importantly, the effects correlate with RON alternative splicing in cancer patients bearing the same mutations. Moreover, we highlight heterogeneous nuclear ribonucleoprotein H (HNRNPH) as a key regulator of RON splicing in healthy tissues and cancer. Using iCLIP and synergy analysis, we pinpoint the functionally most relevant HNRNPH binding sites and demonstrate how cooperative HNRNPH binding facilitates a splicing switch of RON exon 11. Our results thereby offer insights into splicing regulation and the impact of mutations on alternative splicing in cancer.

Freiburg RNA tools: a central online resource for RNA-focused research and teaching.

The Freiburg RNA tools webserver is a well established online resource for RNA-focused research. It provides a unified user interface and comprehensive result visualization for efficient command line tools. The webserver includes RNA-RNA interaction prediction (IntaRNA, CopraRNA, metaMIR), sRNA homology search (GLASSgo), sequence-structure alignments (LocARNA, MARNA, CARNA, ExpaRNA), CRISPR repeat classification (CRISPRmap), sequence design (antaRNA, INFO-RNA, SECISDesign), structure aberration evaluation of point mutations (RaSE), and RNA/protein-family models visualization (CMV), and other methods. Open education resources offer interactive visualizations of RNA structure and RNA-RNA interaction prediction as well as basic and advanced sequence alignment algorithms. The services are freely available at http://rna.informatik.uni-freiburg.de.