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.

K6-linked ubiquitylation marks formaldehyde-induced RNA-protein crosslinks for resolution.

Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA-protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA (mRNA) are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-between-RING (RBR) E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and they suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.

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.

In vitro iCLIP-based modeling uncovers how the splicing factor U2AF2 relies on regulation by cofactors.

Alternative splicing generates distinct mRNA isoforms and is crucial for proteome diversity in eukaryotes. The RNA-binding protein (RBP) U2AF2 is central to splicing decisions, as it recognizes 3' splice sites and recruits the spliceosome. We establish "in vitro iCLIP" experiments, in which recombinant RBPs are incubated with long transcripts, to study how U2AF2 recognizes RNA sequences and how this is modulated by trans-acting RBPs. We measure U2AF2 affinities at hundreds of binding sites and compare in vitro and in vivo binding landscapes by mathematical modeling. We find that trans-acting RBPs extensively regulate U2AF2 binding in vivo, including enhanced recruitment to 3' splice sites and clearance of introns. Using machine learning, we identify and experimentally validate novel trans-acting RBPs (including FUBP1, CELF6, and PCBP1) that modulate U2AF2 binding and affect splicing outcomes. Our study offers a blueprint for the high-throughput characterization of in vitro mRNP assembly and in vivo splicing regulation.

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.

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.

Bacterial and Fungal Toll-Like Receptor Activation Elicits Type I IFN Responses in Mast Cells.

Next to their role in IgE-mediated allergic diseases and in promoting inflammation, mast cells also have antiinflammatory functions. They release pro- as well as antiinflammatory mediators, depending on the biological setting. Here we aimed to better understand the role of mast cells during the resolution phase of a local inflammation induced with the Toll-like receptor (TLR)-2 agonist zymosan. Multiple sequential immunohistology combined with a statistical neighborhood analysis showed that mast cells are located in a predominantly antiinflammatory microenvironment during resolution of inflammation and that mast cell-deficiency causes decreased efferocytosis in the resolution phase. Accordingly, FACS analysis showed decreased phagocytosis of zymosan and neutrophils by macrophages in mast cell-deficient mice. mRNA sequencing using zymosan-induced bone marrow-derived mast cells (BMMC) revealed a strong type I interferon (IFN) response, which is known to enhance phagocytosis by macrophages. Both, zymosan and lipopolysaccharides (LPS) induced IFN-ß synthesis in BMMCs in similar amounts as in bone marrow derived macrophages. IFN-ß was expressed by mast cells in paws from naïve mice and during zymosan-induced inflammation. As described for macrophages the release of type I IFNs from mast cells depended on TLR internalization and endosome acidification. In conclusion, mast cells are able to produce several mediators including IFN-ß, which are alone or in combination with each other able to regulate the phagocytotic activity of macrophages during resolution of inflammation.

CXCL5 as Regulator of Neutrophil Function in Cutaneous Melanoma.

Chemokines mold the tumor microenvironment by recruiting distinct immune cell populations, thereby strongly influencing disease progression. Previously, we showed that CXCL5 expression is upregulated in advanced stages of primary melanomas, which correlates with the presence of neutrophils in the tumor. The analysis of neutrophil populations in various tissues revealed a distinct phenotype of tumor-associated neutrophils. Tumor-associated neutrophils expressed PD-L1, CXCR4, CCR5, Adam17, and Nos2 and were immunosuppressive in a T-cell proliferation assay. To investigate the impact of CXCL5 and neutrophils in vivo, we established a syngeneic mouse tumor transplantation model using CXCL5-overexpressing and control melanoma cell lines. Growth behavior or vascularization of primary tumors was not affected by CXCL5 expression and neutrophils alone. However, in combination with Poly(I:C), tumor-associated neutrophils were able to attenuate induced antitumoral T-cell responses. CXCL5-overexpressing tumors had reduced lung metastasis compared with control tumors. Neutrophil depletion reversed this effect. In vitro, unstimulated lung-derived neutrophils had higher levels of reactive oxygen species compared with tumor-associated neutrophils, and CXCL5 stimulation further increased reactive oxygen species levels. In summary, in melanoma, neutrophils play a context-dependent role that is influenced by local or systemic factors, and interfere with therapies activating the acquired immune system. Actively switching neutrophils into antitumorigenic mode might be a new therapeutic strategy.

Apoptotic tumor cell-derived microRNA-375 uses CD36 to alter the tumor-associated macrophage phenotype.

Tumor-immune cell interactions shape the immune cell phenotype, with microRNAs (miRs) being crucial components of this crosstalk. How they are transferred and how they affect their target landscape, especially in tumor-associated macrophages (TAMs), is largely unknown. Here we report that breast cancer cells have a high constitutive expression of miR-375, which is released as a non-exosome entity during apoptosis. Deep sequencing of the miRome pointed to enhanced accumulation of miR-375 in TAMs, facilitated by the uptake of tumor-derived miR-375 via CD36. In macrophages, miR-375 directly targets TNS3 and PXN to enhance macrophage migration and infiltration into tumor spheroids and in tumors of a xenograft mouse model. In tumor cells, miR-375 regulates CCL2 expression to increase recruitment of macrophages. Our study provides evidence for miR transfer from tumor cells to TAMs and identifies miR-375 as a crucial regulator of phagocyte infiltration and the subsequent development of a tumor-promoting microenvironment.

A combined computational pipeline to detect circular RNAs in human cancer cells under hypoxic stress.

Hypoxia is associated with several diseases, including cancer. Cells that are deprived of adequate oxygen supply trigger transcriptional and post-transcriptional responses, which control cellular pathways such as angiogenesis, proliferation, and metabolic adaptation. Circular RNAs (circRNAs) are a novel class of mainly non-coding RNAs, which have been implicated in multiple cancers and attract increasing attention as potential biomarkers. Here, we characterize the circRNA signatures of three different cancer cell lines from cervical (HeLa), breast (MCF-7), and lung (A549) cancer under hypoxia. In order to reliably detect circRNAs, we integrate available tools with custom approaches for quantification and statistical analysis. Using this consolidated computational pipeline, we identify ~12000 circRNAs in the three cancer cell lines. Their molecular characteristics point to an involvement of complementary RNA sequences as well as trans-acting factors in circRNA biogenesis, such as the RNA-binding protein HNRNPC. Notably, we detect a number of circRNAs that are more abundant than their linear counterparts. In addition, 64 circRNAs significantly change in abundance upon hypoxia, in most cases in a cell type-specific manner. In summary, we present a comparative circRNA profiling in human cancer cell lines, which promises novel insights into the biogenesis and function of circRNAs under hypoxic stress.

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.

CXCL5 Facilitates Melanoma Cell-Neutrophil Interaction and Lymph Node Metastasis.

Chemokines influence tumor metastasis by targeting tumor, stromal, and hematopoietic cells. Characterizing the chemokine mRNA expression profile of human primary melanoma samples, we found CXCL5 significantly up-regulated in stage T4 primary melanomas when compared to thin melanomas (T1 stage). To characterize the role of CXCL5 in melanoma progression, we established a metastasizing murine xenograft model using CXCL5-overexpressing human melanoma cells. CXCL5 had no effect on melanoma proliferation in vitro and on primary tumor growth in vivo, but CXCL5-overexpressing tumors recruited high amounts of neutrophils and exhibited significantly increased lymphangiogenesis in our severe combined immune-deficient mouse model. Recruited neutrophils were found in close proximity to or within lymphatic vessels, often in direct contact with melanoma cells. Clinically, CXCL5-overexpressing melanomas had significantly increased lymph node metastases. We were able to translate these findings to human patient samples and found a positive correlation between CXCL5 expression, numbers of neutrophils in stage T4 primary melanoma, and the occurrence of subsequent locoregional metastasis.

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.