P23 Acts as Functional RBP in the Macrophage Inflammation Response

Macrophages exert the primary cellular immune response. Pathogen components like bacterial lipopolysaccharides (LPS) stimulate macrophage migration, phagocytotic activity and cytokine expression. Previously, we identified the poly(A)+ RNA interactome of RAW 264.7 macrophages. Of the 402 RNA-binding proteins (RBPs), 32 were classified as unique in macrophages, including nineteen not reported to interact with nucleic acids before. Remarkably, P23 a HSP90 co-chaperone, also known as cytosolic prostaglandin E2 synthase (PTGES3), exhibited differential poly(A)+ RNA binding in untreated and LPS-induced macrophages. To identify mRNAs bound by P23 and to elucidate potential regulatory RBP functions in macrophages, we immunoprecipitated P23 from cytoplasmic extracts of cross-linked untreated and LPS-induced cells. RNAseq revealed that enrichment of 44 mRNAs was reduced in response to LPS. Kif15 mRNA, which encodes kinesin family member 15 (KIF15), a motor protein implicated in cytoskeletal reorganization and cell mobility was selected for further analysis. Noteworthy, phagocytic activity of LPS-induced macrophages was enhanced by P23 depletion. Specifically, in untreated RAW 264.7 macrophages, decreased P23 results in Kif15 mRNA destabilization, diminished KIF15 expression and accelerated macrophage migration. We show that the unexpected RBP function of P23 contributes to the regulation of macrophage phagocytotic activity and migration

Die Auswirkung der posttranslationellen Aktivierung des Transkriptionsfaktors Prf1 auf die Pheromonantwort in Ustilago maydis

Voraussetzung für eine erfolgreiche Infektion des phytopathogenen Pilzes Ustilago maydis ist die Erkennung und Fusion zweier kompatibler Sporidien auf der Pflanzenoberfläche. Als entscheidendes Signal wirkt dabei die gegenseitige Pheromonstimulation. Diese wird in der Zelle über mindestens zwei Signalwege weitergeleitet, eine MAP-Kinase-Kaskade (MAPK) sowie eine cAMP-abhängige Proteinkinase A (PKA). Zielprotein der beiden pheromonaktivierten Kinasen ist der Transkriptionsfaktor Prf1, welcher die Expression vieler in den folgenden Entwicklungsschritten entscheidender Faktoren reguliert. In Mutationsanalysen konnte gezeigt werden, dass die entsprechenden MAPK- und PKA-Phosphorylierungsstellen in Prf1 für dessen Aktivierung von essentieller Bedeutung sind. Die beiden Kinasen beeinflussen die Funktion des Transkriptionsfaktors in unterschiedlicher Weise, wobei das Phosphorylierungsmuster auf Prf1 insbesondere eine Unterscheidung der Promotoren der Gene der beiden Kreuzungstyploci zu erlauben schien. In der vorliegenden Arbeit konnte zunächst das tetrazyklinregulierte Expressionssystem als wichtige Ergänzung des Promotorrepertoires in U. maydis etabliert werden. Die tetrazyklinvermittelte Regulation von prf1 belegte Einsatzfähigkeit und Effizienz des heterologen Systems. Erste Hinweise auf die Mechanismen, welche der pheromonvermittelten Aktivierung von Prf1 zugrunde liegen, konnten durch die Lokalisierung des Proteins sowie die Entkopplung funktioneller Module gewonnen werden. Dabei deutete sich an, dass die DNA-Bindeeigenschaften wie auch das Transaktivierungspotential von Prf1 Angriffspunkte dieser Regulation darstellen könnten. Die Charakterisierung konstitutiv aktivierter Prf1-Varianten erlaubte es, die physiologische Relevanz der PKA-Phosphorylierung zu bestätigen. AnschließendeMicroarray-Analysen ermöglichten eine transkriptomweite Untersuchung der Auswirkung von PKA- wie auch MAPK-Modifikationen. Dabei konnten innerhalb der pheromonregulierten Gene unterschiedliche Regulationsklassen definiert werden, deren Expression durch den Phosphorylierungsstatus von Prf1 bestimmt wird. Während die transkriptionelle Induktion von prf1 ausreicht, um u.a. die Expression von Faktoren der Pheromonreifung und -sekretion zu induzieren, bestimmen MAPK- oder PKA-Phosphorylierung die Regulation destinkter Gengruppen. Diese Ergebnisse unterstreichen, dass der Transkriptionsfaktor Prf1 auf posttranslationeller Ebene zu einer Veränderung der Zielpromotorspezifität in der Lage ist, welche ein neues Konzept der Vernetzung unterschiedlicher Signalwege in der pilzlichen Pheromonantwort darstellt

racoon_clip – a complete pipeline for single-nucleotide analyses of iCLIP and eCLIP data

Here, we introduce racoon_clip, a sustainable and fully automated pipeline for the complete processing of iCLIP and eCLIP data to extract RNA binding signal at single-nucleotide resolution. racoon_clip is easy to install and execute, with multiple pre-settings and fully customizable parameters, and outputs a conclusive summary report with visualizations and statistics for all analysis steps

Die Auswirkung der posttranslationellen Aktivierung des Transkriptionsfaktors Prf1 auf die Pheromonantwort in Ustilago maydis

Voraussetzung für eine erfolgreiche Infektion des phytopathogenen Pilzes Ustilago maydis ist die Erkennung und Fusion zweier kompatibler Sporidien auf der Pflanzenoberfläche. Als entscheidendes Signal wirkt dabei die gegenseitige Pheromonstimulation. Diese wird in der Zelle über mindestens zwei Signalwege weitergeleitet, eine MAP-Kinase-Kaskade (MAPK) sowie eine cAMP-abhängige Proteinkinase A (PKA). Zielprotein der beiden pheromonaktivierten Kinasen ist der Transkriptionsfaktor Prf1, welcher die Expression vieler in den folgenden Entwicklungsschritten entscheidender Faktoren reguliert. In Mutationsanalysen konnte gezeigt werden, dass die entsprechenden MAPK- und PKA-Phosphorylierungsstellen in Prf1 für dessen Aktivierung von essentieller Bedeutung sind. Die beiden Kinasen beeinflussen die Funktion des Transkriptionsfaktors in unterschiedlicher Weise, wobei das Phosphorylierungsmuster auf Prf1 insbesondere eine Unterscheidung der Promotoren der Gene der beiden Kreuzungstyploci zu erlauben schien. In der vorliegenden Arbeit konnte zunächst das tetrazyklinregulierte Expressionssystem als wichtige Ergänzung des Promotorrepertoires in U. maydis etabliert werden. Die tetrazyklinvermittelte Regulation von prf1 belegte Einsatzfähigkeit und Effizienz des heterologen Systems. Erste Hinweise auf die Mechanismen, welche der pheromonvermittelten Aktivierung von Prf1 zugrunde liegen, konnten durch die Lokalisierung des Proteins sowie die Entkopplung funktioneller Module gewonnen werden. Dabei deutete sich an, dass die DNA-Bindeeigenschaften wie auch das Transaktivierungspotential von Prf1 Angriffspunkte dieser Regulation darstellen könnten. Die Charakterisierung konstitutiv aktivierter Prf1-Varianten erlaubte es, die physiologische Relevanz der PKA-Phosphorylierung zu bestätigen. AnschließendeMicroarray-Analysen ermöglichten eine transkriptomweite Untersuchung der Auswirkung von PKA- wie auch MAPK-Modifikationen. Dabei konnten innerhalb der pheromonregulierten Gene unterschiedliche Regulationsklassen definiert werden, deren Expression durch den Phosphorylierungsstatus von Prf1 bestimmt wird. Während die transkriptionelle Induktion von prf1 ausreicht, um u.a. die Expression von Faktoren der Pheromonreifung und -sekretion zu induzieren, bestimmen MAPK- oder PKA-Phosphorylierung die Regulation destinkter Gengruppen. Diese Ergebnisse unterstreichen, dass der Transkriptionsfaktor Prf1 auf posttranslationeller Ebene zu einer Veränderung der Zielpromotorspezifität in der Lage ist, welche ein neues Konzept der Vernetzung unterschiedlicher Signalwege in der pilzlichen Pheromonantwort darstellt

Direct long-read RNA sequencing identifies a subset of questionable exitrons likely arising from reverse transcription artifacts

Resistance to CD19-directed immunotherapies in lymphoblastic leukemia has been attributed, among other factors, to several aberrant CD19 pre-mRNA splicing events, including recently reported excision of a cryptic intron embedded within CD19 exon 2. While “exitrons” are known to exist in hundreds of human transcripts, we discovered, using reporter assays and direct long-read RNA sequencing (dRNA-seq), that the CD19 exitron is an artifact of reverse transcription. Extending our analysis to publicly available datasets, we identified dozens of questionable exitrons, dubbed “falsitrons,” that appear only in cDNA-seq, but never in dRNA-seq. Our results highlight the importance of dRNA-seq for transcript isoform validation

Phenotypic plasticity of fibroblasts during mammary carcinoma development

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment contribute to all stages of tumorigenesis and are usually considered to be tumor-promoting cells. CAFs show a remarkable degree of heterogeneity, which is attributed to developmental origin or to local environmental niches, resulting in distinct CAF subsets within individual tumors. While CAF heterogeneity is frequently investigated in late-stage tumors, data on longitudinal CAF development in tumors are lacking. To this end, we used the transgenic polyoma middle T oncogene-induced mouse mammary carcinoma model and performed whole transcriptome analysis in FACS-sorted fibroblasts from early- and late-stage tumors. We observed a shift in fibroblast populations over time towards a subset previously shown to negatively correlate with patient survival, which was confirmed by multispectral immunofluorescence analysis. Moreover, we identified a transcriptomic signature distinguishing CAFs from early- and late-stage tumors. Importantly, the signature of early-stage CAFs correlated well with tumor stage and survival in human mammary carcinoma patients. A random forest analysis suggested predictive value of the complete set of differentially expressed genes between early- and late-stage CAFs on bulk tumor patient samples, supporting the clinical relevance of our findings. In conclusion, our data show transcriptome alterations in CAFs during tumorigenesis in the mammary gland, which suggest that CAFs are educated by the tumor over time to promote tumor development. Moreover, we show that murine CAF gene signatures can harbor predictive value for human cancer

Cyclin CLB2 mRNA localization determines efficient protein synthesis to orchestrate bud growth and cell cycle progression

mRNA localization to subcellular compartments has been reported across all kingdoms of life and it is generally believed to promote asymmetric protein synthesis and localization. In striking contrast to previous observations, we show that in S. cerevisiae the B-type cyclin CLB2 mRNA is localized and translated in the yeast bud, while the Clb2 protein, a key regulator of mitosis progression, is concentrated in the mother nucleus. Using single-molecule RNA imaging in fixed (smFISH) and living cells (MS2 system), we show that the CLB2 mRNA is transported to the yeast bud by the She2-She3 complex, via an mRNA ZIP-code situated in the coding sequence. In CLB2 mRNA localization mutants, Clb2 protein synthesis in the bud is decreased resulting in changes in cell cycle distribution and genetic instability. Altogether, we propose that CLB2 mRNA localization acts as a sensor for bud development to couple cell growth and cell cycle progression, revealing a novel function for mRNA localization

A high-resolution map of functional miR-181 response elements in the thymus reveals the role of coding sequence targeting and an alternative seed match

MicroRNAs (miRNAs) are critical post-transcriptional regulators in many biological processes. They act by guiding RNA-induced silencing complexes to miRNA response elements (MREs) in target mRNAs, inducing translational inhibition and/or mRNA degradation. Functional MREs are expected to predominantly occur in the 3' untranslated region and involve perfect base-pairing of the miRNA seed. Here, we generate a high-resolution map of miR-181a/b-1 (miR-181) MREs to define the targeting rules of miR-181 in developing murine T-cells. By combining a multi-omics approach with computational high-resolution analyses, we uncover novel miR-181 targets and demonstrate that miR-181 acts predominantly through RNA destabilization. Importantly, we discover an alternative seed match and identify a distinct set of targets with repeat elements in the coding sequence which are targeted by miR-181 and mediate translational inhibition. In conclusion, deep profiling of MREs in primary cells is critical to expand physiologically relevant targetomes and establish context-dependent miRNA targeting rules

SRSF3 and SRSF7 modulate 3′UTR length through suppression or activation of proximal polyadenylation sites and regulation of CFIm levels

Background: Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3′ untranslated regions (3′UTRs). We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, connect APA with mRNA export. The mechanism underlying APA regulation by SRSF3 and SRSF7 remained unknown. Results: Here we combine iCLIP and 3′-end sequencing and find that SRSF3 and SRSF7 bind upstream of proximal PASs (pPASs), but they exert opposite effects on 3′UTR length. SRSF7 enhances pPAS usage in a concentration-dependent but splicing-independent manner by recruiting the cleavage factor FIP1, generating short 3′UTRs. Protein domains unique to SRSF7, which are absent from SRSF3, contribute to FIP1 recruitment. In contrast, SRSF3 promotes distal PAS (dPAS) usage and hence long 3′UTRs directly by counteracting SRSF7, but also indirectly by maintaining high levels of cleavage factor Im (CFIm) via alternative splicing. Upon SRSF3 depletion, CFIm levels decrease and 3′UTRs are shortened. The indirect SRSF3 targets are particularly sensitive to low CFIm levels, because here CFIm serves a dual function; it enhances dPAS and inhibits pPAS usage by binding immediately downstream and assembling unproductive cleavage complexes, which together promotes long 3′UTRs. Conclusions; We demonstrate that SRSF3 and SRSF7 are direct modulators of pPAS usage and show how small differences in the domain architecture of SR proteins can confer opposite effects on pPAS regulation

Prediction of m6A and m5C at single-molecule resolution reveals a cooccurrence of RNA modifications across the transcriptome

The epitranscriptome embodies many new and largely unexplored functions of RNA. A significant roadblock hindering progress in epitranscriptomics is the identification of more than one modification in individual transcript molecules. We address this with CHEUI (CH3 (methylation) Estimation Using Ionic current). CHEUI predicts N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual molecules from the same sample, the stoichiometry at transcript reference sites, and differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals to achieve high single-molecule, transcript-site, and stoichiometry accuracies in multiple tests using synthetic RNA standards and cell line data. CHEUI’s capability to identify two modification types in the same sample reveals a co-occurrence of m6A and m5C in individual mRNAs in cell line and tissue transcriptomes. CHEUI provides new avenues to discover and study the function of the epitranscriptome