Biology subjects

Klostermann, M.

Publications and source records attributed to Klostermann, M..

7 recordsLinked to original sources

bioRxiv2026

iCLIP3: A streamlined, non-radioactive protocol for mapping protein-RNA interactions in cellular transcripts at single-nucleotide resolution

UV-C crosslinking and immunoprecipitation (CLIP)-based methods are the gold standard for identifying direct RNA binding protein (RBP) interaction sites on cellular RNA in vivo. Here, we describe individual-nucleotide resolution CLIP version 3 (iCLIP3), an optimized protocol for generating transcriptome-wide maps of RBP-RNA interaction sites at single-nucleotide resolution from low-input material. iCLIP3 introduces several key improvements over previous iCLIP variants, including rapid and safe infrared-based visualization of RBP-RNA complexes, silica column-based RNA isolation, and the incorporation of TruSeq adapter sequences with unique dual indexing. These modifications streamline library preparation, facilitate multiplexing, and enable concurrent sequencing of iCLIP3 libraries alongside unrelated RNA-seq libraries. In addition, we provide a detailed bioinformatics workflow for identifying crosslinking events and defining RBP binding sites. The complete protocol can be performed within 4-5 days, offering a robust and efficient approach for high-resolution mapping of RBP-RNA interactions. HighlightsO_LI3' RNA labeling with pCp-IR750 enables rapid infrared visualization of RBP-RNA complexes C_LIO_LISilica column-based RNA isolation improves efficiency and reproducibility C_LIO_LITruSeq adapters with unique dual indexing enable flexible and scalable multiplexing C_LIO_LIiCLIP3 generates high-quality libraries from ultra-low amounts of cellular material C_LI

genomics↗

bioRxiv2024

How to choose the optimal RNA-Seq library characteristics for alternative splicing analysis

Alternative splicing (AS) is a key layer of regulation in eukaryotic gene expression that is investigated in all areas of life sciences. Differences in AS between conditions can be quantified from transcriptome-wide short-read RNA sequencing (RNA-Seq) data with designated computational tools. However, not all short-read RNA-Seq data are equally suited for AS analysis. Here, we perform an exemplary AS analysis to showcase the impact of the RNA-Seq library characteristics on the obtained results. Using three standard ENCODE datasets with widespread AS changes, we modulate read length, read depth and the number of replicates and compare their influence on the detection, quantification and classification of AS events with the state-of-the-art AS algorithm MAJIQ. We find that longer reads and a higher read depth are the most effective measures to improve the sensitivity and precision of the analysis. From our results, we provide a recommendation on how to best choose the short-read RNA-Seq library specifications for an AS analysis.

genomics↗

bioRxiv2024

Intrinsically disordered RNA-binding motifs cooperate to catalyze RNA folding and drive phase separation

RNA-binding proteins are essential for gene regulation and the spatial organization of cells. Here, we report that the yeast ribosome biogenesis factor Loc1p is an intrinsically disordered RNA-binding protein with eight repeating positively charged, unstructured nucleic acid binding (PUN) motifs. While a single of these previously undefined motifs stabilizes folded RNAs, multiple copies strongly cooperate to catalyze RNA folding. In the presence of RNA, these multivalent PUN motifs drive phase separation. Proteome-wide searches in pro-and eukaryotes for proteins with similar arrays of PUN motifs reveal a strong enrichment in RNA-mediated processes and DNA remodeling. Thus, PUN motifs are potentially involved in a large variety of RNA-and DNA-related processes by concentrating them in membrane-less organelles. The general function and wide distribution of PUN motifs across species suggests that in an ancient "RNA world" PUN-like motifs may have supported the correct folding of early ribozymes.

biochemistry↗

bioRxiv2024

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

SummaryHere, 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. Availability and Implementationracoon_clip is implemented as a snakemake-powered command line tool (snakemake version [≥] 7.22, Python version [≥] 3.9). The latest release can be downloaded from GitHub (https://github.com/ZarnackGroup/racoon_clip/tree/main) and installed via pip. A detailed documentation, including installation, usage and customization, can be found at https://racoon-clip.readthedocs.io/en/latest/. The example datasets can be downloaded from the Short Read Archive (SRA; iCLIP: SRR5646576, SRR5646577, SRR5646578) or the ENCODE Project (eCLIP: ENCSR202BFN). ContactKathi Zarnack, kathi.zarnack@bmls.de Issue SectionGenome analysis

bioinformatics↗

bioRxiv2023

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. Key Points* Deep profiling identifies novel targets of miR-181 associated with global gene regulation. * miR-181 MREs in repeat elements in the coding sequence act through translational inhibition. * High-resolution analysis reveals an alternative seed match in functional MREs.

immunology↗

bioRxiv2022

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 decrease. 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/479353v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@5712f9org.highwire.dtl.DTLVardef@9eb133org.highwire.dtl.DTLVardef@83b7b2org.highwire.dtl.DTLVardef@1912e5c_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

Modulation of pre-mRNA structure by hnRNP proteins regulates alternative splicing of MALT1

Alternative splicing is controlled by differential binding of trans-acting RNA binding proteins (RBPs) to cis-regulatory elements in intronic and exonic pre-mRNA regions 1-3. How secondary structure in the pre-mRNA transcripts affects recognition by RBPs and determines alternative exon usage is poorly understood. The MALT1 paracaspase is a key component of signaling pathways that mediate innate and adaptive immune responses 4. Alternative splicing of MALT1 exon7 is critical for controlling optimal T cell activation 5,6. Here, we demonstrate that processing of the MALT1 pre-mRNA depends on RNA structural elements that shield the 5 and 3 splice sites of the alternatively spliced exon7. By combining biochemical analyses with chemical probing and NMR we show that the RBPs hnRNP U and hnRNP L bind competitively and with comparable affinities to identical stem-loop RNA structures flanking the 5 and 3 splice sites of MALT1 exon7. While hnRNP U stabilizes RNA stem-loop conformations that maintain exon7 skipping, hnRNP L unwinds these RNA elements to facilitate recruitment of the essential splicing factor U2AF2 to promote exon7 inclusion. Our data represent a paradigm for the control of splice site selection by differential RBP binding and modulation of pre-mRNA structure.

biochemistry↗