Transcriptome-wide functions of the RNA-binding protein PURA in health and disease DOI Creative Commons
Melina Klostermann

Published: Jan. 1, 2024

The attention on the protein PURA has increased recently following discovery of rare Syndrome. This neurodevelopmental disorder is caused by de novo mutations in gene. Notably, our collaborators could show that can bind DNA and RNA vitro. As a result, I was motivated to explore PURA's cellular RNAbinding activity. Furthermore, inquired connection PURA-RNA binding effect reduction functional as present Syndrome patients. To investigate impact ciency expression, performed an integrative computational analysis multimodal data from complementary high-throughput experiments. An essential component examination UV Crosslinking immunoprecipitation (CLIP) experiments, which query global RNA-binding behaviour given context. processing CLIP are rather complex, introduce automated command line tool for named racoon_clip part this dissertation. Therefore, dissertation comprises two major segments. Firstly, describe implementation usage racoon clip analysis. Secondly, discuss my research PURA, demonstrating its properties, effects depletion association with neuronal functions P-bodies, among others. application have developed individualnucleotide resolution (iCLIP) enhanced (eCLIP) experiments - most commonly used types comparable user-friendly way. For this, built work how encompasses all steps raw single-nucleotide crosslink events. available users run single command. implemented Snakemake management providing advantage tages including parallelisation, scalability portability how. main task extract events iCLIP, iCLIP2, eCLIP similar types. strike balance between being highly customisable easy use, supplies pre-set options common Additionally, it possible create custom setup barcode adapter architectures, allows them use software other data. While accounting different architectures reads, central remain same. leads high degree comparability experiment types, demonstrate exemplary U2AF2 iCLIP Taken together, am confident will be beneficial numerous researchers interested RNA-Protein interactions offers easily accessible enhances multiple datasets across di erent In second dissertation, focus function PURA. Through in-depth one set endogenous sets overexpressed HeLa cells, establish protein. It preferentially binds RNAs either coding sequence (CDS) or 3' untranslated region (3'UTR) mature protein-coding transcripts recognising Purine-rich degenerated motif. Even though overexpression results less specific behaviour, same overall patterns persist. Overall characteristics three distinct without overexpression. learn about molecular consequences context, 50% cells model heterozygous loss evaluated expression. globally ects integrate changes expression proteins context depletion. reveals 234 targets bound impacted at both levels change abundance upon enriched development factors, lifecycle regulators, mitochondrial Consistent role transport, there considerable overlap transcripts, transported dendritic end neurons. link documented enrichment PURA-bound P-body stress granule transcriptome. Further, found localised within P-bodies numbers were strongly reduced depleted absence might attributed downregulation encoded LSM14A DDX6 previously identified formation. Overall, depletion, mitochondria regulation may indicate foundation related diseases. summary, versatile Subsequently, conduct thorough valuable insights into These advance understanding disease contexts.

Language: Английский

Large-scale evaluation of the ability of RNA-binding proteins to activate exon inclusion DOI Creative Commons
Jonathan C. Schmok,

Manya Jain,

Lena Annika Street

et al.

Nature Biotechnology, Journal Year: 2024, Volume and Issue: 42(9), P. 1429 - 1441

Published: Jan. 2, 2024

RNA-binding proteins (RBPs) modulate alternative splicing outcomes to determine isoform expression and cellular survival. To identify RBPs that directly drive exon inclusion, we developed tethered function luciferase-based reporters provide rapid, scalable robust readouts of inclusion changes used these evaluate 718 human RBPs. We performed enhanced cross-linking immunoprecipitation, RNA sequencing affinity purification-mass spectrometry investigate a subset candidates with no prior association splicing. Integrative analysis assays indicates surprising roles for TRNAU1AP, SCAF8 RTCA in the modulation hundreds endogenous events. also leveraged our tethering top potent compact activation domains applications. Using identified domains, engineered programmable fusion outperform current artificial factors at manipulating reporter exons. This approach characterizes ability induce yields new molecular parts control.

Language: Английский

Citations

19
Integrated multi-omics analysis of zinc-finger proteins uncovers roles in RNA regulation DOI Creative Commons
Maya L. Gosztyla, Lijun Zhan, Sara Olson

et al.

Molecular Cell, Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 1, 2024

Language: Английский

Citations

6
Decoding protein–RNA interactions using CLIP-based methodologies DOI
Joy S. Xiang,

Danielle Schafer,

Katherine Rothamel

et al.

Nature Reviews Genetics, Journal Year: 2024, Volume and Issue: 25(12), P. 879 - 895

Published: July 9, 2024

Language: Английский

Citations

5
Phenylpyrazoles as Inhibitors of the m6A RNA-Binding Protein YTHDF2 DOI Creative Commons

Xiaqiu Qiu,

Claus Kemker,

Georg L. Goebel

et al.

JACS Au, Journal Year: 2025, Volume and Issue: 5(2), P. 618 - 630

Published: Feb. 10, 2025

The N6-methyladenosine (m6A) modification, which is the most common RNA modification in eukaryotes, regulated by "writer" methyltransferases, "reader" m6A binding proteins, and "eraser" demethylases. plays a multifunctional role physiological pathological processes, regulating all aspects of metabolism function, including splicing, translation, transportation, degradation. Accumulating evidence suggests that YT521-B homology domain family 2 (YTHDF2), one "readers," associated with various biological processes cancers noncancerous disorders, impacting migration, invasion, metastasis, proliferation, apoptosis, cell cycle. Here, we describe our work identification series functionalized pyrazoles, such as CK-75, new YTHDF2 inhibitors, potentially bind to small hydrophobic pocket on YTH domain. Cellular evaluations revealed small-molecule inhibitors induced cycle arrest, significantly inhibited viability cancer cells. Furthermore, evaluated transcriptome-wide change global RNA-binding protein patterns CK-75 via an enhanced cross-linking immunoprecipitation assay. Our demonstrated feasibility targeting molecules. phenylpyrazoles studied this provided lead structure for further development molecules both therapeutic applications.

Language: Английский

Citations

0
ePRINT: exonuclease assisted mapping of protein-RNA interactions DOI Creative Commons
Sophie Hawkins, Alexandre Mondaini, Seema C. Namboori

et al.

Genome biology, Journal Year: 2024, Volume and Issue: 25(1)

Published: May 28, 2024

Abstract RNA-binding proteins (RBPs) regulate key aspects of RNA processing including alternative splicing, mRNA degradation and localization by physically binding molecules. Current methods to map these interactions, such as CLIP, rely on purifying single at a time. Our new method, ePRINT, maps RBP-RNA interaction networks global scale without individual RBPs. ePRINT uses exoribonuclease XRN1 precisely the 5′ end RBP site uncovers direct indirect targets an interest. Importantly, can also uncover RBPs that are differentially activated between cell fate transitions, neural progenitor differentiation into neurons.

Language: Английский

Citations

3
Large-scale map of RNA-binding protein interactomes across the mRNA life cycle DOI Creative Commons
Lena Annika Street, Katherine Rothamel, Kristopher W. Brannan

et al.

Molecular Cell, Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 1, 2024

Language: Английский

Citations

3
From computational models of the splicing code to regulatory mechanisms and therapeutic implications DOI
Charlotte Capitanchik, Oscar G. Wilkins, Nils Wagner

et al.

Nature Reviews Genetics, Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 2, 2024

Language: Английский

Citations

2
Seryl-tRNA synthetase promotes translational readthrough by mRNA binding and involvement of the selenocysteine incorporation machinery DOI Creative Commons
Ze Liu, Justin Wang, Yi Shi

et al.

Nucleic Acids Research, Journal Year: 2023, Volume and Issue: 51(19), P. 10768 - 10781

Published: Sept. 22, 2023

Abstract Translational readthrough of UGA stop codons by selenocysteine-specific tRNA (tRNASec) enables the synthesis selenoproteins. Seryl-tRNA synthetase (SerRS) charges tRNASec with serine, which is modified into selenocysteine and delivered to ribosome a designated elongation factor (eEFSec in eukaryotes). Here we found that components human incorporation machinery (SerRS, tRNASec, eEFSec) also increased translational non-selenocysteine genes, including VEGFA, create C-terminally extended isoforms. SerRS recognizes target mRNAs through stem-loop structure resembles variable loop its cognate tRNAs. This function depends on both enzymatic activity vertebrate-specific domain. Through eCLIP-seq, identified additional SerRS-interacting as potential genes. Moreover, overexpression was sufficient reverse premature termination caused pathogenic nonsense mutation. Our findings expand repertoire selenoprotein biosynthesis suggest an avenue for therapeutic targeting mutations using endogenous factors.

Language: Английский

Citations

6
Large-scale map of RNA binding protein interactomes across the mRNA life-cycle DOI Creative Commons
Lena Annika Street, Katherine Rothamel, Kristopher W. Brannan

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: June 8, 2023

Messenger RNAs (mRNAs) interact with RNA-binding proteins (RBPs) in diverse ribonucleoprotein complexes (RNPs) during distinct life-cycle stages for their processing and maturation. While substantial attention has focused on understanding RNA regulation by assigning proteins, particularly RBPs, to specific substrates, there been considerably less exploration leveraging protein-protein interaction (PPI) methodologies identify study the role of mRNA stages. To address this gap, we generated an RNA-aware RBP-centric PPI map across immunopurification (IP-MS) ~100 endogenous RBPs presence or absence RNase, augmented size exclusion chromatography (SEC-MS). Aside from confirming 8,700 known discovering 20,359 novel interactions between 1125 determined that 73% our IP are regulated RNA. Our data enables us link stage functions, highlighting nearly half participate at least two We show one most highly interconnected ERH, engages multiple processes, including via nuclear speckles export machinery. also demonstrate spliceosomal protein SNRNP200 participates stress granule-associated RNPs occupies different target regions cytoplasm stress. comprehensive RBP-focused network is a resource identifying multi-stage exploring RBP

Language: Английский

Citations

4
nf-core/clipseq - a robust Nextflow pipeline for comprehensive CLIP data analysis DOI Creative Commons
Charlotte West, Charlotte Capitanchik,

Chris Cheshire

et al.

Wellcome Open Research, Journal Year: 2023, Volume and Issue: 8, P. 286 - 286

Published: July 4, 2023

Crosslinking and immunoprecipitation (CLIP) technologies have become a central component of the molecular biologists' toolkit to study protein-RNA interactions thus uncover core principles RNA biology. There has been proliferation CLIP-based experimental protocols, as well computational tools, especially for peak-calling. Consequently, there is an urgent need well-documented bioinformatic pipeline that enshrines robustness, reproducibility, scalability, portability flexibility while embracing diversity CLIP tools. To address this, we present nf-core/clipseq - robust Nextflow quality control analysis sequencing data. It part international nf-core community effort develop curate best-practice, gold-standard set pipelines data analysis. The standards enabled by nf-core, including workflow management, version control, continuous integration containerisation ensure these key needs are met. Furthermore, multiple tools implemented (

Language: Английский

Citations

4