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

Опубликована: Янв. 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.

Язык: 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

и другие.

Nature Biotechnology, Год журнала: 2024, Номер 42(9), С. 1429 - 1441

Опубликована: Янв. 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.

Язык: Английский

Процитировано

20
Integrated multi-omics analysis of zinc-finger proteins uncovers roles in RNA regulation DOI Creative Commons
Maya L. Gosztyla, Lijun Zhan, Sara Olson

и другие.

Molecular Cell, Год журнала: 2024, Номер unknown

Опубликована: Сен. 1, 2024

Язык: Английский

Процитировано

7
Decoding protein–RNA interactions using CLIP-based methodologies DOI
Joy S. Xiang,

Danielle Schafer,

Katherine Rothamel

и другие.

Nature Reviews Genetics, Год журнала: 2024, Номер 25(12), С. 879 - 895

Опубликована: Июль 9, 2024

Язык: Английский

Процитировано

6
From computational models of the splicing code to regulatory mechanisms and therapeutic implications DOI
Charlotte Capitanchik, Oscar G. Wilkins, Nils Wagner

и другие.

Nature Reviews Genetics, Год журнала: 2024, Номер unknown

Опубликована: Окт. 2, 2024

Язык: Английский

Процитировано

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

и другие.

Molecular Cell, Год журнала: 2024, Номер unknown

Опубликована: Сен. 1, 2024

Язык: Английский

Процитировано

5
ePRINT: exonuclease assisted mapping of protein-RNA interactions DOI Creative Commons
Sophie Hawkins, Alexandre Mondaini, Seema C. Namboori

и другие.

Genome biology, Год журнала: 2024, Номер 25(1)

Опубликована: Май 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.

Язык: Английский

Процитировано

3
Phenylpyrazoles as Inhibitors of the m6A RNA-Binding Protein YTHDF2 DOI Creative Commons

Xiaqiu Qiu,

Claus Kemker,

Georg L. Goebel

и другие.

JACS Au, Год журнала: 2025, Номер 5(2), С. 618 - 630

Опубликована: Фев. 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.

Язык: Английский

Процитировано

0
Neuronal aging causes mislocalization of splicing proteins and unchecked cellular stress DOI Creative Commons
Kevin Rhine,

Rachel Li,

Hema Kopalle

и другие.

Nature Neuroscience, Год журнала: 2025, Номер unknown

Опубликована: Июнь 2, 2025

Abstract Aging is one of the most prominent risk factors for neurodegeneration, yet molecular mechanisms underlying deterioration old neurons are mostly unknown. To efficiently study neurodegeneration in context aging, we transdifferentiated primary human fibroblasts from aged healthy donors directly into neurons, which retained their aging hallmarks, and verified key findings mouse brain tissue. Here show that broadly depleted RNA-binding proteins, especially spliceosome components. Intriguingly, splicing proteins—like dementia- ALS-associated protein TDP-43—mislocalize to cytoplasm leads widespread alternative splicing. Cytoplasmic components typically recruited stress granules, but suffer chronic cellular prevents this sequestration. We link malfunctioning ubiquitylation machinery, poor HSP90α chaperone activity failure respond new events. Together, our data demonstrate aging-linked RNA biology a driver resiliency neurons.

Язык: Английский

Процитировано

0
Inhibition of RNA splicing triggers CHMP7 nuclear entry, impacting TDP-43 function and leading to the onset of ALS cellular phenotypes DOI Creative Commons
Norah Al-Azzam,

Jenny H To,

Vaishali Gautam

и другие.

Neuron, Год журнала: 2024, Номер unknown

Опубликована: Окт. 1, 2024

Amyotrophic lateral sclerosis (ALS) is linked to the reduction of certain nucleoporins in neurons. Increased nuclear localization charged multivesicular body protein 7 (CHMP7), a involved pore surveillance, has been identified as key factor damaging pores and disrupting transport. Using CRISPR-based microRaft, followed by gRNA identification (CRaft-ID), we discovered 55 RNA-binding proteins (RBPs) that influence CHMP7 localization, including SmD1, survival motor neuron (SMN) complex component. Immunoprecipitation-mass spectrometry (IP-MS) enhanced crosslinking immunoprecipitation (CLIP) analyses revealed CHMP7's interactions with small RNAs, splicing mRNAs neurons (MNs). ALS induced pluripotent stem cell (iPSC)-MNs show reduced SmD1 expression, inhibiting SmD1/SMN increased localization. Crucially, overexpressing iPSC-MNs restored cytoplasmic corrected STMN2 splicing. Our findings suggest early pathogenesis driven SMN dysregulation.

Язык: Английский

Процитировано

3
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

и другие.

Nucleic Acids Research, Год журнала: 2023, Номер 51(19), С. 10768 - 10781

Опубликована: Сен. 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.

Язык: Английский

Процитировано

7