Biology subjects

Koertel, N.

Publications and source records attributed to Koertel, N..

5 recordsLinked to original sources

bioRxiv2026

Isoform-Specific Localization Diversifies Human MSI2 Function

Musashi-2 (MSI2) is an RNA-binding protein implicated in stem cell regulation and cancer, yet reports of its regulatory activity span translational activation, repression, and effects on mRNA stability, sometimes within the same cellular context. The basis for this diversity has remained unclear. Here, we identify alternative splicing as a determinant of MSI2 regulatory activity, acting through isoform-specific subcellular localization. Using a tethered reporter assay, we show that the canonical MSI2-328 isoform promotes translation without changing mRNA abundance. Truncation and mutation analysis identify a 40-amino-acid region (residues 194-234) as necessary and sufficient for cytoplasmic localization and necessary for translational activation. In contrast, the alternatively spliced MSI2-324 isoform localizes predominantly to the nucleus and fails to promote translation. We map this difference to an 18-amino-acid sequence introduced by an alternative 3 splice acceptor in exon 12; this sequence is sufficient to direct nuclear localization, and its removal restores MSI2-328-like cytoplasmic localization and translational activation. Consistent with these compartmental differences, the two isoforms associate with distinct protein networks, MSI2-328 with translation factors and ribosomal proteins, and MSI2-324 with chromatin and pre-mRNA processing factors. These isoform-specific activities are conserved across cell types, and the relative abundance of MSI2 isoforms shifts toward MSI2-324 in several cancers. Altogether, alternative splicing controls MSI2 subcellular localization, interaction networks, and regulatory output, providing a mechanistic framework for the context-dependent roles of MSI2 in gene regulation.

molecular biology↗

bioRxiv2026

RNA G-quadruplexes mediate cooperativity in HNRNPH binding and splicing regulation

Alternative splicing, regulated by RNA-binding proteins (RBPs), enables the generation of diverse transcript isoforms critical for cellular function. However, how RNA secondary structure impacts RBP binding and function remains poorly understood. Here, we unravel how RNA G-quadruplexes (rG4s) facilitate cooperativity in splicing regulation by the RBP heterogeneous nuclear ribonucleoprotein H (HNRNPH). Through high-throughput in vivo and in vitro studies combined with theoretical modeling, we dissect how rG4s mediate cooperative HNRNPH binding to RNA, ultimately modulating the splicing of hundreds of exons. rG4 unfolding by HNRNPH exposes multiple G-rich binding sites, thereby establishing indirect cooperativity, which is further amplified to achieve switch-like splicing regulation. HNRNPH-mediated regulation is evident in breast cancer patients, with tumors showing rG4-disrupting variants and global HNRNPH alterations, driving distinct splicing patterns that distinguish tumor subtypes. Overall, our findings offer valuable insights into the mechanistic role of RNA secondary structures in cooperative RBP binding and splicing regulation and highlight the clinical relevance of HNRNPH-dependent splicing in cancer. HighlightsO_LIHundreds of cassette exons are cooperatively regulated by HNRNPH. C_LIO_LIUnfolding of RNA G-quadruplexes (rG4s) at HNRNPH binding sites facilitates indirect cooperativity in RNA binding. C_LIO_LIMulti-step splicing amplifies the response into highly switch-like regulation. C_LIO_LIrG4-disrupting variants and changing HNRNPH expression are associated with breast cancer phenotypes. C_LI

molecular biology↗

bioRxiv2024

Microbial iCLIP2: Enhanced mapping of RNA-Protein interaction by promoting protein and RNA stability

The entire RNA lifecycle, spanning from transcription to decay, is intricately regulated by RNA-binding proteins (RBPs). To understand their precise functions, it is crucial to identify direct targets, pinpoint their exact binding sites, and unravel the underlying specificity in vivo. Individual-nucleotide resolution UV crosslinking and immunoprecipitation 2 (iCLIP2) is a state-of-the-art technique that enables the identification of RBP binding sites at single-nucleotide resolution. However, in the field of microbiology, optimized iCLIP protocols compared to mammalian systems are lacking. Here, we present the first microbial iCLIP2 approach using the multi-RRM domain protein Rrm4 from the fungus Ustilago maydis as an example. Key challenges such as inherently high RNase and protease activity in fungi were addressed by improving mechanical cell disruption and lysis buffer composition. Our modifications increased the yield of crosslink events and improved the identification of Rrm4 binding sites. Thus, we were able to pinpoint that Rrm4 binds the stop codons of nuclear-encoded mRNAs of mitochondrial respiratory complex I, III and V - revealing an intimate link between endosomal mRNA transport and mitochondrial physiology. Thus, our study serves as a paradigm for optimizing iCLIP2 procedures in challenging organisms or tissues under high RNase/ protease conditions.

molecular biology↗

bioRxiv2024

Comparative analysis of 43 distinct RNA modifications by nanopore tRNA sequencing

Transfer RNAs are the fundamental adapter molecules of protein synthesis and the most abundant and heterogeneous class of noncoding RNA molecules in cells. The study of tRNA repertoires remains challenging, complicated by the presence of dozens of post transcriptional modifications. Nanopore sequencing is an emerging technology with promise for both tRNA sequencing and the detection of RNA modifications; however, such studies have been limited by the throughput and accuracy of direct RNA sequencing methods. Moreover, detection of the complete set of tRNA modifications by nanopore sequencing remains challenging. Here we show that recent updates to nanopore direct RNA sequencing chemistry (RNA004) combined with our own optimizations to tRNA sequencing protocols and analysis workflows enable high throughput coverage of tRNA molecules and characterization of nanopore signals produced by 43 distinct RNA modifications. We share best practices and protocols for nanopore sequencing of tRNA and further report successful detection of low abundance mitochondrial and viral tRNAs, providing proof of concept for use of nanopore sequencing to study tRNA populations in the context of infection and organelle biology. This work provides a roadmap to guide future efforts towards de novo detection of RNA modifications across multiple organisms using nanopore sequencing.

molecular biology↗

bioRxiv2020

Deep and accurate detection of m6A RNA modifications using miCLIP2 and m6Aboost machine learning

N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic mRNAs and influences many aspects of RNA processing. miCLIP (m6A individual-nucleotide resolution UV crosslinking and immunoprecipitation) is an antibody-based approach to map m6A sites with single-nucleotide resolution. However, due to broad antibody reactivity, reliable identification of m6A sites from miCLIP data remains challenging. Here, we present miCLIP2 in combination with machine learning to significantly improve m6A detection. The optimised miCLIP2 results in high-complexity libraries from less input material. Importantly, we established a robust computational pipeline to tackle the inherent issue of false positives in antibody-based m6A detection. The analyses are calibrated with Mettl3 knockout cells to learn the characteristics of m6A deposition, including m6A sites outside of DRACH motifs. To make our results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m6A sites in miCLIP2 data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m6A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m6A identification. HighlightsO_LImiCLIP2 produces complex libraries to map m6A RNA modifications C_LIO_LIMettl3 KO miCLIP2 allows to identify Mettl3-dependent RNA modification sites C_LIO_LIMachine learning predicts genuine m6A sites from human and mouse miCLIP2 data without Mettl3 KO C_LIO_LIm6A modifications occur outside of DRACH motifs and associate with alternative splicing C_LI

genomics↗