Biology subjects

Mueller-McNicoll, M.

Publications and source records attributed to Mueller-McNicoll, M..

9 recordsLinked to original sources

bioRxiv2026

Prediction and evaluation of Split-ORFs using Ribo-seq data

Split Open Reading frames (Split-ORFs) occur in transcripts containing at least two open reading frames, each encoding a part of the same full-length protein. These multiple open reading frames arise from alternatively spliced transcript isoforms. Understanding which genes make Split-ORFs, and in which cell types and under which conditions, would generate new insights into gene regulation. We previously published the Split-ORF pipeline, a computational tool that predicts candidate Split-ORFs from transcript sequences. Here, we present a new and improved version of the Split-ORF pipeline adding modules to analyze Ribo-seq data, calculate regions unique to the Split-ORF candidates, quantitatively assess Ribo-seq coverage in these regions, and perform candidate prioritization. Using this pipeline, we predicted more than 14,000 candidate Split-ORF transcripts from alternatively spliced human transcripts containing premature termination codons or retained introns. Hundreds of candidate Split-ORFs show significant Ribo-seq coverage across diverse cell types and diseases in at least one of the Split-ORFs, and 120 transcripts in both Split-ORFs. The candidate Split-ORF genes with significant Ribo-seq coverage are enriched for RNA-binding and RNA-processing functions and the majority of them encode RNA-binding proteins.

bioinformatics↗

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↗

bioRxiv2025

Regulation of early-stage tomato fruit growth by the splicing factor RS2Z36

Fruit growth is mediated by cell division and expansion. In tomato, the model for fleshy fruit development, both processes are tightly linked to changes in gene expression, including transcriptional regulation and RNA processing. While several transcription factors are implicated in fruit developmental programs, the role of splicing regulators remains largely unexplored. Expression profiling of splicing-related genes revealed expression patterns. The serine/arginine-rich splicing factor RS2Z36 is expressed in ovaries and during early fruit development. Loss-of-function mutations in RS2Z36 result in ovaries with altered patterning and in smaller, ellipsoid fruits. rs2z36 mutants display elongated pericarp cells along the longitudinal axis of pre-anthesis ovaries, indicating that RS2Z36-dependent expansion patterns are established before anthesis. RNA-seq uncovered widespread alternative splicing of genes across diverse biological processes, while proteome analysis revealed altered protein abundance and peptides derived from novel splice variants. In addition, rs2z36-1 pericarps show increased deposition of LM6-recognized arabinan and AGP epitopes. Together, these findings identify RS2Z36 as a regulator of ovary and fruit development and highlight a previously underappreciated role for splicing control in shaping early fruit morphology.

developmental biology↗

bioRxiv2024

Smart 3D super-resolution microscopy reveals the architecture of the RNA scaffold in a nuclear body

Small subcellular organelles orchestrate key cellular functions. How biomolecules are spatially organized within these assemblies is poorly understood. Here, we report an automated super- resolution imaging and analysis workflow that integrates confocal microscopy, morphological object screening, targeted 3D super-resolution STED microscopy and quantitative image analysis. Using this smart microscopy workflow, we targeted the 3D organization of an architectural RNA that constitutes the structural backbone of paraspeckles, a membraneless nuclear organelle. Using site-specific labeling, morphological sorting and particle averaging, we reconstructed the morphological space of paraspeckles along their development cycle from over 10,000 individual particles. Applying spherical harmonics analysis, we report so-far unknown heterotypes of RNA organization. By integrating multi- positional labeling, we determined the coarse conformation of the RNA within the organelle and found the 3 end forming a loop-like structure at the surface of the paraspeckle. Our study reveals key structural features of nuclear paraspeckle structure and growth, as well as on the molecular organization of the scaffold RNA.

biophysics↗

bioRxiv2024

ERH regulates type II interferon immune signaling through post-transcriptional regulation of JAK2 mRNA

Type II interferon (IFN{gamma}) signaling is essential for innate immunity and critical for effective immunological checkpoint blockade in cancer immunotherapy. Genetic screen identification of post-transcriptional regulators of this pathway has been challenging since such factors are often essential for cell viability. Here, we utilize our inducible CRISPR/Cas9 approach to screen for key post-transcriptional regulators of IFN{gamma} signaling, and in this way identify ERH and the ERH-associated splicing and RNA export factors MAGOH, SRSF1, and ALYREF. Loss of these factors impairs post-transcriptional mRNA maturation of JAK2, a crucial kinase for IFN{gamma} signaling, resulting in abrogated JAK2 protein levels and diminished IFN{gamma} signaling. Further analysis highlights a critical role for ERH in preventing intron retention in AU-rich regions in specific transcripts, such as JAK2. This regulation is markedly different from previously described retention of GC-rich introns. Overall, these findings reveal that post-transcriptional JAK2 processing is a critical rate-limiting step for the IFN{gamma}-driven innate immune response.

cell biology↗

bioRxiv2024

Rapid depletion and super-resolution microscopy reveal an unexpected role of the nuclear-speckle protein SRSF5 in paraspeckle assembly and dynamics during cellular stress

Nuclear speckles (NS) and paraspeckles (PS) are adjacent condensates with distinct protein composition, with serine-arginine-rich splicing factors (SRSFs) concentrated in NS. Surprisingly, we find that SRSF5 is present in both. Combining super-resolution imaging, proximity proteomics and iCLIP, we show that SRSF5 binds with PS core proteins to the PS-scaffold RNA NEAT1 and locates between PS spheres. Acute SRSF5 depletion results in reduced PS with differently packaged NEAT1. Under stress, SRSF5s association with PS increases, and without SRSF5, PS cluster assembly is impaired. Interfering with binding to purine-rich RNAs even causes PS-NS fusion. In an intriguing over-compensation, longer SRSF5 depletion reduces TDP-43 levels via premature polyadenylation, leading to NEAT1 isoform switching and more PS. We propose that SRSF5 forms a stress-specific PS shell and acts as a glue for PS clusters. Additionally, we uncover SRSF5 as a novel regulator of TDP-43 and demonstrate how acute depletion distinguishes direct from compensatory effects. HighlightsO_LINS protein SRSF5 associates with PS shells and enriches between PS spheres C_LIO_LISRSF5 binds the PS-scaffold RNA NEAT1 and ensures proper NEAT1 packaging C_LIO_LISRSF5 association with PS increases under stress and promotes cluster formation C_LIO_LIInterfering with binding to purine-rich RNAs causes the fusion of PS and NS C_LIO_LIAcute SRSF5 depletion reveals compensatory effects on PS assembly and dynamics C_LIO_LISRSF5 regulates TDP-43 levels via premature polyadenylation C_LI

cell biology↗

bioRxiv2024

A plant-specific clade of serine/arginine-rich proteins regulates RNA splicing homeostasis and thermotolerance in tomato

High temperatures cause heat stress (HS), which has negative effects on plant growth and development and affects many cellular processes including pre-mRNA splicing. In tomato plants the splicing profile of many of genes is altered under HS, including that of HSFA2, a central transcriptional regulator of thermotolerance. To identify the core splicing regulators of HS-sensitive alternative splicing, we used HSFA2 as bait and identified two plant-specific members of the serine/arginine-rich family of splicing factors, namely RS2Z35 and RS2Z36, that inhibit HSFA2 intron splicing. Single and double CRISPR mutants of these proteins show dysregulated splicing of many genes and exhibit lower basal and acquired thermotolerance. Individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) of tomato leaves revealed that the majority of HS-sensitive alternatively spliced RNAs are bound by RS2Z35 and RS2Z36 and this interaction occurs at purine-rich RNA motifs. Phenotypic and transcriptome analyses revealed that RS2Z35 and RS2Z36 are important players in the stress response and thermotolerance in plants that mitigate the negative effects of HS on RNA splicing homeostasis.

plant biology↗

bioRxiv2024

Arid5a uses disordered extensions of its core ARID domain for distinct DNA- and RNA-recognition and gene regulation

AT-rich interacting domain (ARID)-containing proteins, Arids, are a heterogeneous DNA-binding protein family involved in transcription regulation and chromatin processing. For the member Arid5a, no exact DNA-binding preference has been experimentally defined so far. Additionally, the protein binds to mRNA motifs for transcript stabilization, supposedly through the DNA-binding ARID domain. To date, however, no unbiased RNA motif definition and clear dissection of nucleic acid-binding through the ARID domain have been undertaken. Using NMR-centered biochemistry, we here define the Arid5a DNA preference. Further, high-throughput in vitro binding (RBNS) reveals a consensus RNA-binding motif engaged by the core ARID domain. Finally, transcriptome-wide binding (iCLIP2) reveals that Arid5a has a weak preference for (A)U-rich regions in pre-mRNA transcripts of factors related to RNA processing. We find that the intrinsically disordered regions (IDR) flanking the ARID domain modulate the specificity and affinity of DNA-binding, while they appear crucial for RNA interactions. Ultimately, our data suggest that Arid5a uses its extended ARID domain for bi-functional gene regulation and that the involvement of IDR extensions is a more general feature of Arids in interacting with different nucleic acids at the chromatin-mRNA interface.

biochemistry↗

bioRxiv2020

A paralog-specific role of the COPI pathway in the neuronal differentiation of murine pluripotent cells

Coat protein complex I (COPI)-coated vesicles mediate membrane trafficking between Golgi cisternae as well as retrieval of proteins from the Golgi to the endoplasmic reticulum. There are several flavors of the COPI coat defined by paralogous subunits of the protein complex coatomer. However, whether paralogous COPI proteins have specific functions is currently unknown. Here we show that the paralogous coatomer subunits {gamma}1-COP and {gamma}2-COP are differentially expressed during the neuronal differentiation of mouse pluripotent cells. Moreover, through a combination of genome editing experiments, we demonstrate that whereas {gamma}-COP paralogs are largely functionally redundant, {gamma}1-COP specifically promotes neurite outgrowth. Our work stresses a role of the COPI pathway in neuronal polarization and provides evidence for distinct functions for coatomer paralogous subunits in this process.

cell biology↗