Biology subjects

Machyna, M.

Publications and source records attributed to Machyna, M..

4 recordsLinked to original sources

bioRxiv2025

Uncovering the isoform-resolution kinetic landscape of nonsense-mediated mRNA decay with EZbakR

Cellular RNA levels are a product of synthesis and degradation kinetics, which can differ among transcripts of the same gene. An important cause of isoform-specific decay is the nonsense-mediated mRNA decay (NMD) pathway, which degrades transcripts with premature termination codons (PTCs) and other features. Understanding NMD functions requires strategies to quantify isoform kinetics; however, current approaches remain limited. Methods like nucleotide-recoding RNA-seq (NR-seq) enable insights into RNA kinetics, but existing bioinformatic tools do not provide robust, isoform-specific degradation rate constant estimates. We extend the EZbakR-suite by implementing a strategy to infer isoform-level kinetics from short-read NR-seq data. This approach uncovers unexpected variability in NMD efficiency among transcripts with conserved PTC-containing exons and rapid decay of a subset of mRNAs lacking PTCs. Our findings highlight the effects of competition between NMD and other decay pathways, provide mechanistic insights into established NMD efficiency correlates, and identify transcript features promoting efficient decay.

molecular biology↗

bioRxiv2024

Expanding and improving analyses of nucleotide recoding RNA-seq experiments with the EZbakR suite

Nucleotide recoding RNA sequencing methods (NR-seq; TimeLapse-seq, SLAM-seq, TUC-seq, etc.) are powerful approaches for assaying transcript population dynamics. In addition, these methods have been extended to probe a host of regulated steps in the RNA life cycle. Current bioinformatic tools significantly constrain analyses of NR-seq data. To address this limitation, we developed EZbakR, an R package to facilitate a more comprehensive set of NR-seq analyses, and fastq2EZbakR, a Snakemake pipeline for flexible preprocessing of NR-seq datasets, collectively referred to as the EZbakR suite. Together, these tools generalize many aspects of the NR-seq analysis workflow. The fastq2EZbakR pipeline can assign reads to a diverse set of genomic features (e.g., genes, exons, splice junctions, etc.), and EZbakR can perform analyses on any combination of these features. EZbakR extends standard NR-seq mutational modeling to support multi-label analyses (e.g., s4U and s6G dual labeling), and implements an improved hierarchical model to better account for transcript-to-transcript variance in metabolic label incorporation. EZbakR also generalizes dynamical systems modeling of NR-seq data to support analyses of premature mRNA processing and flow between subcellular compartments. Finally, EZbakR implements flexible and well-powered comparative analyses of all estimated parameters via design matrix-specified generalized linear modeling. The EZbakR suite will thus allow researchers to make full, effective use of NR-seq data.

bioinformatics↗

bioRxiv2021

Coilin oligomerization and remodeling by Nopp140 reveals a hybrid assembly mechanism for Cajal bodies

Cajal bodies (CBs) are ubiquitous nuclear membraneless organelles (MLOs) that promote efficient biogenesis of RNA-protein complexes. Depletion of the CB scaffolding protein coilin is lethal for vertebrate embryogenesis, making CBs a strong model for understanding the structure and function of MLOs. Although it is assumed that CBs form through biomolecular condensation, the biochemical and biophysical principles that govern CB dynamics have eluded study. Here, we identify features of the coilin protein that drive CB assembly and shape. Focusing on coilins N-terminal domain (NTD), we discovered its unexpected capacity for oligomerization in vivo. Single amino acid mutational analysis of coilin revealed distinct molecular interactions required for oligomerization and binding to the Nopp140 ligand, which facilitates CB assembly. We demonstrate that the intrinsically disordered regions of Nopp140 have substantial condensation properties and suggest that Nopp140 binding thereby remodels stable coilin oligomers to form a particle that recruits other functional components.

cell biology↗

bioRxiv2021

Multi-omics profiling of U2AF1 mutants dissects pathogenic mechanisms affecting RNA granules in myeloid malignancies

Somatic mutations in splicing factors are of significant interest in myeloid malignancies and other cancers. U2AF1, together with U2AF2, is essential for 3 splice site recognition. U2AF1 mutations result in aberrant splicing, but the molecular mechanism and the full spectrum of consequences on RNA biology have not been fully elucidated to date. We performed multi-omics profiling of in vivo RNA binding, splicing and turnover for U2AF1 S34F and Q157R mutants. We dissected specific binding signals of U2AF1 and U2AF2 and showed that U2AF1 mutations individually alter U2AF1-RNA binding, resulting in defective U2AF2 recruitment. We demonstrated a complex relationship between differential binding and splicing, expanding upon the currently accepted loss-of-binding model. Finally, we observed that U2AF1 mutations increase the formation of stress granules in both cell lines and primary acute myeloid leukemia samples. Our results uncover U2AF1 mutation-dependent pathogenic RNA mechanisms and provide the basis for developing targeted therapeutic strategies.

cancer biology↗