Biology subjects

Hogg, J. R.

Publications and source records attributed to Hogg, J. R..

8 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↗

bioRxiv2023

UPF1 ATPase autoinhibition and activation modulate RNA binding kinetics and NMD efficiency

The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited "closed" state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active "open" state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower nucleic acid binding kinetics and enhanced ATP-stimulated nucleic acid dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2, and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.

molecular biology↗

bioRxiv2022

UPF1 mutants with intact ATPase but deficient helicase activities promote efficient nonsense-mediated mRNA decay

The conserved RNA helicase UPF1 coordinates nonsense-mediated mRNA decay (NMD) by engaging with mRNAs, RNA decay machinery, and the terminating ribosome. UPF1 ATPase activity is necessary for mRNA target discrimination and completion of decay, but the mechanisms through which UPF1 enzymatic activities such as helicase, translocase, RNP remodeling, and ATPase-stimulated dissociation influence NMD remain poorly defined. Using high-throughput biochemical assays to quantify UPF1 enzymatic activities, we show that UPF1 is only moderately processive (< 200 nt) in physiological contexts and undergoes ATPase-stimulated dissociation from RNA. We combine an in silico screen with these assays to identify and characterize known and novel UPF1 mutants with altered helicase, ATPase, and RNA binding properties. We find that UPF1 mutants with substantially impaired processivity, slower unwinding rate, and reduced mechanochemical coupling (i.e. the ability to harness ATP hydrolysis for work) still support efficient NMD in human cells. These data are consistent with a central role for UPF1 ATPase activity in driving cycles of RNA binding and dissociation to ensure accurate NMD target selection.

molecular biology↗

bioRxiv2022

A network of DZF proteins controls alternative splicing regulation and fidelity

Proteins containing DZF (domain associated with zinc fingers) modules play important roles throughout gene expression, from transcription to translation. Derived from nucleotidyltransferases but lacking catalytic residues, DZF domains serve as heterodimerization surfaces between DZF protein pairs. Three DZF proteins are widely expressed in mammalian tissues, ILF2, ILF3, and ZFR, which form mutually exclusive ILF2-ILF3 and ILF2-ZFR heterodimers. Using eCLIP-Seq, we find that ZFR binds across broad intronic regions to regulate the alternative splicing of cassette and mutually exclusive exons. ZFR preferentially binds dsRNA in vitro and is enriched on introns containing conserved dsRNA elements in cells. Many splicing events are similarly altered upon depletion of any of the three DZF proteins; however, we also identify independent and opposing roles for ZFR and ILF3 in alternative splicing regulation. Along with widespread involvement in cassette exon splicing, the DZF proteins control the fidelity and regulation of over a dozen highly validated mutually exclusive splicing events. Our findings indicate that the DZF proteins form a complex regulatory network that leverages dsRNA binding by ILF3 and ZFR to modulate splicing regulation and fidelity.

molecular biology↗

bioRxiv2021

A UPF1 variant drives conditional remodeling of nonsense-mediated mRNA decay

The nonsense-mediated mRNA decay (NMD) pathway monitors translation termination to degrade transcripts with premature stop codons and regulate thousands of human genes. Here we show that an alternative mammalian-specific isoform of the core NMD factor UPF1, termed UPF1LL, enables condition-dependent remodeling of NMD specificity. Previous studies indicate that the extension of a conserved regulatory loop in the UPF1LL helicase core confers a decreased propensity to dissociate from RNA upon ATP hydrolysis relative to the major UPF1 isoform, designated UPF1 SL. Using biochemical and transcriptome-wide approaches, we find that UPF1LL overcomes the protective RNA binding proteins PTBP1 and hnRNP L to preferentially bind and down-regulate long 3UTRs normally shielded from NMD. Unexpectedly, UPF1LL supports induction of NMD on new populations of substrate mRNAs in response to activation of the integrated stress response and impaired translation efficiency. Thus, while canonical NMD is abolished by moderate translational repression, UPF1LL activity is enhanced, providing a mechanism to rapidly rewire NMD specificity in response to cellular stress.

molecular biology↗

bioRxiv2019

Activation and inhibition of nonsense-mediated mRNA decay controls the abundance of alternative polyadenylation products

Alternative polyadenylation (APA) produces transcript 3 untranslated regions (3UTRs) with distinct sequences, lengths, stability, and functions. We show here that APA products include a class of cryptic nonsense-mediated mRNA decay (NMD) substrates with extended 3UTRs that gene- or transcript-level analyses of NMD often fail to detect. Transcriptome-wide, the core NMD factor UPF1 preferentially recognizes long 3UTR products of APA, leading to their systematic downregulation. Further, we find that many APA events consistently observed in multiple tumor types are controlled by NMD. Additionally, PTBP1, previously implicated in direct modulation of co-transcriptional polyA site choice, regulates the balance of short and long 3UTR isoforms by inhibiting NMD. Our data suggest that PTBP1 binding near polyA sites can drive production of long 3UTR APA products in the nucleus and/or protect them from decay in the cytoplasm. Together, our findings reveal a widespread role for NMD in shaping the outcomes of APA.

molecular biology↗

bioRxiv2019

Targeting destabilized DNA G-quadruplexes and aberrant splicing in drug-resistant glioblastoma

Temozolomide (TMZ) is a chemotherapeutic agent that has been the first-line standard of care for the aggressive brain cancer glioblastoma (GBM) since 2005. Though initially beneficial, TMZ- resistance is universal and second-line interventions are an unmet clinical need. Here we took advantage the mechanism of action of TMZ to target guanines (G) and investigated G-rich g- quadruplex (G4) and splice site changes that occur upon TMZ-resistance. We report TMZ-resistant GBM has guanine mutations that disrupt the G-rich DNA G4s and splice sites that lead to deregulated alternative splicing. These alterations create vulnerabilities, which are selectively targeted by either the G4 stabilizing drug TMPyP4 or a novel splicing kinase inhibitor of cdc2- like kinase. Finally, we show that the G4 and RNA-binding protein EWSR1 aggregates in the cytoplasm in TMZ-resistant GBM cells and patient samples. Together, our findings provide insight into targetable vulnerabilities of TMZ-resistant GBM and present cytoplasmic EWSR1 as a putative biomarker. TeaserTargeting temozolomide mutations in drug resistant glioma via g-quadruplex and splicing modulators with a putative biomarker.

cancer biology↗