Biology subjects

Donovan, B. T.

Publications and source records attributed to Donovan, B. T..

5 recordsLinked to original sources

bioRxiv2024

U2AF regulates the translation and localization of nuclear-encoded mitochondrial mRNAs

The mechanisms underlying molecular targeting to mitochondria remain enigmatic, yet this process is crucial for normal cellular function. The RNA binding proteins U2AF1/2 form a heterodimer (U2AF) that shuttles between the nucleus and cytoplasm, regulating splicing in the nucleus and translation in the cytoplasm. Our study identifies an unexpected role for U2AF in mitochondrial function. We demonstrate that U2AF interacts with nuclear-transcribed mitochondrial mRNAs and proteins, inhibits translation, localizes to the outer mitochondrial membrane, and regulates mRNA localization to mitochondria. Moreover, an oncogenic point-mutation in U2AF1(S34F) disrupts this regulation, leading to altered mitochondrial structure, increased translation, and OXPHOS-dependent metabolic rewiring, recapitulating changes observed in bone marrow progenitors from patients with myelodysplastic syndromes. These findings reveal a non-canonical role for U2AF, where it modulates multiple aspects of mitochondrial function by regulating the translation and mitochondrial targeting of nuclear-encoded mRNAs.

molecular biology↗

bioRxiv2024

REAL-TIME VISUALIZATION OF SPLICEOSOME ASSEMBLY REVEALS BASIC PRINCIPLES OF SPLICE SITE SELECTION

The spliceosome is a megadalton protein-RNA complex which removes introns from pre-mRNA, yet the dynamic early assembly steps have not been structurally resolved. Specifically, how the spliceosome selects the correct 3 splice site (3SS) amongst highly similar non-functional sites is not known. Here, we develop a kinetic model of splice site selection based on single-molecule U2AF heterodimer imaging in vitro and in vivo. The model successfully predicts alternative splicing patterns and indicates that 3SS selection occurs while U2AF is in complex with the spliceosome, not during initial binding. This finding indicates the spliceosome operates in a partial kinetic proofreading regime, catalyzed in part by the helicase DDX42, which increases selectivity to the underlying U2AF binding site while still allowing for efficient forward progression. ONE-SENTENCE SUMMARYWe apply a kinetic proofreading model to elucidate how transient U2AF binding leads to high fidelity splice site selection.

biophysics↗

bioRxiv2022

Basic helix-loop-helix pioneer factors interact with the histone octamer to invade nucleosomes and generate nucleosome depleted regions

Nucleosomes drastically limit transcription factor (TF) occupancy, while pioneer transcription factors (PFs) somehow circumvent this nucleosome barrier. In this study, we compare nucleosome binding of two conserved S. cerevisiae basic helix-loop-helix (bHLH) TFs, Cbf1 and Pho4. A Cryo-EM structure of Cbf1 in complex with the nucleosome reveals that the Cbf1 HLH region can electrostatically interact with exposed histone residues within a partially unwrapped nucleosome. Single molecule fluorescence studies show that the Cbf1 but not the Pho4 HLH region facilitates efficient nucleosome invasion by slowing its dissociation rate relative to DNA through interactions with histones. In vivo studies show that this enhanced binding provided by the Cbf1 HLH region enables nucleosome invasion and ensuing repositioning. These structural, single molecule, and in vivo studies reveal the mechanistic basis of dissociation rate compensation by pioneer factors and how this translates to facilitating chromatin opening inside cells.

biophysics↗

bioRxiv2018

Dissociation rate compensation mechanism for budding yeast pioneer transcription factors

Nucleosomes restrict the occupancy of most transcription factors (TF) by reducing binding and accelerating dissociation, while a small group of TFs have high affinities to nucleosome-embedded sites and facilitate nucleosome displacement. To mechanistically understand this process, we investigated two S. cerevisiae TFs, Reb1 and Cbf1. We show these factors bind their sites within nucleosomes with similar affinities to naked DNA, trapping a partially unwrapped nucleosome without histone eviction. Both the binding and dissociation rates of Reb1 and Cbf1 are significantly slower at the nucleosomal sites relative to DNA, demonstrating that the high affinities are achieved by increasing the dwell time on nucleosomes to compensate for reduced binding. Reb1 also shows slow migration rate in the yeast nuclei. These properties are similar to human pioneer factors (PFs), suggesting the mechanism of nucleosome targeting is conserved from yeast to human.

molecular biology↗

bioRxiv2018

Single-molecule imaging reveals the interplay between transcription factors, nucleosomes, and transcriptional bursting

Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single-molecule imaging approaches, we directly correlated binding of the transcription factor Gal4 with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell times sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model where multiple polymerases initiate during a burst as long as the transcription factor is bound to DNA, and a burst terminates upon transcription factor dissociation.

molecular biology↗