Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2024.07.08.601072

Tom1p ubiquitin ligase structure, interaction with Spt6p, and function in maintaining normal transcript levels and the stability of chromatin in promoters

Madrigal, J. A.·Schubert, H. L.·Sdano, M. A.·McCullough, L.·Connell, Z.·Formosa, T.·Hill, C. P.
View original source ↗Export metadata ↓

Abstract

Phosphorylation-dependent binding of the S. cerevisiae Spt6p tSH2 domain (Spt6ptSH2) to the Rbp1p subunit of RNA polymerase II supports efficient transcription. Here, we report that Spt6ptSH2 also binds the HECT-family E3 ubiquitin ligase Tom1p, a homolog of human HUWE1. Tom1p/HUWE1 have been implicated in targeting many small basic proteins for degradation, including excess ribosomal subunits and histones, although the mechanism of substrate recognition is not known. Our cryo-EM data revealed that Tom1p can adopt a compact -solenoidal "basket" similar to the previously described structure of HUWE1, with the central cavity partially occupied by a disordered acidic domain. Sub-regions of this acidic domain supported binding to Spt6p or histones/nucleosomes in vitro, and the histone-binding region was important for Tom1p function in vivo. We also visualized Tom1p in more extended forms, and speculate that transitions among these forms could be important for substrate selection and ubiquitylation. Genomic analyses provided additional support for the previously observed role for Tom1p in maintaining ribosomal protein pools, and also demonstrated a role in maintaining chromatin structure near genes. This suggests that the interaction with Spt6ptSH2 affects substrate specificity by anchoring Tom1p to localized environments where histone ubiquitylation alters chromatin architecture.

Explore related subjects

molecular biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Madrigal, J. A., Schubert, H. L., Sdano, M. A., McCullough, L., Connell, Z., Formosa, T., Hill, C. P.. 2024-07-08. Tom1p ubiquitin ligase structure, interaction with Spt6p, and function in maintaining normal transcript levels and the stability of chromatin in promoters. https://doi.org/10.1101/2024.07.08.601072

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Genomic context tunes sequence-encoded RNA processing

Sequence and genomic position jointly determine RNA processing, but at native loci the two are difficult to separate. Here, we uncoupled them by integrating RNA-processing elements into endogenous transcription units at tens of thousands of mapped positions and different elements at shared positions. Genomic position dominated RNA production levels but differentially influenced RNA processing events. A cryptic polyadenylation site, used natively only upon U1 inhibition, was rarely used ectopically. In contrast, introns partially retained at their native loci remained so across thousands of ectopic positions, with a substantial fraction of transcripts reaching the cytoplasm unspliced. SON depletion reduced splicing of two such introns, preferentially near speckles. Chromatin state, GC content, and speckle proximity tuned this behavior without overriding it, and in opposite directions: speckle proximity improved the splicing of one intron and reduced that of another. Specialized introns are therefore portable modules carrying their own processing information.

molecular biology↗
bioRxiv2026

Single-probe, amplification-free multiplex mRNA PNA-FISH maps bacterial gene expression in intact three-dimensional biofilms

Biofilms are spatially structured communities in which local microenvironments drive physiological differentiation, yet mapping gene expression inside intact three-dimensional biofilms remains challenging. Bacterial spatial transcriptomics methods typically rely on tiled probe sets, sequential hybridization rounds, or signal amplification. Here, we show that a single short, directly labelled peptide nucleic acid (PNA) probe per target is sufficient to quantify bacterial mRNA in situ without amplification. With all probes designed for a common hybridization condition, a transcript panel is resolved in one hybridization on a standard confocal microscope, in unmodified cells while preserving biofilm architecture. Benchmarked against the biphasic cycle of Legionella pneumophila, multiplex mRNA PNA-FISH recovers the expected transcriptional switch and agrees quantitatively with RT-qPCR in planktonic and biofilm populations. In flow-cell biofilms, it shows that sessile populations do not recapitulate the planktonic program and that transcriptional differences follow cellular aggregation state rather than depth.

molecular biology↗
bioRxiv2026

Autophagy activator AA-20 engages CDK5 to promote lipophagy and improve neurodegeneration-associated phenotypes

Lipophagy, the selective degradation of lipid droplets (LDs) by autophagy, has the potential to ameliorate neurodegenerative diseases such as Parkinson's (PD) and Alzheimer's (AD), wherein LD accumulation has been linked to the exacerbation of the proteinopathy component of neurodegeneration. Pharmacological lipophagy inducers remain scarce. We previously identified AA-20 as a novel autophagy activator that acts independently of mTORC1 inhibition, thereby providing a means of stimulating autophagy while avoiding the immunosuppressive side effects associated with direct mTORC1 inhibition. Prior work also showed that AA-20 treatment reduces PolyQ aggregate levels in several C. elegans tissues. Here, we demonstrate that AA-20 enhances LD recruitment to autophagosomes and promotes LD degradation via lysosomal acid lipase activation in murine hippocampal neuronal cells, consistent with lipophagy activation. In human Swedish Amyloid Precursor Protein mutated cerebral organoids, two weeks of AA-20 treatment normalized aberrant lipidomic profiles by selectively reducing various disease-associated lipids, while also correcting the AD-associated electrical hyperexcitability phenotype. Moreover, we identified cyclin-dependent kinase 5 (CDK5), a neuronal kinase linked to AD hyperexcitability and PD pathology, as a target of AA-20. AA-20-induced autophagy and lipid clearance in HT22 cells require CDK5. In a neuronal seeding model of -synuclein pathology, AA-20 reduced pS129--synuclein levels. Collectively, these findings indicate AA-20 can promote lipophagic clearance of LDs and mitigate multiple cellular phenotypes associated with lipidopathy and proteinopathy in neurodegenerative disease models.

molecular biology↗