Biology subjects

Weidenhausen, J.

Publications and source records attributed to Weidenhausen, J..

3 recordsLinked to original sources

bioRxiv2025

High-resolution mapping of human RNA polymerase III reveals transcription termination as a rate-limiting step

Transfer RNA (tRNA) molecules play a central role in the flow of genetic information, translating nucleic acid sequences into the functional protein portfolio of an organism. Despite the high abundance of tRNAs in cells, studying their biology remains challenging due to the repetitive nature of genomic sequences and the numerous modifications of mature molecules. tRNA genes (tDNAs) are transcribed exclusively by RNA polymerase III (RNAPIII), which has been recently linked to rare genetic disorders but also longevity. Here, we present the first mapping of actively transcribing RNAPIII in the human, using K562 cell line and UV-crosslinking followed by stringent purification. Our data reveals high variability in expression across tDNAs, common occurrences of transcriptional read-through, and unique transcription dynamics across transcription units, with the lowest kinetics associated with transcription termination. Further analysis revealed that release of the nascent transcript is a critical step in tRNA transcription. Unexpectedly, shorter terminators promote more efficient termination, which becomes the rate-limiting step for human RNAPIII transcription of highly expressed tDNAs. Our dataset provides insights into actively transcribing RNAPIII dynamics in human cells, allowing for a comprehensive analysis of genomic loci transcribed by RNAPIII. Our high resolution data and resulting kinetic information reveal that human RNAPIII contradicts the paradigm that initial steps of transcription are the main determinants of its output. This is significant as RNAPIII is commonly used to transcribe synthetic RNA constructs, such as short hairpin RNAs or guide RNAs used in gene editing technologies. With our study we offer a functional framework for the analysis of RNAPIII activity, addressing the need for comprehensive understanding of RNAPIII transcription fueled by a growing number of described genetic disorders caused by mutations in this enzyme.

molecular biology↗

bioRxiv2024

A photoswitchable HaloTag for spatiotemporal control of fluorescence in living cells

Photosensitive fluorophores, which emission can be controlled using light, are essential for advanced biological imaging, enabling precise spatiotemporal tracking of molecular features, and facilitating super-resolution microscopy techniques. While irreversibly photoactivatable fluorophores are well established, reversible reporters which can be re-activated multiple times remain scarce, and only few have been applied in living cells using generalizable protein labelling methods. To address these limitations, we introduce chemigenetic photoswitchable fluorophores, leveraging the self-labelling HaloTag protein with fluorogenic rhodamine dye ligands. By incorporating a light-responsive protein domain into HaloTag, we engineer a tunable, photoswitchable HaloTag (psHaloTag), which can reversibly modulate the fluorescence of a bound dye-ligand via a light-induced conformational change. Our best performing psHaloTag variants show high performance in vitro and in living cells, with large, reversible, far-red fluorescence turn-on upon 450 nm illumination across various biomolecular targets. Together, this work establishes the chemigenetic approach as a versatile platform for the design of photoswitchable reporters, tunable through both genetic and synthetic modifications, with promising applications for dynamic imaging.

biochemistry↗

bioRxiv2022

Trim-Away degrades lysine-less substrates without requiring ligase autoubiquitination

TRIM proteins are the largest family of E3 ligases in mammals. They include the intracellular antibody receptor TRIM21, which is responsible for mediating targeted protein degradation during Trim-Away. Despite their importance, the ubiquitination mechanism of TRIM ligases has remained elusive. Here we show that while TRIM21 activation results in ubiquitination of both ligase and substrate, autoubiquitination is regulatory and not required for substrate degradation. Substrate binding stimulates N-terminal RING autoubiquitination by the E2 Ube2W, but when inhibited by N-terminal acetylation this prevents neither substrate ubiquitination nor degradation and has no impact on TRIM21 antiviral activity. Instead, uncoupling ligase and substrate degradation prevents ligase recycling and extends functional persistence in cells. Substrate ubiquitination mediates degradation but Trim-Away efficiently degrades lysine-less substrates, suggesting a non-canonical ubiquitination mechanism explains its broad substrate specificity.

molecular biology↗