Biology subjects

Bothe, A.

Publications and source records attributed to Bothe, A..

3 recordsLinked to original sources

bioRxiv2024

An in vitro assay of MCTS1-DENR-dependent re-initiation and ribosome profiling uncover the activity of MCTS2 and distinct function of eIF2D

Ribosomes scanning from the mRNA 5' cap to the start codon may initiate at upstream open reading frames (uORFs), decreasing protein biosynthesis. Termination at a uORF can lead to re-initiation, where the 40S subunit resumes scanning and initiates another translation event downstream. In mammals, the noncanonical translation factors MCTS1-DENR participate in re-initiation at specific uORFs, but knowledge of other trans-acting factors and uORF features influencing re-initiation is limited. Here, we describe a cell-free re-initiation assay using HeLa cell lysates. Comparing in vivo and in vitro re-initiation activities on uORF-containing model reporters, we validate that MCTS1-DENR-dependent re-initiation is accurately recapitulated in vitro. Using this system and ribosome profiling in cultured cells, we found that knockdown of the homolog eIF2D causes widespread gene expression deregulation unrelated to uORF translation, suggesting distinct functions from MCTS1-DENR. Additionally, we identified MCTS2, encoded by a retrogene copy of Mcts1, as an alternative DENR partner that promotes re-initiation in vitro, providing a plausible explanation for the striking clinical differences associated with Denr vs. Mcts1 mutations in humans. Our findings on re-initiation and the new assay provide valuable insights and a powerful tool for future research on uORF features and trans-acting factors.

molecular biology↗

bioRxiv2024

ZNF574 is a Quality Control Factor For Defective Ribosome Biogenesis Intermediates

Eukaryotic ribosome assembly is an intricate process that involves four ribosomal RNAs, 80 ribosomal proteins, and over 200 biogenesis factors that take part in numerous interdependent steps. This complexity creates a large genetic space in which pathogenic mutations can occur. "Dead-end" ribosome intermediates that result from biogenesis errors are rapidly degraded, affirming the existence of quality control pathway(s) that monitor ribosome assembly. However, the factors that differentiate between on-path and dead-end intermediates are unknown. We engineered a system to perturb ribosome assembly in human cells and discovered that faulty ribosomes are degraded via the ubiquitin proteasome system. We identified ZNF574 as a key component of a novel quality control pathway, which we term the Ribosome Assembly Surveillance Pathway (RASP). Loss of ZNF574 results in the accumulation of faulty biogenesis intermediates that interfere with global ribosome production, further emphasizing the role of RASP in protein homeostasis and cellular health.

cell biology↗

bioRxiv2018

Electron cryo-microscopy structure of the canonical TRPC4 ion channel

Canonical transient receptor channels (TRPC) are non-selective cation channels. They are involved in receptor-operated Ca2+ signaling and have been proposed to act as store-operated channels (SOC). Their malfunction is related to cardiomyopathies and their modulation by small molecules has been shown to be effective against renal cancer cells. The molecular mechanism underlying the complex activation and regulation is poorly understood. Here, we report the electron cryo-microscopy structure of zebrafish TRPC4 in its unliganded (apo), closed state at an overall resolution of 3.6 [A]. The structure reveals the molecular architecture of the cation conducting pore, including the selectivity filter and lower gate. The cytoplasmic domain contains two key hubs that have been shown to interact with modulating proteins. Structural comparisons with other TRP channels give novel insights into the general architecture and domain organization of this superfamily of channels and help to understand their function and pharmacology.

biochemistry↗