Biology subjects

Winz, M.-L.

Publications and source records attributed to Winz, M.-L..

4 recordsLinked to original sources

bioRxiv2025

Jlp2 is an RQC complex-independent release factor acting on aberrant peptidyl-tRNA, protecting cells against translation elongation stress

Ribosome stalling generates aberrant nascent peptides that remain tethered to the large ribosomal subunit following ribosome splitting. Such peptidyl tRNA:60S complexes are processed by the Ribosome associated Quality Control (RQC) pathway, where Ltn1 and Rqc1 mediate K48 linked ubiquitination and Rqc2 adds CAT tails to the nascent peptides.Subsequently, Vms1 releases the peptides from tRNA, enabling their proteasomal degradation. However, alternative mechanisms that process these stalled intermediates remain poorly defined. In this study, we identify Jlp2 as a novel factor involved in translation quality control. We find that Jlp2 is a release factor that catalyzes peptide release from tRNA and suppresses excessive CAT tailing when Ltn1-dependent ubiquitination is compromised. We define its ribosome binding properties, substrate scope, and critical residues required for peptide release. Our data support a model in which Jlp2-mediated peptide release constitutes an alternative quality control mechanism to safeguard cells during ribosome stalling and conditions of RQC failure or insufficiency.

molecular biology↗

bioRxiv2024

Yeast elongation factor homolog New1 protects a subset of mRNAs from degradation by no-go decay

New1 is a homologue of the essential yeast translation elongation factor eEF3. Lack of New1 has previously been shown to induce queueing of ribosomes upstream of the stop codon on mRNAs encoding specific C-terminal amino acids, primarily lysine and arginine. Here, we used UV crosslinking and analysis of cDNA, long-read nanopore sequencing and proteomics to address the open question of what consequences such queues have for the yeast cell. We show that these queues are ribosomal collisions, which are recognized by the collision sensor and E3 ubiquitin ligase Hel2, marking these collided ribosome complexes for mRNA degradation via canonical no-go decay. Decay is initiated by Cue2-mediated cleavage upstream of the stop codon. Ultimately, this leads to downregulation of encoded proteins, including highly abundant and important metabolic enzymes Pgk1 and Gpm1, as well as translation elongation factors eEF1-alpha and eEF1-beta. Collisions and resulting downstream effects are codon-, rather than amino acid dependent. E.g., for C-terminal lysine and arginine, only specific codons induce collisions upon lack of New1. Our study shows that New1 protects highly abundant and essential genes from degradation by no-go decay thatwould otherwise occur in the absence of translation inhibitors or other direct perturbations of translation.

molecular biology↗

bioRxiv2024

Real-time transcriptomic profiling in distinct experimental conditions

Nanopore technology offers real-time sequencing opportunities, providing rapid access to sequenced data and allowing researchers to manage the sequencing process efficiently, resulting in cost-effective strategies. Here, we present focused case studies demonstrating the versatility of real-time transcriptomics analysis in rapid quality control for long-read RNA-seq. We illustrate its utility through four experimental setups: 1) transcriptome profiling of distinct human cellular populations, 2) identification of experimentally enriched transcripts, 3) transcriptional analysis of cells under heat shock conditions and 4) identification of experimentally manipulated genes (knockout and overexpression) in several yeast strains. We show how to perform multiple layers of quality control as soon as sequencing has started, addressing both the quality of the experimental and sequencing traits. Real-time quality control measures assess sample/condition variability and determine the number of identified genes per sample/condition. Furthermore, real-time differential gene/transcript expression analysis can be conducted at various time points post-sequencing initiation (PSI), revealing dynamic changes in gene/transcript expression between two conditions. Using real-time analysis, which occurs in parallel to the sequencing run, we identified differentially expressed genes/transcripts as early as 1hr PSI. These changes were consistently observed throughout the entire sequencing process. We discuss the new possibilities offered by real-time data analysis, which have the potential to serve as a valuable tool for rapid and cost-effective quality checks in specific experimental settings and can be potentially integrated into clinical applications in the future.

bioinformatics↗

bioRxiv2023

Transcription termination by RNA polymerase I

Transcription elongation is stochastic and driven by a Brownian ratchet mechanism, making it subject to changes in velocity. However, on regions occupied by multiple polymerases, notably the rDNA, DNA rotation plus torsion constrain polymerase molecules to proceed at the same rate generating "torsional entrainment". We report that release of entrainment, by co-transcriptional 3-end cleavage, is permissive for relative movement between polymerases, promoting pausing and backtracking. Subsequent termination (polymerase release) is facilitated by the 5-exonuclease Rat1 (Xrn2) and backtracked transcript cleavage by RNAPI subunit Rpa12. These activities were reproduced in vitro. Short nascent transcripts close to the transcriptional start site, combined with nascent transcript folding energy, similarly facilitate RNAPI pausing. Nascent, backtracked transcripts at pause sites, are targeted by both the exosome cofactor TRAMP and Rat1, promoting termination. Topoisomerase 2 localizes adjacent to RNAPI pause sites, potentially allowing continued elongation by downstream polymerases. Biophysical modeling supported substantial ([~]10%) premature termination. HighlightsNascent pre-rRNA 3 cleavage promotes RNAPI deceleration and termination RNAPI undergoes early, start-site proximal termination at sites of polymerase pausing Biophysical modeling indicates [~]10% early termination - or [~]100 events per minute Model presented for overall organization of pre-rRNA transcription

molecular biology↗