Biology subjects

Huch, S.

Publications and source records attributed to Huch, S..

5 recordsLinked to original sources

bioRxiv2026

Puf3 contributes to changes in mRNA solubility, translation elongation dynamics at rare arginine codons and loss of protein homeostasis in cells lacking Not4

The Not proteins of the Ccr4-Not complex regulate translation elongation dynamics, essential for proper folding and assembly of new proteins. In yeast, ribosomes with non-optimal codons in the A-site are enriched within the pool of ribosomes bound by Not4 and Not5. Such ribosomes accumulate in cells lacking Not4 or Not5 that show defects in co-translational assembly and aggregation of new proteins. Recently we observed that depletion of Not1 and Not4 inversely regulate changes in mRNA solubility, correlating with inverse codon-specific changes in A-site ribosome dwelling occupancies (RDOs). Here we describe that mRNAs less soluble upon Not4 depletion are enriched for targets of the RNA-binding protein Puf3. We determine that Puf3 contributes to inverse changes of A-site RDOs upon Not1 and Not4 depletion, in particular at rare arginine codons, and it contributes to changes in mRNA solubility in not4{Delta}. Moreover, deletion of Puf3 suppresses temperature sensitivity and protein aggregation in the not4{Delta} strain, while overexpression of Puf3 is toxic. Puf3 post-translational modifications and the Puf3 interactome are altered in not4{Delta}. Taken together, our results associate alterations in Puf3 post-translational status and function, including contribution to translation elongation dynamics, with not4{Delta} mutant phenotypes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/695943v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@855afdorg.highwire.dtl.DTLVardef@118addaorg.highwire.dtl.DTLVardef@13dd015org.highwire.dtl.DTLVardef@12824c6_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

A streamlined nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing

Antimicrobial resistance (AMR) poses a significant threat to public health. Rapid and accurate antimicrobial sensitivity testing is essential to guide effective treatment. Here, we present "simplified 5PSeq" (s5PSeq), a streamlined protocol for profiling 5 monophosphorylated (5P) mRNA degradation intermediates that reflect ribosome dynamics in vivo. By capturing antibiotic-induced, context-specific ribosome stalling events, s5PSeq provides a molecular proxy for bacterial growth inhibition--offering a molecular phenotypic readout without the need for culturing. s5PSeq reduces library preparation time to under four hours and incorporates a novel rRNA blocking strategy. We demonstrated its clinical utility by identifying erythromycin-resistant and sensitive Clostridioides difficile clinical isolates. Combining s5PSeq with real-time nanopore sequencing enables fast AMR diagnosis with as few as 3000 reads. In addition to simplifying the study of 5P co-translational mRNA decay, our work suggests that utilizing information-rich phenotypic molecular readouts can significantly improve AMR diagnostics. HighlightsO_LIs5PSeq is a streamlined protocol for profiling 5P mRNA degradation intermediates. C_LIO_LIContext-specific ribosome stalls can be used to assess phenotypic antimicrobial sensitivity at the molecular level. C_LIO_LIBlocking rRNA sequencing at the ligation step streamlines library preparation and lowers costs and hands-on time. C_LIO_LIIntegration with nanopore sequencing allows same-day antimicrobial sensitivity testing in species with 5-3 exonuclease. C_LI

microbiology↗

bioRxiv2024

Ribosomes modulate transcriptome abundance via generalized frameshift and out-of-frame mRNA decay.

Cells need to adapt their transcriptome to quickly match cellular needs in changing environments. mRNA abundance can be controlled by altering both its synthesis and decay. Here we show how, in response to poor nutritional conditions, the bulk of the S. cerevisiae transcriptome undergoes -1 ribosome frameshifts and experiences an accelerated out-of-frame co-translational mRNA decay. Using RNA metabolic labelling, we demonstrate that in poor nutritional conditions, NMD-dependent degradation represents at least one third of the total mRNA decay. We further characterize this mechanism and identify low codon optimality as a key factor for ribosomes to induce out-of-frame mRNA decay. Finally, we show that this phenomenon is conserved from bacteria to humans. Our work provides evidence for a direct regulatory feedback mechanism coupling protein demand with the control of mRNA abundance to limit cellular growth and expands the functional role of mRNA quality control.

molecular biology↗

bioRxiv2022

Not1 and Not4 inversely determine mRNA solubility that sets the dynamics of co-translational events

BackgroundThe Ccr4-Not complex is most well known as the major eukaryotic deadenylase. However, several studies have uncovered roles of the complex, in particular of the Not subunits, unrelated to deadenylation and relevant for translation. In particular, the existence of Not condensates that regulate translation elongation dynamics have been reported. Typical studies that evaluate translation efficiency rely on soluble extracts obtained after disruption of cells and ribosome profiling. Yet cellular mRNAs in condensates can be actively translated and may not be present in such extracts. ResultsIn this work, by analyzing soluble and insoluble mRNA decay intermediates in yeast, we determine that insoluble mRNAs are enriched for ribosomes dwelling at non-optimal codons compared to soluble mRNAs. mRNA decay is higher for soluble RNAs, but the proportion of co-translational degradation relative to the overall mRNA decay is higher for insoluble mRNAs. We show that depletion of Not1 and Not4 inversely impact mRNA solubilities and, for soluble mRNAs, ribosome dwelling according to codon optimality. Depletion of Not4 solubilizes mRNAs with lower non-optimal codon content and higher expression that are rendered insoluble by Not1 depletion. By contrast, depletion of Not1 solubilizes mitochondrial mRNAs, which are rendered insoluble upon Not4 depletion. ConclusionOur results reveal that mRNA solubility defines dynamics of co-translation events and is oppositely regulated by Not1 and Not4, a mechanism that we additionally determine may already be set by Not1 promoter association in the nucleus.

molecular biology↗

bioRxiv2021

RNA degradation analysis reveals ribosome dynamics in complex microbiome samples

Post-transcriptional regulation is essential for life, yet we are currently unable to investigate its role in complex microbiome samples. Here we discover that co-translational mRNA degradation, where the degradation machinery follows the last translating ribosome, is conserved across prokaryotes. By investigating 5P mRNA decay intermediates, we obtain in vivo ribosome protection information that allows the study of codon and gene specific ribosome stalling in response to stress and drug treatment at single nucleotide resolution. We use this approach to investigate in vivo species-specific ribosome footprints of clinical and environmental microbiomes and show for the first time that ribosome protection patterns can be used to phenotype microbiome perturbations. Our work paves the way for the study of the metatranslatome, and enables the investigation of fast, species-specific, post-transcriptional responses to environmental and chemical perturbations in unculturable microbial communities.

genomics↗