Project description:mRNAs are generally assumed to be loaded instantly with ribosomes upon entry into the cytoplasm. To measure ribosome density on nascent mRNA, we developed nascent Ribo-Seq (nRibo-Seq) by combining Ribo-Seq with progressive 4-thiouridine labelling. In mouse macrophages, we experimentally determined, for the first time, the lag between the appearance of nascent RNA and its association with ribosomes, which was calculated to be 20 - 22 min for bulk mRNA, and approximated the time it takes for mRNAs to be fully loaded with ribosomes to be 41 - 44 min. Notably, ribosomal loading time is adapted to gene function as rapid loading was observed with highly regulated genes. The lag and ribosomal loading time correlate positively with ORF size and mRNA half-life, and negatively with tRNA adaptation index. Similar results were obtained in mouse embryonic stem cells, where the lag in ribosome loading was even more pronounced with 35 - 38 min. We validated our measurements after stimulation of macrophages with lipopolysaccharide, where the lag between cytoplasmic and translated mRNA leads to uncoupling between input and ribosome-protected fragments. Uncoupling is stronger for mRNAs with long ORFs or half-lives, a finding we also confirmed at the level of protein production by nascent chain proteomics. As a consequence of the lag in ribosome loading, ribosome density measurements are distorted when performed under conditions where mRNA levels are far from steady state expression, and transcriptional changes affect ribosome density in a passive way. This study uncovers an unexpected and considerable lag in ribosome loading, and provides guidelines for the interpretation of Ribo-Seq data taking passive effects on ribosome density into account.
Project description:Grad-seq in Clostridium difficile 630. Cell lysate is analyzed in a gradient and fractionated into 21 fractions which are analysed for proteins by MS and for transcripts by RNA-sequencing.
Project description:Proteomic data of ScSUGAR-seq samples: 1)TMT experiment to confirm changes in glycosylation due to Swainsonine 2)LFQ biotin pull down experiment to assess lectin binding partners 3)LFQ of MGAT1 cell lines assessing glycopeptide and glycoproteome level changes
Project description:Kilian2024 - Immune cell dynamics in Cue-Induced Extended Human Colitis Model
Single-cell technologies such as scRNA-seq and flow cytometry provide critical insights into immune cell behavior in inflammatory bowel disease (IBD). However, integrating these datasets into computational models for dynamic analysis remains challenging. Here, Kilian et al., (2024) developed a deterministic ODE-based model that incorporates these technologies to study immune cell population changes in murine colitis. The model parameters were optimized to fit experimental data, ensuring an accurate representation of immune cell behavior over time. It was then validated by comparing simulations with experimental data using Pearson’s correlation and further tested on independent datasets to confirm its robustness. Additionally, the model was applied to clinical bulk RNA-seq data from human IBD patients, providing valuable insights into immune system dynamics and potential therapeutic strategies.
Figure 4c, obtained from the simulation of human colitis model is highlighted here.
This model is described in the article:
Kilian, C., Ulrich, H., Zouboulis, V.A. et al. Longitudinal single-cell data informs deterministic modelling of inflammatory bowel disease. npj Syst Biol Appl 10, 69 (2024). https://doi.org/10.1038/s41540-024-00395-9
Abstract:
Single-cell-based methods such as flow cytometry or single-cell mRNA sequencing (scRNA-seq) allow deep molecular and cellular profiling of immunological processes. Despite their high throughput, however, these measurements represent only a snapshot in time. Here, we explore how longitudinal single-cell-based datasets can be used for deterministic ordinary differential equation (ODE)-based modelling to mechanistically describe immune dynamics. We derived longitudinal changes in cell numbers of colonic cell types during inflammatory bowel disease (IBD) from flow cytometry and scRNA-seq data of murine colitis using ODE-based models. Our mathematical model generalised well across different protocols and experimental techniques, and we hypothesised that the estimated model parameters reflect biological processes. We validated this prediction of cellular turnover rates with KI-67 staining and with gene expression information from the scRNA-seq data not used for model fitting. Finally, we tested the translational relevance of the mathematical model by deconvolution of longitudinal bulk mRNA-sequencing data from a cohort of human IBD patients treated with olamkicept. We found that neutrophil depletion may contribute to IBD patients entering remission. The predictive power of IBD deterministic modelling highlights its potential to advance our understanding of immune dynamics in health and disease.
This model was curated during the Hackathon hosted by BioMed X GmbH in 2024.
Project description:Estrogen Receptor alpha (ERα) is a key driver of most breast cancers, and it is the target of endocrine therapies used in the clinic to treat women with ERα positive (ER+) breast cancer. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used to pull down the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06-935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments.
Project description:The regulation of translation elongation plays a vital role in protein folding; an adequate translational pause provides time and cellular environments for the co-translational folding of nascent peptides. However, the genomic landscape, sequence determinants, and molecular consequences of translational pausing remain mostly unknown. In this study, we performed disome-seq that sequenced mRNA fragments protected by two consecutive ribosomes – a product of severe translational pauses during which the upstream ribosome collides into the paused one. We detected severe translational pauses on ~75% of yeast genes. These pauses were often explained by one of the three mechanisms: 1) slow ribosome releasing at stop codons, 2) slow peptide formation from proline, glycine, asparagine, and cysteine, and 3) slow leaving of polylysine from the exit tunnel of ribosomes. Notably, these amino acids also terminate the α-helical conformation. Such dual roles of amino acids establish an inborn coupling between the synthetic completion of a structural motif and a translational pause. Furthermore, paused ribosomes often recruit chaperones to assist protein folding. As a consequence, emergent protein structures during evolution should be ready to be correctly folded. Collectively, our study shows widespread translational pauses and sheds lights on a better understanding of the regulation of co-translational protein folding.