Biology subjects

Frese, C. K.

Publications and source records attributed to Frese, C. K..

4 recordsLinked to original sources

bioRxiv2024

Novel RNA-binding protein YebC enhances translation of proline-rich amino acid stretches in bacteria

The ribosome employs a set of highly conserved translation factors to efficiently synthesise proteins. Some translation factors interact with the ribosome in a transient manner and are thus challenging to identify. However, proteins involved in translation can be specifically identified by their interaction with ribosomal RNAs. Using a combination of proteomics approaches, we identified novel RNA binding proteins in the pathogenic bacterium Streptococcus pyogenes. One of these, a universally conserved protein YebC, was shown to transiently interact with 23S rRNA near the peptidyl-transferase centre. Deletion of yebC moderately affected the physiology and virulence of S. pyogenes. We performed ribosome profiling and detected increased pausing at proline-rich amino acid stretches in the absence of functional YebC. Further results obtained with in vivo reporters and in vitro translation system suggest that YebC is a novel translation factor required for efficient translation of proteins with proline-rich motifs.

molecular biology↗

bioRxiv2022

Bacillus subtilis remains translationally active after CRISPRi-mediated replication initiation arrest

Initiation of bacterial DNA replication takes place at the origin of replication, a region characterized by the presence of multiple DnaA boxes that serve as the binding sites for the master initiator protein DnaA. The absence or failure of DNA replication can result in bacterial cell growth arrest or death. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium Bacillus subtilis. For this purpose, DNA replication was blocked using a CRISPRi approach specifically targeting DnaA boxes 6 and 7, which are essential for replication initiation. We characterized the phenotype of these cells and analyzed the overall changes in the proteome using quantitative mass spectrometry. Cells with arrested replication were elongating and not dividing but showed no evidence of DNA damage response. Moreover, these cells did not cease translation over time. This study sets the ground for future research on non-replicating but translationally active B. subtilis, which might be a valuable tool for biotechnological applications. ImportanceEven though bacteria are constantly replicating under laboratory conditions, natural environments expose them to various stresses like lack of nutrients, high salinity, and pH changes, which can keep them in non-replicating states. Non-replicating states can allow bacteria to become less sensitive or tolerant to antibiotics (persisters), remain inactive in specific niches for an extended period (dormancy), and adapt to some hostile ecosystems. Non-replicating states have been studied due to the possibility of repurposing energy to produce additional metabolites or proteins. Using CRISPRi targeting bacterial replication initiation sequences, we successfully arrested the replication of B. subtilis. We observed that non-replicating cells continued growing but not dividing, and the initial arrest did not induce global stress conditions such as SOS or stringent response. Notably, these cells continued their metabolic activity and translation. This study provides comprehensive insights into the physiological response of replication initiation blockage in B. subtilis.

microbiology↗

bioRxiv2020

Expanding the depth and sensitivity of cross-link identification by differential ion mobility using FAIMS

In cross-linking mass spectrometry, the depth and sensitivity is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of low abundant cross-links, here we present a gas-phase separation strategy using high field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in gas phase using the FAIMS device, we increase the number of cross-link identification by 23% for a medium complex sample and 56% for strong cation exchange-fractionated HEK293 cell lysate. Furthermore, we show that for medium complex samples, FAIMS enables the collection of single-shot cross-linking data with comparable depth to the corresponding sample fractionated by chromatography-based approaches. Altogether, we demonstrate FAIMS is highly beneficial for XL-MS studies by expanding the proteome coverage of cross-links while improving the efficiency and confidence of cross-link identification.

biochemistry↗

bioRxiv2019

CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex

The exon junction complex (EJC) is an essential constituent and regulator of spliced messenger ribonucleoprotein particles (mRNPs) in metazoans. As a core component of the EJC, CASC3 was described to be pivotal for EJC-dependent nuclear and cytoplasmic processes. However, recent evidence suggests that CASC3 functions differently from other EJC core proteins. Here, we have established human CASC3 knockout cell lines to elucidate the cellular role of CASC3. In the knockout cells, overall EJC composition and EJC-dependent splicing are unchanged. A transcriptome-wide analysis reveals that hundreds of mRNA isoforms targeted by nonsense-mediated decay (NMD) are upregulated. Mechanistically, recruiting CASC3 to reporter mRNAs by direct tethering or via binding to the EJC stimulates mRNA decay and endonucleolytic cleavage at the termination codon. Building on existing EJC-NMD models, we propose that CASC3 equips the EJC with the ability to communicate with the NMD machinery in the cytoplasm. Collectively, our results characterize CASC3 as a peripheral EJC protein that tailors the transcriptome by promoting the degradation of EJC-dependent NMD substrates.

molecular biology↗