Biology subjects

Kopp, J.

Publications and source records attributed to Kopp, J..

5 recordsLinked to original sources

bioRxiv2024

Structure of fungal tRNA ligase with RNA reveals conserved substrate binding principles

RNA ligases play a vital role in RNA processing and maturation including tRNA splicing, RNA repair and the unfolded protein response (UPR). In fungi and plants, the tripartite tRNA ligase Trl1 catalyzes the joining of TSEN-cleaved pre-tRNA exon halves. Trl1 also functions as ligase in the non-conventional HAC1 mRNA splicing during the UPR. The final ligation step is performed by the N-terminal adenylyltransferase domain (LIG). The spatial arrangement of the exon ends during the ligation reaction has remained elusive. Here, we report the crystal structure of Trl1-LIG in complex with a tRNA-derived substrate. Our structure represents a snapshot of the activated RNA intermediate and defines the conserved substrate binding interface. The underlying enzyme-substrate interplay reveals a general substrate binding principle shared by adenylyltransferases. Moreover, we identify the determinants of RNA end specificity as well as the specific roles of Trl1-LIGs subdomains during ligase activation, substrate binding and phosphoryltransfer.

biochemistry↗

bioRxiv2023

Structural and mechanistic insights into activation of the human RNA ligase RTCB by Archease

RNA ligases of the RTCB-type play an essential role in tRNA splicing, the unfolded protein response and RNA repair. RTCB is the catalytic subunit of the pentameric human tRNA ligase complex. RNA ligation by the tRNA-ligase complex requires GTP-dependent activation of RTCB. This active site guanylylation reaction relies on the activation factor Archease. The mechanistic interplay between both proteins has remained unknown. Here, we report a biochemical and structural analysis of the human RTCB-Archease complex in the pre- and post-activation state. Archease reaches into the active site of RTCB and promotes the formation of a covalent RTCB-GMP intermediate through coordination of GTP and metal ions. During the activation reaction, Archease prevents futile RNA substrate binding to RTCB. Moreover, monomer structures of Archease and RTCB reveal additional states within the RNA ligation mechanism. Taken together, we present structural snapshots along the reaction cycle of the tRNA ligase complex.

biochemistry↗

bioRxiv2022

Insulin receptors in pancreatic acinar cells contribute to KrasG12D-driven cancer initiation in the context of diet-induced obesity

The rising pancreatic cancer incidence due to obesity and type 2 diabetes is closely tied to hyperinsulinemia, an independent cancer risk factor. Previous studies demonstrated reducing insulin production suppressed pancreatic intraepithelial neoplasia (PanIN) pre-cancerous lesions in Kras-mutant mice. However, the pathophysiological and molecular mechanisms remained unknown and in particular it was unclear whether hyperinsulinemia affected PanIN precursor cells directly or indirectly. Here, we demonstrate that insulin receptors (Insr) in KrasG12D-expressing pancreatic acinar cells are dispensable for glucose homeostasis, but necessary for hyperinsulinemia-driven PanIN formation in the context of diet-induced hyperinsulinemia and obesity. Mechanistically, this was attributed to amplified digestive enzyme protein translation, triggering of local inflammation and PanIN metaplasia in vivo. In vitro, insulin dose-dependently increased acinar-to-ductal metaplasia formation in a trypsin- and Insr-dependent manner. Collectively, our data shed light on the mechanisms connecting obesity-driven hyperinsulinemia and pancreatic cancer development.

cancer biology↗

bioRxiv2022

Coordination between ECM and cell-cell adhesion regulates the development of islet aggregation, architecture, and functional maturation

Pancreatic islets are 3-dimensional cell aggregates consisting of unique cellular composition, cell-to-cell contacts, and interactions with blood vessels. Cell aggregation is essential for islet endocrine function; however, it remains unclear how developing islets establish aggregation. By combining genetic animal models, imaging tools, and gene expression profiling, we demonstrate that islet aggregation is regulated by extracellular matrix signaling and cell-cell adhesion. Islet endocrine cell-specific inactivation of extracellular matrix receptor Integrin {beta}1 disrupted blood vessel interactions but promoted cell-cell adhesion and the formation of larger islets. In contrast, ablation of cell-cell adhesion molecule -Catenin promoted blood vessel interactions yet compromised islet clustering. Simultaneous removal of Integrin {beta}1 and -Catenin disrupts islet aggregation and the endocrine cell maturation process, demonstrating that establishment of islet aggregates is essential for functional maturation. Our study provides new insights into understanding the fundamental self-organizing mechanism for islet aggregation, architecture, and functional maturation. HIGHLIGHTSO_LIIslet vascularization and aggregation are regulated via ECM-Itgb1 signaling C_LIO_LIECM-Itgb1 signaling negatively controls islet aggregation via regulation of cell-cell adhesion during development C_LIO_LICell-cell adhesion negatively regulates the interaction of endocrine cell-vasculature in islets C_LIO_LIDifferential cell adhesion regulates the establishment of islet architecture C_LIO_LIEndocrine functional maturation depends on islet aggregation regulated by the coordination of ECM-Itgb1 signaling and cell-cell adhesion C_LI

developmental biology↗

bioRxiv2021

Reducing phenotypic and genotypic instabilities of microbial population during continuous cultivation based on stochastic switching dynamics

Predicting the fate of a microbial population (i.e., growth, gene expression...) remains a challenge, especially when this population is exposed to very dynamic environmental conditions, such as those encountered during continuous cultivation. Indeed, the dynamic nature of continuous cultivation process implies the potential deviation of the microbial population involving genotypic and phenotypic diversification. This work has been focused on the induction of the arabinose operon in Escherichia coli as a model system. As a preliminary step, the GFP level triggered by an arabinose-inducible ParaBAD promoter has been tracked by flow cytometry in chemostat with glucose-arabinose co-feeding. For a large range of glucose-arabinose co-feeding, the simultaneous occurrence of GFP positive and negative subpopulation was observed. In a second set of experiments, continuous cultivation was performed by adding either glucose or arabinose, based on the ability of individual cells for switching from low GFP to high GFP states, according to a technology called segregostat. In segregostat mode of cultivation, on-line flow cytometry analysis was used for adjusting the arabinose/glucose transitions based on the phenotypic switching capabilities of the microbial population. This strategy allowed finding an appropriate arabinose pulsing frequency, leading to a prolonged maintenance of the induction level with limited impact on phenotypic diversity for more than 60 generations. This result suggests that constraining individual cells into a given phenotypic trajectory is maybe not the best strategy for directing cell population. Instead, allowing individual cells switching around a predefined threshold seems to be a robust strategy leading to oscillating, but predictable, cell population behavior.

microbiology↗