Biology subjects

Jungers, C. F.

Publications and source records attributed to Jungers, C. F..

4 recordsLinked to original sources

bioRxiv2026

Protein expression is amplified by stabilizing mRNA using gene-specific antisense oligonucleotides

Effective and safe strategies to restore gene expression in haploinsufficiency disorders or to augment protective protein signaling have long been attractive but challenging therapeutic goals. Here, we demonstrate the potential of antisense oligonucleotides (ASOs) to upregulate gene targets through mRNA stabilization. The 3 untranslated regions (UTRs) of mRNAs regulate stability via activating and repressive elements. One prominent destabilizing cis-element found in ~30% of transcripts is the Pumilio RNA-binding protein recognition element (PRE), which can be targeted to enable gene-specific protein upregulation. A scan for PREs highlights hundreds of disease-relevant transcripts that may be targetable via ASO-mediated gene-specific blocking of Pumilio binding. We show that masking specific PREs with ASOs produces sustained protein upregulation through mRNA stabilization using multiple reporter and endogenous gene targets. Using PRE-masking ASOs, we neutralized PRE-mediated destabilization of TANK-binding kinase-1 (TBK1) mRNA and restored TBK1 protein expression in participant-derived fibroblasts with TBK1 haploinsufficiency, causal for amyotrophic lateral sclerosis. Additionally, by targeting PRE sites in the myoprotective gene follistatin (FST), we achieved effective FST mRNA upregulation in tested mouse tissues. These findings suggest that ASO strategies that neutralize destabilizing 3UTR sequences like PREs may provide a powerful approach to upregulate haploinsufficiency-related genes or enhance protective protein levels for therapeutic benefit.

molecular biology↗

bioRxiv2024

A rapid, facile, and economical method for the isolation of ribosomes and translational machinery for structural and functional studies

Short abstractRibosomes are essential RNA-protein complexes involved in protein synthesis and quality control. Traditional methods for ribosome isolation are labor intensive, expensive, and require substantial biological material. In contrast, our new method, RAPPL (RNA Affinity Purification using Poly-Lysine), offers a rapid, simple, and cost-effective alternative. This method enriches ribosomes and associated factors from various species and sample types, including cell lysates, whole cells, organs, and whole organisms, and is compatible with traditional isolation techniques. Here, we use RAPPL to facilitate the rapid isolation, functional screening, and structural analysis of ribosomes with associated factors. We demonstrate ribosome-associated resistance mechanisms from patient uropathogenic Escherichia coli samples and generate a 2.7[A] cryoEM structure of ribosomes from Cryptococcus neoformans. By significantly reducing the amount of the starting biological material and the time required for isolation, the RAPPL approach improves the study of ribosomal function, interactions, and antibiotic resistance, providing a versatile platform for academic, clinical, and industrial research on ribosomes. Long abstractRibosomes are macromolecular RNA-protein complexes that constitute the central machinery responsible for protein synthesis and quality control in the cell. Ribosomes also serve as a hub for multiple non-ribosomal proteins and RNAs that control protein synthesis. However, the purification of ribosomes and associated factors for functional and structural studies requires a large amount of starting biological material and a tedious workflow. Current methods are challenging as they combine ultracentrifugation, the use of sucrose cushions or gradients, expensive equipment, and multiple hours to days of work. Here, we present a rapid, facile, and cost-effective method to isolate ribosomes from in vivo or in vitro samples for functional and structural studies using single-step enrichment on magnetic beads - RAPPL (RNA Affinity Purification using Poly-Lysine). Using mass spectrometry and western blot analyses, we show that poly-lysine coated beads incubated with E. coli and HEK-293 cell lysates enrich specifically for ribosomes and ribosome-associated factors. We demonstrate the ability of RAPPL to isolate ribosomes and translation-associated factors from limited material quantities, as well as a wide variety of biological samples: cell lysates, cells, organs, and whole organisms. Using RAPPL, we characterized and visualized the different effects of various drugs and translation inhibitors on protein synthesis. Our method is compatible with traditional ribosome isolation. It can be used to purify specific complexes from fractions of sucrose gradients or in tandem affinity purifications for ribosome-associated factors. Ribosomes isolated using RAPPL are functionally active and can be used for rapid screening and in vitro characterization of ribosome antibiotic resistance. Lastly, we demonstrate the structural applications of RAPPL by purifying and solving the 2.7[A] cryo-EM structure of ribosomes from the Cryptococcus neoformans, an encapsulated yeast causing cryptococcosis. Ribosomes and translational machinery purified with this method are suitable for subsequent functional or structural analyses and provide a solid foundation for researchers to carry out further applications - academic, clinical, or industrial - on ribosomes.

biochemistry↗

bioRxiv2024

Role of U11/U12 minor spliceosome gene ZCRB1 in Ciliogenesis and WNT Signaling

Despite the fact that 0.5% of human introns are processed by the U11/U12 minor spliceosome, the latter influences gene expression across multiple cellular processes. The ZCRB1 protein is a recently described core component of the U12 mono-snRNP minor spliceosome, but its functional significance to minor splicing, gene regulation, and biological signaling cascades is poorly understood. Using CRISPR-Cas9 and siRNA targeted knockout and knockdown strategies, we show that human cell lines with a partial reduction in ZCRB1 expression exhibit significant dysregulation of the splicing and expression of U12-type genes, primarily due to dysregulation of U12 mono-snRNA. RNA-Seq and targeted analyses of minor intron-containing genes indicate a downregulation in the expression of genes involved in ciliogenesis, and consequentially an upregulation in WNT signaling. Additionally, zcrb1 CRISPR-Cas12a knockdown in zebrafish embryos led to gross developmental and body axis abnormalities, disrupted ciliogenesis, and upregulated WNT signaling, complementing our human cell studies. This work highlights a conserved and essential biological role of the minor spliceosome in general, and the ZCRB1 protein specifically in cellular and developmental processes across species, shedding light on the multifaceted relationship between splicing regulation, ciliogenesis, and WNT signaling.

cell biology↗

bioRxiv2018

Starvation-dependent regulation of eIF6 through multisite phosphorylation by GSK3β

Eukaryotic translation initiation factor 6 is essential for the synthesis of 60S ribosomal subunits and for regulating the association of 60S and 40S subunits. A mechanistic understanding of how eIF6 modulates translation in response to stress, specifically starvation-induced stress, is lacking. Our studies have uncovered a novel mode of eIF6 regulation by GSK3 that is predominantly active in response to serum starvation. Human eIF6 is phosphorylated by GSK3{beta} at a multisite motif in the C-terminus. Sequential phosphorylation by GSK3{beta} requires phosphorylation at a priming site. In response to serum starvation, eIF6 accumulates in the cytoplasm and this altered localization is dependent on GSK3. Disruption of eIF6 phosphorylation enhances translation inhibition and markedly sensitizes the cells to serum starvation. These results suggest that eIF6 regulation by GSK3{beta} contributes to the attenuation of global protein synthesis that is critical for adaptation to starvation-induced stress.

molecular biology↗