Biology subjects

Vicencio, J.

Publications and source records attributed to Vicencio, J..

5 recordsLinked to original sources

bioRxiv2026

Declines in mRNA synthesis set the rate of organismal aging

The abundance of mRNA sets a ceiling on a cells capacity to produce protein and carry out its functions. Here, we describe a pathological decline in absolute mRNA abundance that occurs in most cell types during invertebrate and mammalian aging, caused by decreases in mRNA synthesis capacity. In C. elegans, decreases in mRNA abundance are tightly coupled to declines in RNA Polymerase II (Pol II) protein abundance. Measuring Pol II abundance dynamics in vivo, we find that individuals enter adulthood with a four-fold excess of Pol II, whose kinetic equilibration towards its homeostatic set point drives reductions in mRNA abundance and, in turn, organismal aging. Briefly accelerating Pol II declines produces permanent, dose-dependent reductions in healthspan and lifespan, whereas deceleration extends both. Although disrupted cellular homeostasis is conventionally seen as a consequence of aging, our results reveal how an out-of-equilibrium state established during development provides a driving force for aging.

systems biology↗

bioRxiv2026

Ether lipid remodeling during neuronal differentiation prevents ferroptosis

Ferroptosis is a form of cell death driven by iron-dependent lipid peroxidation, with specific lipid species playing key roles in modulating susceptibility. Among these, ether lipids have shown conflicting effects, being linked to both protection and sensitization. Here, we dissect the relationship between lipid structure and ferroptosis sensitivity and explain how ether lipids exert context-dependent effects. Ether lipids can promote ferroptosis through a metabolic bias towards the accumulation of polyunsaturated acyl chains and ethanolamine head groups, whereas this pro-ferroptotic tendency is counterbalanced by the anti-ferroptotic vinyl ether moiety introduced by plasmanylethanolamine desaturase 1. We show that this protective effect is critical for preventing ferroptosis in hiPSC-derived neurons, which accumulate otherwise pro-ferroptotic ether lipids during differentiation. This effect is not solely due to its antioxidant properties but also stems from the reprogramming of mitochondrial respiration. The lack of vinyl ether bonds leads to multiple mitochondrial defects, including increased mitochondrial reactive oxygen species (ROS), lower membrane potential, and abnormal cristae structures. These findings indicate that vinyl ether bonds in ether lipids offer dual ferroptosis resistance by scavenging ROS and minimizing its production at the mitochondrial level. The disruption of this system in Caenorhabditis elegans leads to iron-induced death and impaired motility. Thus, our study reveals ether lipid structural remodeling as a key regulator of ferroptosis sensitivity in neurons.

cell biology↗

bioRxiv2024

Engineering the auxin-inducible degron system for tunable in vivo control of organismal physiology

The physiological mechanisms governing health and disease exhibit complex interactions between multiple genes and gene products. To study the dynamics of living systems, researchers need experimental methods capable of producing calibrated, quantitative perturbations in vivo -- perturbations that cannot be obtained using classical genetics, RNAi interference, or small molecule drugs. Recently, an auxin-inducible degron (AID) system has been developed to allow targeted degradation of proteins using small-molecule activators, providing spatiotemporal control of protein abundance. However, a better understanding of the biochemical activities of AID system components in their physiological context is needed to design quantitative interventions. Here, we apply engineering approaches to characterize and understand the performance of several AID technologies and then improve this performance in the multicellular animal Caenorhabditis elegans. We 1) develop new technologies that allow for a careful calibration of AID activity for specific purposes; 2) develop new TIR1 enzyme constructs with improved performance over existing constructs; 3) develop an approach to simultaneously and independently degrade target proteins in distinct tissues; and finally, 4) develop an approach for pan-organismal protein degradation by re-engineering the TIR1 enzyme. Taken together, these advances enable new quantitative experimental approaches to study the cellular and systems dynamics of animals.

systems biology↗

bioRxiv2021

Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes

The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAM, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. We have characterized and optimized the activity of SpG and SpRY in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to efficient mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm can predict SpG and SpRY activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.

genetics↗

bioRxiv2018

Efficient generation of endogenous fluorescent reporters by Nested CRISPR in Caenorhabditis elegans

CRISPR-based genome editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is still inefficient. The use of plasmids with selection markers is an effective methodology, but often requires laborious and complicated cloning steps. We have established a cloning-free ribonucleoprotein-driven Nested CRISPR method that robustly produces endogenous fluorescent reporters. This methodology is based on the division of the GFP and mCherry sequences in three fragments. In the first step we use ssDNA donors ([≤]200 bp) to insert 5 and 3 fragments in the place of interest. In the second step, we use these sequences as homology regions for Homology Directed Repair (HDR) with a dsDNA donor (PCR product, {approx}700 bp) including the middle fragment, thus completing the fluorescent protein sequence. This method is advantageous because the first step with ssDNA donors is known to be very efficient, and the second step, uses universal reagents, including validated PCR products and crRNAs, to create fluorescent reporters reaching reliable editing efficiencies as high as 40%. We have also used Nested CRISPR in a non-essential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step.\n\nIn the search of modifications to optimize the method, we tested synthetic sgRNAs, but we did not observe a significant increase in the efficacy compared to independently adding tracrRNA and crRNA to the injection mix. Conveniently, we also found that both steps of Nested CRISPR could be performed in a single injection. Finally, we discuss the utility of Nested CRISPR for targeted insertion of long DNA fragments in other systems and prospects of this method in the future.

genetics↗