Biology subjects

Sass, E.

Publications and source records attributed to Sass, E..

5 recordsLinked to original sources

bioRxiv2025

Creation and Validation of a Proteome-Wide Yeast Library for Protein Detection and Analysis

A significant challenge in cell biology is to uncover the function of uncharacterized proteins. Surprisingly a quarter of the proteome is still poorly understood even in the most well studied model organisms. Systematic methodologies, including the use of tagged protein collections, have emerged as a powerful approach to address this gap. Despite the availability of proteome- wide collections featuring various fused proteins, the impact of tag size on protein function highlighted the need for using minimally disruptive tags for functional genomic studies. To rise to this challenge, we have created a proteome-wide collection of yeast strains in which proteins are N-terminally tagged with the Hemagglutinin (HA) epitope. The library leverages the compact size of the HA tag to minimize drawbacks associated with larger tags while enabling efficient functional analysis. We showcase the potential uses of our library for systematically evaluating protein size, abundance and localization using an in vivo labeling approach. Our characterization underscores the potential utility of a proteome-wide HA-tagged library in revealing novel aspects of cell biology, providing an additional powerful tool for functional genomics.

molecular biology↗

bioRxiv2024

A proteome-wide yeast degron collection for the dynamic study of protein function.

Genome-wide collections of yeast strains, known as libraries, revolutionized the way systematic studies are carried out. Specifically, libraries that involve a cellular perturbation, such as the deletion collection, have facilitated key biological discoveries. However, short-term rewiring and long-term accumulation of suppressor mutations often obscure the functional consequences of such perturbations. We present the AID library which supplies "on demand" protein depletion to overcome these limitations. Here, each protein is tagged with a Green Fluorescent Protein (GFP) and an Auxin inducible degron (AID), enabling rapid protein depletion that can be quantified systematically using the GFP element. We characterized the degradation response of all strains and demonstrated its utility by revisiting seminal yeast screens for genes involved in cell cycle progression as well as mitochondrial distribution and morphology. In addition to recapitulating known phenotypes, we also uncovered proteins with previously unrecognized roles in these central processes. Hence, our tool expands our knowledge of cellular biology and physiology by enabling access to phenotypes that are central to cellular physiology and therefore rapidly equilibrated.

cell biology↗

bioRxiv2018

Pooled clone collections by multiplexed CRISPR/Cas12a-assisted gene tagging in yeast

Clone collections of modified strains ( libraries) are a major resource for systematic studies with the yeast Saccharomyces cerevisiae. Construction of such libraries is time-consuming, costly and confined to the genetic background of a specific yeast strain. To overcome these limitations, we developed CRISPR/Cas12a (Cpf1)-assisted tag library engineering (CASTLING) for multiplexed strain construction. CASTLING uses microarray-synthesized oligonucleotide pools and in vitro recombineering to program the genomic insertion of long DNA constructs via homologous recombination. One simple transformation yields pooled libraries with >90% of correctly tagged clones. Up to several hundred genes can be tagged in a single step and, on a genomic scale, approximately half of all genes are tagged with only ~10-fold oversampling. We report several parameters that affect tagging success and provide a quantitative targeted next-generation sequencing method to analyze such pooled collections. Thus, CASTLING unlocks new avenues for increased throughput in functional genomics and cell biology research. (max 150 words)

genomics↗

bioRxiv2018

Reprogramming protein abundance fluctuations in single cells by degradation

Isogenic cells living in the same environment show a natural heterogeneity associated with fluctuations in gene expression. When these fluctuations propagate through cellular regulatory networks, they can give rise to noise regulons, whereby multiple genes fluctuate in a coordinated fashion in single cells. The propagation of these fluctuations has been extensively characterized at the transcriptional level. For example, variations in transcription factor concentration induce correlated fluctuations in the abundance of target gene products. Here, we find that such noise regulons can also stem from protein degradation. We expressed pairs of yellow and red fluorescent proteins, subjected them to differential translation or degradation, and analyzed their fluctuations in single cells. While differential translation had little impact on fluctuations, protein degradation was found to be a dominant contributor. A mathematical model to decompose fluctuations arising from multiple sources of regulation revealed that cells with higher protein production capacity also exhibited higher protein degradation capacity. This association uncouples fluctuations in protein abundance from fluctuations in production rate, and can generate orthogonal noise regulons even for proteins relying on the same transcriptional program.

systems biology↗

bioRxiv2017

A genome-wide resource for high-throughput genomic tagging of yeast ORFs

Here we describe a C-SWAT library for high-throughput tagging of Saccharomyces cerevisiae ORFs. It consists of 5661 strains with an acceptor module inserted after each ORF, which can be efficiently replaced with tags or regulatory elements. We validate the library with targeted sequencing and demonstrate its use by tagging the yeast proteome with bright fluorescent proteins, determining how sequences downstream of ORFs influence protein expression and localizing previously undetected proteins.

genomics↗