Biology subjects

Muller, R. Y.

Publications and source records attributed to Muller, R. Y..

5 recordsLinked to original sources

bioRxiv2026

The molecular determinants of PABPC-mediated deadenylation rate

Deadenylation, the enzymatic shortening of the poly(A) tail, is typically the first committed step of mRNA decay. Deadenylation rates span nearly a 1000-fold range between transcripts and are governed by protein-RNA interactions, including those involving cytoplasmic poly(A)-binding protein (PABPC). Previous work shows that PABPC can straddle the junction between the poly(A) tail and the 3' untranslated region (UTR), but whether this conformation influences deadenylation has not been tested. To investigate how straddling influences deadenylation kinetics, we designed a library of tailed RNA substrates and measured both in vitro deadenylation rates in the presence of PABPC and PABPC binding propensity for each substrate. We found that 3' UTR sequences influence deadenylation through two mechanisms. First, structured UTRs are deadenylated more slowly in the absence of PABPC1, an effect that is alleviated by PABPC1. Second, sequences upstream of the poly(A) tail modulate PABPC1 binding propensity, with tighter binding correlating with slower deadenylation. This relationship is abolished with a PABPC1 mutant lacking UTR-binding capacity. Together, these results show that sequences upstream of the poly(A) tail tune PABPC1 binding and deadenylation rates, likely contributing to the range of deadenylation rates observed for cellular mRNAs.

biochemistry↗

bioRxiv2026

Dissection of Poly(A)-binding protein (PABPC) cellular function using degron-mediated depletion with replacement

Cytoplasmic poly(A)-binding proteins (PABPCs) are essential and highly abundant regulators of mRNA stability and translation, but their cellular functions have been difficult to dissect due to slow turnover and the lethality observed upon loss of both major paralogs, PABPC1 and PABPC4. To enable structure-function analysis of PABPC in human cells, we developed a protein-replacement platform that couples rapid auxin-inducible degradation of endogenous PABPC1 and PABPC4 with doxycycline-controlled expression of engineered PABPC variants. This approach enables acute removal of native PABPC and real-time assessment of how specific domains, paralogs, and sequence alterations support cellular fitness, shape transcriptome profiles, and regulate poly(A)-tail length. Using this system, we show that the RRM4 domain of PABPC1 is essential for growth, whereas post-translationally modified lysines within RRM4 are individually dispensable. Paralogs and variants with heterologous RRM4 domains vary in their ability to substitute for PABPC1, revealing functional divergence among PABPCs. Transcriptome profiling identifies variant-specific regulatory signatures, and dose-controlled rescue further delineates the relationship between PABPC variant abundance and global poly(A)-tail lengths in vivo. Together, this platform provides a generalizable strategy for dissecting PABPC biology in the cellular context using rationally designed variants.

molecular biology↗

bioRxiv2024

Mapping the Genetic Architecture of the Adaptive Integrated Stress Response in S. cerevisiae

The integrated stress response (ISR) is a conserved eukaryotic signaling pathway that responds to diverse stress stimuli to restore proteostasis. The strength and speed of ISR activation must be tuned properly to allow protein synthesis while maintaining proteostasis. Here, we describe how genetic perturbations change the dynamics of the ISR in budding yeast. We treated ISR dynamics, comprising timecourses of ISR activity across different levels of stress, as a holistic phenotype. We profiled changes in ISR dynamics across thousands of genetic perturbations in parallel using CRISPR interference with barcoded expression reporter sequencing (CiBER-seq). We treated cells with sulfometuron methyl, a titratable inhibitor of branched-amino acid synthesis, and measured expression of an ISR reporter. Perturbations to translation such as depletion of aminoacyl-tRNA synthetases or tRNA biogenesis factors reduced cell growth and caused a strikingly proportionate activation of the ISR activation. In contrast, impaired ribosome biogenesis reduced basal ISR activity and weakened ISR dynamics. Reduced ribosome capacity may lower the demand for amino acids and thereby explain these changes. Our work illustrates how CiBER-seq enables high-throughput measurements of complex and dynamic phenotypes that shed light on adaptive and homeostatic mechanisms.

genomics↗

bioRxiv2024

CRISPRi with barcoded expression reporters dissects regulatory networks in human cells

Genome-wide CRISPR screens have emerged as powerful tools for uncovering the genetic underpinnings of diverse biological processes. Incisive screens often depend on directly measuring molecular phenotypes, such as regulated gene expression changes, provoked by CRISPR-mediated genetic perturbations. Here, we provide quantitative measurements of transcriptional responses in human cells across genome-scale perturbation libraries by coupling CRISPR interference (CRISPRi) with barcoded expression reporter sequencing (CiBER-seq). To enable CiBER-seq in mammalian cells, we optimize the integration of highly complex, barcoded sgRNA libraries into a defined genomic context. CiBER-seq profiling of a nuclear factor kappa B (NF-{kappa}B) reporter delineates the canonical signaling cascade linking the transmembrane TNF-alpha receptor to inflammatory gene activation and highlights cell-type-specific factors in this response. Importantly, CiBER-seq relies solely on bulk RNA sequencing to capture the regulatory circuit driving this rapid transcriptional response. Our work demonstrates the accuracy of CiBER-seq and its potential for dissecting genetic networks in mammalian cells with superior time resolution.

genomics↗

bioRxiv2023

Codon optimality modulates protein output by tuning translation initiation

The impact of synonymous codon choice on protein output has important implications for understanding endogenous gene expression and design of synthetic mRNAs. Synonymous codons are decoded at different speeds, but simple models predict that this should not drive protein output. Instead, translation initiation should be the rate limiting step for production of protein per mRNA, with little impact of codon choice. Previously, we used a neural network model to design a series of synonymous fluorescent reporters and showed that their protein output in yeast spanned a seven-fold range corresponding to their predicted translation elongation speed. Here, we show that this effect is not due primarily to the established impact of slow elongation on mRNA stability, but rather, that slow elongation further decreases the number of proteins made per mRNA. We combine simulations and careful experiments on fluorescent reporters to show that translation is limited on non-optimally encoded transcripts. Using a genome-wide CRISPRi screen, we find that impairing translation initiation attenuates the impact of slow elongation, showing a dynamic balance between rate limiting steps of protein production. Our results show that codon choice can directly limit protein production across the full range of endogenous variability in codon usage.

molecular biology↗