Translation initiation events on structured eukaryotic mRNAs generate gene expression noise

Gene expression stochasticity plays a major role in biology, creating non-genetic cellular individuality and influencing multiple processes, including differentiation and stress responses. We have addressed the lack of knowledge about posttranscriptional contributions to noise by determining cell-to-cell variations in the abundance of mRNA and reporter protein in yeast. Two types of structural element, a stem–loop and a poly(G) motif, not only inhibit translation initiation when inserted into an mRNA 5΄ untranslated region, but also generate noise. The noise-enhancing effect of the stem–loop structure also remains operational when combined with an upstream open reading frame. This has broad significance, since these elements are known to modulate the expression of a diversity of eukaryotic genes. Our findings suggest a mechanism for posttranscriptional noise generation that will contribute to understanding of the generally poor correlation between protein-level stochasticity and transcriptional bursting. We propose that posttranscriptional stochasticity can be linked to cycles of folding/unfolding of a stem–loop structure, or to interconversion between higher-order structural conformations of a G-rich motif, and have created a correspondingly configured computational model that generates fits to the experimental data. Stochastic events occurring during the ribosomal scanning process can therefore feature alongside transcriptional bursting as a source of noise

Stochastic scanning events on the GCN4 mRNA 5’ untranslated region generate cell-to-cell heterogeneity in the yeast nutritional stress response

Gene expression stochasticity is inherent in the functional properties and evolution of biological systems, creating non-genetic cellular individuality and influencing multiple processes, including differentiation and stress responses. In a distinct form of non-transcriptional noise, we find that interactions of the yeast translation machinery with the GCN4 mRNA 5’UTR, which underpins starvation-induced regulation of this transcriptional activator gene, manifest stochastic variation across cellular populations. We use flow cytometry, fluorescence-activated cell sorting and microfluidics coupled to fluorescence microscopy to characterize the cell-to-cell heterogeneity of GCN4-5’UTR-mediated translation initiation. GCN4-5’UTR-mediated translation is generally not de-repressed under non-starvation conditions; however, a sub-population of cells consistently manifests a stochastically enhanced GCN4 translation (SETGCN4) state that depends on the integrity of the GCN4 uORFs. This sub-population is eliminated upon deletion of the Gcn2 kinase that phosphorylates eIF2α under nutrient-limitation conditions, or upon mutation to Ala of the Gcn2 kinase target site, eIF2α-Ser51. SETGCN4 cells isolated using cell sorting spontaneously regenerate the full bimodal population distribution upon further growth. Analysis of ADE8::ymRuby3/ GCN4::yEGFP cells reveals enhanced Gcn4-activated biosynthetic pathway activity in SETGCN4 cells under non-starvation conditions. Computational modeling interprets our experimental observations in terms of a novel translational noise mechanism underpinned by natural variations in Gcn2 kinase activity

Minimum-noise production of translation factor eIF4G maps to a mechanistically determined optimal rate control window for protein synthesis

Gene expression noise influences organism evolution and fitness. The mechanisms determining the relationship between stochasticity and the functional role of translation machinery components are critical to viability. eIF4G is an essential translation factor that exerts strong control over protein synthesis. We observe an asymmetric, approximately bell-shaped, relationship between the average intracellular abundance of eIF4G and rates of cell population growth and global mRNA translation, with peak rates occurring at normal physiological abundance. This relationship fits a computational model in which eIF4G is at the core of a multi-component– complex assembly pathway. This model also correctly predicts a plateau-like response of translation to super-physiological increases in abundance of the other cap-complex factors, eIF4E and eIF4A. Engineered changes in eIF4G abundance amplify noise, demonstrating that minimum stochasticity coincides with physiological abundance of this factor. Noise is not increased when eIF4E is overproduced. Plasmid-mediated synthesis of eIF4G imposes increased global gene expression stochasticity and reduced viability because the intrinsic noise for this factor influences total cellular gene noise. The naturally evolved eIF4G gene expression noise minimum maps within the optimal activity zone dictated by eIF4G’s mechanistic role. Rate control and noise are therefore interdependent and have co-evolved to share an optimal physiological abundance point

Exploring the Impact of PQN-75 and GLH-1/Vasa on Germline Development, Maintenance, and GSC Reprogramming Using Caenorhabditis elegans as a Model

This thesis combines research on PQN-75 expression, functional motifs of GLH-1/Vasa, and germ granule components in Caenorhabditis elegans to provide a comprehensive understanding of germline development, maintenance, and reprogramming, while also examining the role of pharyngeal gland cells in stress resistance and thermotolerance. In C. elegans, pharyngeal gland cells secrete mucin-like proteins, such as PQN-75, with similarities to human PRB2. The expression of PQN-75 in gland cells confers stress resistance and thermotolerance but does not affect fertility, instead it plays a role in the organism\u27s ability to adapt to varying environmental conditions. While, GLH-1/Vasa, an ATP-dependent DEAD-box helicase, plays a critical role in safeguarding the germline by regulating translation and amplifying piwi-interacting RNAs. To elucidate the functions of GLH-1 and its role in germline development, CRISPR/Cas9 technology was employed to investigate its functional motifs in C. elegans by analyzing 28 endogenous mutant alleles. Results demonstrate that helicase activity is essential for GLH-1\u27s association with P granules, and removing glycine-rich repeats diminishes P-granule interactions at the nuclear periphery. Additional, mass spectrometry reveals an affinity between GLH-1 and three structurally conserved PCI complexes, along with a reciprocal aversion for assembled ribosomes and the 26S proteasome. Suggesting that P granules compartmentalize the cytoplasm to exclude large protein assemblies, effectively shielding associated transcripts from translation, contributing to germline maintenance. Germ granules are essential for maintaining germline integrity and stem cell totipotency. Depletion of core germ granule components in C. elegans leads to germ cell reprogramming and sterility. To better understand the initiation of somatic reprogramming and the role of GLH-1 in this process, total mRNA (transcriptome) and polysome-associated mRNA (translatome) changes in a precision full-length deletion of glh-1 where examined. Here two significant changes were observed: first, GLH-1 suppresses the expression of neuropeptide-encoding transcripts, suggesting a role in repressing somatic reprogramming and maintaining germline integrity; second, GLH-1 promotes Major Sperm Proteins levels, repressing spermatogenic expression during oogenesis and promoting MSP expression to drive spermiogenesis and sperm motility, highlighting its importance in fertility. Our findings contribute to understanding the roles of PQN-75 and GLH-1/Vasa in C. elegans germline development, maintenance, and germline stem cell reprogramming, while also shedding light on the organism\u27s stress resistance and thermotolerance mechanisms. With broader implications identifying early stem cell reprogramming processes and provides a platform for future research on germline biology in C. elegans

RNA, the Epicenter of Genetic Information

The origin story and emergence of molecular biology is muddled. The early triumphs in bacterial genetics and the complexity of animal and plant genomes complicate an intricate history. This book documents the many advances, as well as the prejudices and founder fallacies. It highlights the premature relegation of RNA to simply an intermediate between gene and protein, the underestimation of the amount of information required to program the development of multicellular organisms, and the dawning realization that RNA is the cornerstone of cell biology, development, brain function and probably evolution itself. Key personalities, their hubris as well as prescient predictions are richly illustrated with quotes, archival material, photographs, diagrams and references to bring the people, ideas and discoveries to life, from the conceptual cradles of molecular biology to the current revolution in the understanding of genetic information. Key Features Documents the confused early history of DNA, RNA and proteins - a transformative history of molecular biology like no other. Integrates the influences of biochemistry and genetics on the landscape of molecular biology. Chronicles the important discoveries, preconceptions and misconceptions that retarded or misdirected progress. Highlights major pioneers and contributors to molecular biology, with a focus on RNA and noncoding DNA. Summarizes the mounting evidence for the central roles of non-protein-coding RNA in cell and developmental biology. Provides a thought-provoking retrospective and forward-looking perspective for advanced students and professional researchers

Development of RNA-based translation modulation tools for synthetic biology applications

The ability to reliably regulate translation of heterologous proteins is of great interest for diverse applications in the field of biological and metabolic engineering. RNA-based tools to control ribosome-dependent synthesis of proteins within E. coli were developed. First, adaptation of the well-studied RNA-IN and RNA-OUT system based on E. coli Tn10 to reliably repress translation regardless of coding sequence context was performed. Using an invariant sequence within the RNA-IN/OUT interacting region does not interfere with the function of the RNAs involved. The same RNA-IN/RNA-OUT system was adapted to activate the translation of a specific target mRNA. Regulation of translation extends to processing of polyproteins. In eukaryotes this can be accomplished by viral 2A peptides, avoiding reinitiating at a downstream start codon but has not been observed in bacteria. Therefore, a strategy for developing libraries of 2A peptides with potential activity across an array of organisms was developed

On the Origin of Phenotypic Variation: Novel Technologies to Dissect Molecular Determinants of Phenotype

This thesis describes the conception, design, and development of novel computational tools, theoretical models, and experimental techniques applied to the dissection of molecular factors underlying phenotypic variation. The first part of my work is focused on finding rare genetic variants in pooled DNA samples, leading to the development of a novel set of algorithms, SNPseeker and SPLINTER, applied to next-generation sequencing data. The second part of my work describes the creation of a reporter system for DNA methylation for the purpose of dissecting the genetic contribution of tissue-specific patterns of DNA methylation across the genome. Finally the last part of my work is focused on understanding the basis of stochastic variation in gene expression with a focus on modeling and dissecting the relationship between single-cell protein variance and mean at a genome-wide scale

The evolutionary genomics of CTCF binding and functional signatures in mouse.

Genetic differences within and between species predominantly lie in the noncoding sequence of the regulatory regions of the genome whose function and significance largely remain poorly understood. Despite significant progress in the field of genomics and the rapid progress in sequencing methods and the subsequent explosion of genomic data, our understanding of the role of the non- coding genetic sequence in the regulation of tissue- and species-specific gene expression is still lagging behind, limiting our comprehension of the evolutionary mechanisms and pressures that shape those expression profiles, and their involvement in the health and disease. The CTCF protein demarcates mammalian genomes into discrete transcriptionally active domains, providing the platform for complex spatial and temporal regulatory processing of genetic information that govern biological processes. In this thesis, I investigate the dynamics and functional implications of evolutionarily novel CTCF binding sites in two Mus genus mouse subspecies, Mus musculus domesticus and Mus musculus castaneus, separated by a short evolutionary time of only one million years. The project investigated the subspecies-specific binding of CTCF in terms of the repeat content, evolution, functional impact and involvement in chromatin conformation. The key findings of this investigation are: (1) the incorporation of young CTCF sites into the non-coding genome via action of transposable elements is followed rapidly with the exhibition of various characteristics of biological function; (2) Unlike other tissue-specific transcription factors, allele- specific CTCF occupancy is affected by cis- and trans-acting regulatory mechanisms that exhibit similar functional characteristics; (3) CTCF evolutionary dynamics support both maintenance of pre-existing structures and functions and provide template for novel ones. In summary, this thesis discusses the evolutionary dynamics of CTCF genomic occupancy and functional signatures in short evolutionary time, and illustrates how either novel species-specific CTCF sites, or common sites with newly-acquired genotypic variants integrate into existing genomic architecture and begin to exert their effects