Dataset Information

Systematic In Vivo Characterization of Fluorescent Protein Maturation in Budding Yeast.

ABSTRACT: Fluorescent protein (FP) maturation can limit the accuracy with which dynamic intracellular processes are captured and reduce the in vivo brightness of a given FP in fast-dividing cells. The knowledge of maturation timescales can therefore help users determine the appropriate FP for each application. However, in vivo maturation rates can greatly deviate from in vitro estimates that are mostly available. In this work, we present the first systematic study of in vivo maturation for 12 FPs in budding yeast. To overcome the technical limitations of translation inhibitors commonly used to study FP maturation, we implemented a new approach based on the optogenetic stimulations of FP expression in cells grown under constant nutrient conditions. Combining the rapid and orthogonal induction of FP transcription with a mathematical model of expression and maturation allowed us to accurately estimate maturation rates from microscopy data in a minimally invasive manner. Besides providing a useful resource for the budding yeast community, we present a new joint experimental and computational approach for characterizing FP maturation, which is applicable to a wide range of organisms.

SUBMITTER: Guerra P

PROVIDER: S-EPMC8938947 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Publications

Systematic In Vivo Characterization of Fluorescent Protein Maturation in Budding Yeast.

Guerra Paolo P Vuillemenot Luc-Alban LA Rae Brady B Ladyhina Valeriia V Milias-Argeitis Andreas A

ACS synthetic biology 20220218 3

Fluorescent protein (FP) maturation can limit the accuracy with which dynamic intracellular processes are captured and reduce the in vivo brightness of a given FP in fast-dividing cells. The knowledge of maturation timescales can therefore help users determine the appropriate FP for each application. However, in vivo maturation rates can greatly deviate from in vitro estimates that are mostly available. In this work, we present the first systematic study of in vivo ma ...[more]

PMID: 35180343

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Project description:Bicistronic transcripts (operon-like transcripts) have occasionally been reported in eukaryotes, including unicellular yeasts, plants, and humans, despite the fact that they lack trans-splice mechanisms. However, the characteristics of eukaryotic bicistronic transcripts are poorly understood, except for those in nematodes. Here, we describe the genomic, transcriptomic, and ribosome profiling features of bicistronic transcripts in unicellular yeasts. By comparing the expression level of bicistronic transcripts with their monocistronic equivalents, we identify two main categories of bicistronic transcripts: highly and lowly expressed. These two categories exhibit quite different features. First, highly expressed bicistronic transcripts have higher conservation within and between strains and shorter intergenic spacers with higher GC content and less stable secondary structure. Second, genes in highly expressed bicistronic transcripts have lower translation efficiency, with the second gene showing statistically significant lower translation efficiency than the first. Finally, the genes found in these highly expressed bicistronic transcripts tend to be younger, with more recent origins. Together, these results suggest that bicistronic transcripts in yeast are heterogeneous. We further propose that at least some highly expressed bicistronic transcripts appear to play a role in modulating monocistronic translation.IMPORTANCE Operons, where a single mRNA transcript encodes multiple adjacent proteins, are a widespread feature of bacteria and archaea. In contrast, the genes of eukaryotes are generally considered monocistronic. However, a number of studies have revealed the presence of bicistronic transcripts in eukaryotes, including humans. The basic features of these transcripts are largely unknown in eukaryotes, especially in organisms lacking trans-splice mechanisms. Our analyses characterize bicistronic transcripts in one such eukaryotic group, yeasts. We show that highly expressed bicistronic transcripts have unusual features compared to lowly expressed bicistronic transcripts, with several features influencing translational modulation.

Project description:It is essential that cells orchestrate gene expression for the specific niche that they occupy, and this often requires coordination of the expression of large sets of genes. There are multiple regulatory systems that exist for modulation of gene expression, including the adjacent-gene coregulation of the rRNA and ribosome biogenesis and ribosomal protein families. Both gene families exhibit a nonrandom genomic distribution, often clustered directly adjacent to another member of the same family, which results in a tighter transcriptional coordination among adjacent paired genes than that of the unpaired genes within each regulon and can result in a shared promoter that coordinates expression of the pairs. This nonrandom genomic distribution has been seen in a few functionally related gene families, and many of these functional pairings are conserved across divergent fungal lineages. To date, the significance of these observations has not been extended in a systematic way to characterize how prevalent the role of adjacent-gene coregulation is in transcriptional regulation. In the present study, we systematically analyzed the transcriptional coherence of the functional pairs compared to the singletons within all gene families defined by the Gene Ontology Slim designation, using Saccharomyces cerevisiae as a model system, finding that clusters exhibit a tighter transcriptional correlation under specific contexts. We found that the longer a functional pairing is conserved the tighter its response to broad stress and nutritional responses, that roughly 25% of gene families exhibit a nonrandom genomic distribution, and that many of these clusters are conserved. This suggests that adjacent-gene coregulation is a widespread, yet underappreciated, transcriptional mechanism.IMPORTANCE The spatial positioning of genes throughout the genome arrangement can alter their expression in many eukaryotic organisms. Often this results in a genomic context-specific effect on transcription. One example of this is through the clustering of functionally related genes, which results in adjacent-gene coregulation in the budding yeast Saccharomyces cerevisiae In the present study, we set out to systematically characterize the prevalence of this phenomenon, finding the genomic organization of functionally related genes into clusters is a characteristic of myriad gene families. These arrangements are found in many evolutionarily divergent fungi and thus represent a widespread, yet underappreciated, layer of transcriptional regulation.

Dataset Information

Systematic In Vivo Characterization of Fluorescent Protein Maturation in Budding Yeast.

Publications

Systematic <i>In Vivo</i> Characterization of Fluorescent Protein Maturation in Budding Yeast.

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