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Design, Construction, and Functional Characterization of a tRNA Neochromosome in Yeast

ABSTRACT: Here we report the design, construction and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ~190 kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporated orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enable an orthogonal SCRaMbLE system capable of adjusting tRNA abundance. Following construction, we obtained evidence of a potent selective force once the neochromosome was introduced into yeast cells, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up new opportunities to directly test hypotheses surrounding these essential non-coding RNAs

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE243449 | GEO | 2023/11/01

REPOSITORIES: GEO

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Design, Construction and Functional Characterization of a tRNA Neochromosome in Yeast

Project description:Here we report the design, construction and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae chromosomes. Intending to address one of the central design principles of the Sc2.0 project, the ~190 kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporated orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enable an orthogonal SCRaMbLE system capable of adjusting tRNA abundance. Following construction, we obtained evidence of a potent selective force once the neochromosome was introduced into yeast cells, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up new opportunities to directly test hypotheses surrounding these essential non-coding RNAs.

2022-10-08 | PXD037209 | iProX

What’s wrong with SwYG? Probing the limits of S. cerevisiae molecular and analytical toolbox

Project description:The construction of powerful cell factories requires extensive remodelling of microbial genomes, entailing many rounds of transformations to perform the large number of desired gene modifications. However, increasing the number of genetic interventions inevitably increases the occurrence of unwanted mutations and effects. Using glycolysis as paradigm, a previous study developed a Saccharomyces cerevisiae strain in which the glycolytic genes, relocalized to a single locus, can be easily swapped by any new design, thereby enabling fast and easy remodelling of the entire pathway. After 27 genetic modifications performed in 43 transformation rounds, the Switchable Yeast Glycolysis (SwYG) strain grew ca. 20% slower than its ancestor with native glycolysis design. Exploring the cause of this slower growth rate, the present study reflects on the genetic and analytical challenges encountered by extensive strain construction programs and provides design guidelines for integration of large constructs in the yeast genome.

2021-12-06 | GSE190122 | GEO

Multiomics of GCN4-dependent replicative lifespan extension models reveals Gcn4 as a regulator of protein turnover in yeast

Project description:We have shown that multiple tRNA synthetase inhibitors can increase lifespan in both the nematode C. elegans and the budding yeast S. cerevisiae by acting through the conserved transcription factor Gcn4 (yeast) / ATF-4 (worms). To further understand the biology downstream of this conserved transcription factor in the yeast model system, we looked at two different yeast models known to have both upregulated Gcn4, and GCN4-dependent increased replicative lifespan. These two models are rpl31aΔ yeast, and yeast treated with the tRNA synthetase inhibitor borrelidin. We used both proteomic and RNAseq analysis of a block experimental design that included both of these models, to identify GCN4-dependent changes in these two longlived strains of yeast. Proteomic analysis of these yeast indicate that the longlived yeast have increased abundance of proteins involved in amino acid biosynthesis. RNASeq of these same yeast uncovered further regulation of protein turnover, identifying the differential expression of genes associated with both autophagy and with the ubiquitin proteasome system. The data presented here further underscore the important role that GCN4 and its orthologs play in the maintenance of protein homeostasis, which is itself an important hallmark of aging. Importantly, these changes could also have wider-ranging implications in the understanding and treatment of diseases of aging characterized by protein aggregation.

2023-11-11 | GSE242739 | GEO

Chimeric design of Pyrrolysyl-tRNA synthetase/tRNA pairs and canonical synthetase/tRNA pairs for genetic code expansion

Project description:An orthogonal aminoacyl-tRNA synthetase/tRNA pair is a key prerequisite for site-specific incorporation of unnatural amino acids. Due to its high codon suppression efficiency and full orthogonality, the pyrrolysyl-tRNA synthetase/pyrrolysyl-tRNA pair is currently the ideal system for genetic code expansion in both eukaryotes and prokaryotes. There is a pressing need to discover or engineer other fully orthogonal translation systems that allow unnatural amino acids with distinct scaffolds and functionalities to be incorporated into a wide range of living organisms efficiently. Here, through rational chimera design by transplanting the key orthogonal components from the pyrrolysine system, we create multiple chimeric tRNA synthetase/chimeric tRNA pairs, including chimera histidine, phenylalanine, and alanine systems. We further show that these engineered chimeric systems are orthogonal and highly efficient with comparable flexibility to the pyrrolysine system. In addition, the chimera phenylalanine system can incorporate a group of phenylalanine, tyrosine and tryptophan analogues efficiently in both E. coli and mammalian cells. These aromatic amino acids analogous exhibit unique properties and characteristics, including fluorescence, post-translation modification. To our knowledge, most of these molecules have never been shown to be incorporated with fully orthogonal pairs. Therefore, these chimera pairs offer the potential for incorporation of de novo unnatural amino acids into target proteins for a variety of applications.

2020-05-25 | PXD018660 | Pride

Non-coding roX RNAs Prevent the Binding of the MSL-complex to Heterochromatic Regions

Project description:Long non-coding RNAs are involved in dosage compensation both in mammals and in Drosophila by inducing changes in the X-chromosome chromatin structure. In Drosophila melanogaster, roX1 and roX2 are long non-coding RNAs that together with proteins form the male-specific lethal (MSL) complex, which coats the entire male X-chromosome and mediates dosage compensation by increased transcriptional output. It has been shown that in polytene chromosomes, in absence of both roX1 and roX2, the MSL-complex is decreased on the male X-chromosome and found relocated to the chromocenter, and the 4th chromosome. Here we address the role of roX RNAs in MSL-complex targeting and in the evolution of dosage compensation in Drosophila. We performed ChIP-seq experiments and show that MSL-complex recruitment to high affinity sites (HAS) on the X-chromosome is independent on roX and that the HAS sequence motif is conserved in D. simulans. Additionally, a complete and enzymatically active MSL-complex is recruited to six specific genes on the 4th chromosome. Interestingly, our sequence analysis shows that in the absence of roX RNAs, the MSL-complex has affinity to regions enriched in Hoppel transposable elements and to repeats in general. We hypothesize that roX mutants reveal an ancient targeting of the MSL-complex and propose that the role of roX RNAs is to restrict MSL-complex from binding to heterochromatin.

2014-11-07 | GSE58768 | GEO

tRNAs marked with CCACCA are targeted for degradation

Project description:The CCA-adding enzyme adds CCA to the 3' ends of transfer RNAs (tRNAs), a critical step in tRNA biogenesis that generates the amino acid attachment site. We found that the CCA-adding enzyme plays a key role in tRNA quality control by selectively marking unstable tRNAs and tRNA-like small RNAs for degradation. Instead of adding CCA to the 3' ends of these transcripts, CCA-adding enzymes from all three kingdoms of life add CCACCA. Here, we report deep sequencing analysis of the 3' ends of tRNA-Ser-CGA and tRNA-Ser-UGA from S. cerevisiae strains and show that hypomodified mature tRNAs are subjected to CCACCA (or poly(A) addition) as part of a rapid tRNA decay pathway in vivo. We conjecture that CCACCA addtion is a universal mechanism for controlling tRNA levels and preventing errors in translation. 121 samples analyzed in total, representing time courses of 10 different yeast strains; Biological replicates for each time point are included

2011-11-17 | E-GEOD-33756 | biostudies-arrayexpress

Deep functional analysis of synII, a 770 kb synthetic yeast chromosome

Project description:Here we report the design, construction and characterization of a 770 Kb synthetic yeast chromosome II (synII). With the UPLC-MS method, we demonstrated synII maintains a nearly wild-type phenotype and fitness despite hundreds of designer editing to it. Our phenomics, transcriptomics, proteomics data futher supports the identity of synII and wild one.

2016-10-13 | MTBLS296 | MetaboLights

RNA sequence (RNA-seq) profiling of SynXI strain

Project description:We describe construction of the 660 kilobase synthetic yeast chromosome XI (synXI) and reveal how synthetic redesign of non-coding DNA elements impact the cell. To aid construction from synthesized 5 to 10 kilobase DNA fragments, we implemented CRISPR-based methods for synthetic crossovers in vivo and used these methods in an extensive process of bug discovery, redesign and chromosome repair, including for the precise removal of 200 kilobases of unexpected repeated sequence. In synXI, the underlying causes of several fitness defects were identified as modifications to non-coding DNA, including defects related to centromere function and mitochondrial activity that were subsequently corrected. As part of synthetic yeast chromosome design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show here that targeted insertion of these sites can be used to create extrachromosomal circular DNA on demand, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI has uncovered effects of non-coding and extrachromosomal circular DNA, contributing to better understanding of these elements and informing future synthetic genome design.

2023-10-09 | GSE244611 | GEO

Comparative Genomic Hybridization of S. cerevisiae YJS329 and BYZ1

Project description:The environmental stresses and inhibitors encounted by Saccharomyces cerevisiae strains are main limiting factors in bioethanol fermentation. Investigation of the molecular mechanisms underlying the stresses-related phenotypes diversities within and between S. cerevisiae populations could guide the construction of yeast strains with improved stresses tolerance and fermentation performances. Here, we explored the genetic characteristics of the bioethanol S. cerevisiae strain YJS329, and elucidated the genetic variations correlated with its advantaged traits (higher ethanol yield under sever conditions and better tolerance to multiple stresses compared to an S288c derived laboratory strain BYZ1). Firstly, pulse-field gel electrophoresis combined with array-comparative genomic hybridization was used to compare the genome structure of YJS329 and the laboratory strain BYZ1. Yeast cells were cultured in YPD medium. Genome DNA of YJS329 and BYZ1 was isolated and sonicated. The average length of DNA fragments was 200-1000bp. The shearing DNA was labeled with Cy5/Cy3 and hybridized to NimbleGen S.cerevisiae Whole-Genome Tiling arrays, which is single array design containing all chromosomes with 32bp median probe spacing and totally covered by ~385,000 probes. Scanning was performed with the Axon GenePix 4000B microarray scanner.

2011-09-14 | E-GEOD-31872 | biostudies-arrayexpress

Experimental relocation of the mitochondrial ATP9 gene to the nucleus reveals forces underlying mitochondrial genome evolution

Project description:Transcriptome profile of S. cerevisiae strains grown in rich ethanol/glycerol media, where the endogenous mitochondrial gene ATP9 has been deleted and relocated to the nucleus by expressing synthetic, codon-optimized versions of the Podospora anserina genes Atp9-5 and Atp9-7. The strains AMY7, AMY8, AMY10, and AMY11 each express one of these genes either from a centromeric or 2-micron plasmid. These strains are derived from RKY26, an atp9 deletion mutant which was not included in this experiment since it does not grow on respiratory media. The isogenic wild-type strain (MR6) was included as a control. Strains were produced for this study, and the arrays used were yeast tiling microarrays from Affymetrix. Note that normalization was performed using genomic DNA hybridizations, the raw data for which can be found in ArrayExpress Accession # E-TABM-1176.

2012-08-28 | E-MTAB-1115 | biostudies-arrayexpress

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