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CellSynthetic BiologyPeer-reviewed journal article

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

Daniel SchindleriD, Roy S. K. WalkeriD, Shuangying JiangiD, Aaron N. BrooksiD, Yun WangiD, Carolin A. MülleriD, Charlotte A. CockramiD, Yisha LuoiD, Alicia GarcíaiD, Daniel SchraivogeliD, Julien MozziconacciiD, Noah PeñaiD, Mahdi AssariiD, María del Carmen Sánchez OlmosiD, Yu ZhaoiD, Alba Ballerini, Benjamin A. BlountiD, Jitong Cai, Lois OgunlanaiD, Wei LiuiD, Katarina Jönsson, Dariusz AbramczykiD, Eva Garcia‐RuiziD, Tomasz W. TurowskiiD, Reem SwidahiD, , Yizhi CaiiD

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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. HighlightsO_LIDe novo design, construction and functional characterization of a neochromosome containing all 275 nuclear tRNA genes of Saccharomyces cerevisiae. C_LIO_LIIncreasing the copy number of the 275 highly expressed tRNA genes causes cellular burden, which the host cell likely buffers either by selecting for partial tRNA neochromosome deletions or by increasing its ploidy. C_LIO_LIThe tRNA neochromosome can be chemically extracted and transformed into new strain backgrounds, enabling its transplantation into multi-synthetic chromosome strains to finalize the Sc2.0 strain. C_LIO_LIComprehensive functional characterization does not pinpoint a singular cause for the cellular burden caused by the tRNA neochromosome, but does reveal novel insights into its tRNA and structural chromosome biology. C_LI

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (3 Oct 2022) · CC BY 4.0

A de novo yeast neochromosome carrying all 275 tRNA genes was built and characterized; it imposes a growth burden, with genome doubling and deletions in haploids possibly reflecting compensation.

Sc2.0 design calls for moving all tRNA genes, which are genomic instability hotspots, onto one dedicated chromosome, but no native template exists for it. This work supplies a de novo design, build and functional characterization that tests whether the added tRNA gene dosage is tolerated.

Key results

  1. ShownA circular tRNA neochromosome carrying all 275 intron-free tRNA genes in largely orthogonal flanking sequence was assembled by recursive in vivo recombination (eSwAP-In), then repaired to match the design after complex structural variants appeared.

    16 tRNA arrays in 8 mega-arrays; ~89% of sequence from non-S. cerevisiae species; 15 rounds of integration. First-pass strain YCy1576 had an inversion, duplication and deletion, repaired via I-SceI to NGS-validated YCy2508; eleven minor nucleotide variations found. · Design of the tRNA Neochromosome; Neochromosome construction; Neochromosome linearization and repair of…; Figure legends

  2. SuggestedDiploid strains carrying the neochromosome grow more slowly, and the defect vanishes when the neochromosome is lost. Heterozygous RPC40 deletion gave an apparent growth recovery, RPC31 a slight one; MAF1 variants and most other subunit deletions had no significant effect.

    Doubling time 110.0 +/- 2.6 min vs 92.3 +/- 2.7 min (19.2% increase); loss isolate YCy2649 reverted. Defect worst on 6-azauracil and hydroxyurea among 22 conditions; hydroxyurea defect also seen in the MATa/a diploid control without neochromosome. · Phenotypic characterization of the tRNA…; Figure legends

  3. SuggestedHost cells spontaneously doubled from haploid to homozygous MATa/a diploid, and the neochromosome was more stable in diploids than in haploids, consistent with ploidy increase as a compensatory response to tRNA gene burden.

    After about 50 generations, internal deletions were found in two out of eight diploid isolates vs 7 out of 8 haploid isolates (BY4741 YCy2837, BY4742 YCy2838); flow cytometry and mating tests confirmed the DNA content doubling. · Spontaneous doubling in yeast genome ploidy and…; tRNA neochromosome stability assay

  4. ShownNeochromosome tRNA genes are transcribed. Aggregate pre-tRNA levels by amino acid or isoacceptor resemble the native genome, although individual tDNA expression correlates less well, and no global tRNA processing defect was evident.

    tRNAseq (three biological replicates, four strains) assigned genomic origin for 255 of 275 tRNAs; no systematic effect of intron removal; no pre-tRNA accumulation; tRNASer-CGA was among the isoacceptors not detected after normalization. · tRNA sequencing to characterize tRNA…; Figure legends

  5. ShownCandida glabrata origins fired and designed termination sites showed coverage troughs. The neochromosome lay away from the nucleolus, and Hi-C showed no structural rearrangement.

    G1 abundance about 0.8 relative to other chromosomes, suggesting copy-number heterogeneity or partial loss; FISH showed approximately half of cells with clear separation from rDNA; Hi-C maps (YCy2508, YCy2670-YCy2672) were smooth. · Deep-sequencing to determine the tRNA…; Intranuclear localization of the tRNA…

Methods, data and reproducibility

Hierarchical de novo design (cassettes, arrays, mega-arrays) with custom Python flanking-sequence assignment, assembled by recursive in vivo recombination (eSwAP-In), then profiled by tRNAseq, RNA-seq, TMT proteomics, MNase nucleosome mapping, sort-seq replication profiling, FISH and Hi-C.

  • Designed and sequenced tRNA neochromosome (275 tRNA cassettes) in BY4741 and synIII/VI/IXR backgrounds; final strain YCy2508 · Saccharomyces cerevisiae · n = 275 tRNA cassettes in 16 tRNA arrays · PRJNA351844
  • WGS, tRNAseq, RNA-seq, nucleosome positioning and Hi-C data from neochromosome strains · Saccharomyces cerevisiae · n = tRNAseq: 4 strains, 3 biological replicates each · PRJNA884518
  • TMT pro 16plex proteomics of circular and linear neochromosome strains versus haploid and diploid controls · Saccharomyces cerevisiae · n = 3 biological replicates
  • Linearized neochromosome variants (YCy2670 to YCy2678) phenotyped under 22 solid media conditions · Saccharomyces cerevisiae · n = one circular and nine linear derivatives
  • Haploid vs diploid neochromosome stability time-course (about 50 generations) with colony PCR of isolates · Saccharomyces cerevisiae · n = eight isolates per ploidy (four per strain, two strains each)
  • FISH and live-cell imaging of neochromosome position relative to rDNA and nucleolus · Saccharomyces cerevisiae · n = FISH: >100 cells per derivative; live imaging: >250 cells per clone
Methods & tools
eSwAP-In, Custom BioPython scripts for flanking sequence assignment, tRNAscan-SE v.2.0, Bowtie2 v2.3.5.1, GATK v2.7, hisat2 v2.1.0, featureCounts v2.0.1, DESeq2 v1.30.1, Salmon v1.2.1, Sleuth v0.30.0, NUCwave, Mascot and IQuant
Software & environment
Bowtie2 v2.3.5.1, GATK v2.7, SAMtools v0.1.19, hisat2 v2.1.0, featureCounts v2.0.1, DESeq2 v1.30.1, Salmon v1.2.1, Sleuth v0.30.0, tRNAscan-SE v.2.0, cutadapt v1.15, FastQC v0.10.1.
Reproducibility gaps
Proteome data (PRIDE) have no accession printed; some results are marked 'data not shown'; further reanalysis information available from the lead contact on request.
Limitations (5)
  • No single cause of the cellular burden was identified, suggesting a multifactorial origin; the final test is pending introduction into the full Sc2.0 strain without native tRNA copies. · stated by the authors
  • Twenty tRNA genes were not detected by tRNAseq, so processing defects (for example for tRNASer-CGA) cannot be excluded for them. · stated by the authors
  • The design was inherently arbitrary given then-current knowledge; tRNA introns were removed although some may have a physiological role, so future variants may restore them. · stated by the authors
  • The stability comparison rests on eight isolates per background, with no statistical test reported; ploidy doubling as a causal compensation mechanism was inferred, not directly tested. · noted in this summary
  • Hydroxyurea sensitivity also appeared in the MATa/a control lacking the neochromosome, so host genome duplication is confounded with neochromosome burden for that phenotype. · noted in this summary

Read this if you work on synthetic genomics, Sc2.0 or tRNA biology and want to see how a designer chromosome was built and what its cellular costs were.

Citations
81
FWCI
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top 1%

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