期刊
出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2017714118
关键词
transcriptome; bicistronic; dicistronic; leaky ribosome scanning; uORFs
资金
- US Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research Awards [DE-SC0018301, DE-FC02-02ER63421]
- Office of Science of the US DOE [DE-AC02-05CH11231]
- US DOE Office of Science [DE-AC05-76RL01830]
- Office of Basic Energy Sciences of the US DOE [DE-SC0017035]
- European Molecular Biology Organization Fellowship [ALTF 653-2013]
- U.S. Department of Energy (DOE) [DE-SC0018301, DE-SC0017035] Funding Source: U.S. Department of Energy (DOE)
The common belief that polycistronic gene expression is rare in eukaryotes has been challenged by the discovery of hundreds of examples in green algae using the Iso-Seq technology. Various approaches confirmed that multiple proteins are translated from a shared transcript at numerous loci, with transcription starting exclusively at upstream genes and ending after downstream genes. The conservation of this mechanism in different green algae species suggests it may be evolutionarily ancient and biologically significant.
Polycistronic gene expression, common in prokaryotes, was thought to be extremely rare in eukaryotes. The development of long-read sequencing of full-length transcript isomers (Iso-Seq) has facilitated a reexamination of that dogma. Using Iso-Seq, we discovered hundreds of examples of polycistronic expression of nuclear genes in two divergent species of green algae: Chlamydomonas reinhardtii and Chromochloris zofingiensis. Here, we employ a range of independent approaches to validate that multiple proteins are translated from a common transcript for hundreds of loci. A chromatin immunoprecipitation analysis using trimethylation of lysine 4 on histone H3 marks confirmed that transcription begins exclusively at the upstream gene. Quantification of polyadenylated [poly(A)] tails and poly(A) signal sequences confirmed that transcription ends exclusively after the downstream gene. Coexpression analysis found nearly perfect correlation for open reading frames (ORFs) within polycistronic loci, consistent with expression in a shared transcript. For many polycistronic loci, terminal peptides from both ORFs were identified from proteomics datasets, consistent with independent translation. Synthetic polycistronic gene pairs were transcribed and translated in vitro to recapitulate the production of two distinct proteins from a common transcript. The relative abundance of these two proteins can be modified by altering the Kozak-like sequence of the upstream gene. Replacement of the ORFs with selectable markers or reporters allows production of such heterologous proteins, speaking to utility in synthetic biology approaches. Conservation of a significant number of polycistronic gene pairs between C. reinhardtii, C. zofingiensis, and five other species suggests that this mechanism may be evolutionarily ancient and biologically important in the green algal lineage.
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