Widespread polycistronic gene expression in green algae.

Area of Science:

• Molecular Biology
• Genomics
• Eukaryotic Gene Expression

Background:

• Polycistronic gene expression, common in prokaryotes, was considered rare in eukaryotes.
• Long-read sequencing technologies like Iso-Seq enable comprehensive analysis of full-length transcript isomers.
• Previous studies suggested limited polycistronic transcription in eukaryotes, particularly in nuclear genes.

Purpose of the Study:

• To reexamine the prevalence of polycistronic gene expression in eukaryotes using advanced sequencing.
• To validate the discovery of hundreds of polycistronic nuclear gene loci in green algae.
• To explore the functional implications and evolutionary conservation of polycistronic expression in algae.

Main Methods:

• Iso-Seq (full-length transcript sequencing) to identify polycistronic transcripts.
• Chromatin immunoprecipitation (ChIP) to confirm transcription start sites.
• Analysis of polyadenylation signals and tails to confirm transcription end sites.
• Coexpression analysis, proteomics, and in vitro transcription/translation assays for validation.

Main Results:

• Hundreds of polycistronic nuclear gene loci were discovered in *Chlamydomonas reinhardtii* and *Chromochloris zofingiensis*.
• Multiple independent methods confirmed that distinct proteins are translated from single polycistronic transcripts.
• Polycistronic gene pairs are conserved across multiple green algal species, suggesting evolutionary importance.
• Synthetic biology applications demonstrated the utility of polycistronic loci for heterologous protein production.

Conclusions:

• Polycistronic gene expression is a significant and validated mechanism in the nuclear genomes of green algae.
• This finding necessitates a revision of the understanding of eukaryotic gene regulation.
• The conserved nature of polycistronic genes highlights their ancient biological importance in green algae and potential in synthetic biology.