PNNL

Abstract

Polycistronic gene expression, while common in prokaryotes, was thought to be extremely rare in eukaryotes. Recently, the development of long-read sequencing of whole transcripts (Iso-Seq) has facilitated a re-examination of that dogma. Using Iso38 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 multiple, independent approaches to validate that multiple genes are translated from a common transcript for hundreds of loci. A chromatin immunoprecipitation analysis confirmed that transcription begins exclusively at the upstream gene. Quantification of poly(A) tails and poly(A) signal sequences confirmed that transcription ends exclusively at the downstream gene. A co-expression analysis found nearly perfect correlation for ORFs within polycistronic loci, consistent with expression in a shared transcript. For many polycistronic loci, proteomics datasets allowed identification of terminal peptides from both ORFs, 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 allowed 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.