Leaky ribosomal scanning enables tunable translation of bicistronic ORFs in green algae

Dueñas, Marco A.; Craig, Rory J.; Gallaher, Sean D.; Moseley, Jeffrey L.; Merchant, Sabeeha S.

doi:10.1073/pnas.2417695122

Title: Leaky ribosomal scanning enables tunable translation of bicistronic ORFs in green algae

Journal Article · 25 February 2025 · Proceedings of the National Academy of Sciences of the United States of America

DOI: https://doi.org/10.1073/pnas.2417695122 · OSTI ID:2524092

^[1];

^[2]

University of California, Berkeley, CA (United States)
University of California, Berkeley, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Advances in sequencing technology have unveiled examples of nucleus-encoded polycistrons, once considered rare. Exclusively polycistronic transcripts are prevalent in green algae, although the mechanism by which multiple polypeptides are translated from a single transcript is unknown. Here, we used bioinformatic and in vivo mutational analyses to evaluate competing mechanistic models for translation of bicistronic mRNAs in green algae. High-confidence manually curated datasets of bicistronic loci from two divergent green algae, Chlamydomonas reinhardtii and Auxenochlorella protothecoides, revealed a preference for weak Kozak-like sequences for ORF 1 and an underrepresentation of potential initiation codons before the ORF 2 start codon, which are suitable conditions for leaky ribosome scanning to allow ORF 2 translation. We used mutational analysis in A. protothecoides to test the mechanism. In vivo manipulation of the ORF 1 Kozak-like sequence and start codon altered reporter expression at ORF 2, with a weaker Kozak-like sequence enhancing expression and a stronger one diminishing it. A synthetic bicistronic dual reporter demonstrated inversely adjustable activity of green fluorescent protein expressed from ORF 1 and luciferase from ORF 2, depending on the strength of the ORF 1 Kozak-like sequence. Our findings demonstrate that translation of multiple ORFs in green algal bicistronic transcripts is consistent with episodic leaky scanning of ORF 1 to allow translation at ORF 2. This work has implications for the potential functionality of upstream open reading frames (uORFs) found across eukaryotic genomes and for transgene expression in synthetic biology applications.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: National Institutes of Health (NIH); USDOE; USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science (BSS)

Grant/Contract Number:: AC02-05CH11231; SC0023027

OSTI ID:: 2524092

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 9 Vol. 122; ISSN 0027-8424

Publisher:: National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: English

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Title: Leaky ribosomal scanning enables tunable translation of bicistronic ORFs in green algae

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