Title: Widespread polycistronic gene expression in green algae

Abstract

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. Replacementmore » 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.« less

Authors:

Gallaher, Sean D.  ^[1];

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• Search DOE PAGES for ORCID "0000-0002-9773-6051"

Craig, Rory J.  ^[2];

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• Search DOE PAGES for ORCID "0000-0002-6262-0008"

Ganesan, Iniyan  ^[3];

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• Search DOE PAGES for ORCID "0000-0002-1082-202X"

Purvine, Samuel O.  ^[4];

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• Search DOE PAGES for ORCID "0000-0002-2257-2400"

McCorkle, Sean R.  ^[5];

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• Search DOE PAGES for ORCID "0000-0003-1126-4071"

Grimwood, Jane  ^[6];

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• Search DOE PAGES for ORCID "0000-0002-8356-8325"

Strenkert, Daniela  ^[7];

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• Search DOE PAGES for ORCID "0000-0002-3420-1332"

Davidi, Lital  ^[7];

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Roth, Melissa S.  ^[8];

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Jeffers, Tim L.  ^[8];

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Lipton, Mary S. ^[4]; Niyogi, Krishna K.  ^[9];

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• Search DOE PAGES for ORCID "0000-0001-7229-2071"

Schmutz, Jeremy  ^[10];

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Theg, Steven M.  ^[3];

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Blaby-Haas, Crysten E.  ^[5];

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• Search DOE PAGES for ORCID "0000-0002-1583-1291"

Merchant, Sabeeha S.  ^[11]

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• Search DOE PAGES for ORCID "0000-0002-2594-509X"

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+ Show Author Affiliations

1. UCLA DOE Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095,, Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095,
2. Joint Genome Institute, United States Department of Energy, Berkeley, CA 94720,, Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FL, United Kingdom,
3. Department of Plant Biology, University of California, Davis, CA 95616,
4. Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99354,
5. Biology Department, Brookhaven National Laboratory, Upton, NY 11973,
6. HudsonAlpha Genome Sequencing Center, HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806,
7. Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095,
8. Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720,
9. Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720,, Division of Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA 94720,, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720-3102,
10. Joint Genome Institute, United States Department of Energy, Berkeley, CA 94720,, HudsonAlpha Genome Sequencing Center, HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806,
11. UCLA DOE Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095,, Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095,, Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720,, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720,, Division of Environmental Genomics and Systems Biology, Lawrence Berkeley National Laboratory, Berkeley, CA 94720

Publication Date:

Fri Feb 12 00:00:00 EST 2021

Research Org.:

Brookhaven National Laboratory (BNL), Upton, NY (United States); Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Laboratory (EMSL); USDOE Joint Genome Institute (JGI), Berkeley, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER); European Molecular Biology Organization

OSTI Identifier:

1779559

Alternate Identifier(s):

OSTI ID: 1756294; OSTI ID: 1768889

Report Number(s):

BNL-220781-2021-JAAM; PNNL-30295
Journal ID: ISSN 0027-8424; e2017714118

Grant/Contract Number:  

SC0018301; FC02-02ER63421; AC02-05CH11231; AC05-76RL01830; SC0017035; SC0012704

Resource Type:

Published Article

Journal Name:

Proceedings of the National Academy of Sciences of the United States of America

Additional Journal Information:

Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 118 Journal Issue: 7; Journal ID: ISSN 0027-8424

Publisher:

Proceedings of the National Academy of Sciences

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; transcriptome; bicistronic; dicistronic; leaky ribosome scanning; uORFs; LYR-motif; respiratory complex