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Leaky ribosomal scanning enables tunable translation of bicistronic ORFs in green algae
Saturday, 2025/03/08 | 07:13:34
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Marco A. Dueñas, Rory J. Craig, Sean D. Gallaher, and Sabeeha S. Merchant PNAS; Feb. 26 2025; 122 (9) e2417695122; https://doi.org/10.1073/pnas.2417695122 SignificanceTextbook dogma states that nucleus-encoded genes are monocistronic, producing transcripts with a single translated open reading frame (ORF). However, highly conserved bicistronic loci are pervasive in green algae that are separated by several hundred million years of evolution, speaking to their ancestral origins and functions within the Chlorophyte lineage. A combination of bioinformatic analysis and in vivo gene manipulation supports leaky ribosomal scanning as the primary mechanism for translation of more than one ORF from bicistronic transcripts. We have successfully tuned synthesis levels of two proteins encoded on one mRNA by modifying the ORF 1 Kozak-like sequence and limiting the number of AUG codons upstream of ORF 2. These findings may have broad applications in synthetic biology. AbstractAdvances 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.
See https://www.pnas.org/doi/10.1073/pnas.2417695122
Figure 1: Bicistronic loci in divergent green algae. (A) 36 bicistronic loci in C. reinhardtii are conserved in field isolates. Translation of both ORFs is supported by Ribo-Seq data. Orthologous bicistronic transcripts were identified in other Chlorophytes. High confidence hits and evidence of colinearity were further evaluated (Materials and Methods). Yellow denotes a colinear pair of ORFs with significant similarity to a pair of bicistronic ORFs from C. reinhardtii. Red and orange, respectively, indicate additional support with Iso-Seq or EST data. Columns are ordered by the phylogenetic tree above each panel. Species abbreviations (From leftmost to rightmost column) are as follows: Mpu, Micromonas. pusilla; Olu, Ostreococcus lucimarinus; Csu, Coccomyxa subellipsoidea; Czo, Chromochloris zofingiensis; Dsa, Dunaliella salina; Vca, Volvox carteri. (B) 43 bicistronic loci were identified in A. protothecoides. A similar search for orthologs was performed as described in panel A but restricted to the Trebouxiophyceae with C. reinhardtii as an outgroup. Species abbreviations (from leftmost to rightmost column) are as follows: Cre, Chlamydomonas reinhardtii; Csu, Coccomyxa subellipsoidea; Nde, Nannochloris desiccata; Cvu, Chlorella vulgaris; Cso, Chlorella sorokiniana; Pcu, Prototheca cutis. |
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