— A new rulebook for eukaryotic gene expression revealed in PNAS —

A joint research team from RIKEN, Yamagata University, the University of Tokyo, Euglena Co., Ltd., Kochi University, Tsuruoka College, and collaborators has uncovered that Euglena employs a widespread, nonconventional intron system alongside the textbook GT–AG rule of eukaryotic RNA splicing. Through genome-wide analysis and precise genome editing assays, the team decoded the DNA sequence signature that enables this unusual form of splicing—and showed it can be programmed with fully synthetic introns.

Why it matters

In most eukaryotes, introns begin with GT and end with AG—a basic rule used in biology textbooks and gene-finding software. The team shows that in Euglena, ~72% of introns break this rule, yet are accurately removed during mRNA processing by following a different sequence logic. This discovery expands our understanding of RNA processing diversity and opens new possibilities for programmable control of gene expression in microalgae used for health foods, biofuels, and carbon capture.

What the team found

• Genome-scale landscape: In Euglena agilis, 71.8% of ~650,000 introns are nonconventional (do not follow GT–AG). Many genes carry both conventional (CV) and nonconventional (NC) introns, implying dual splicing systems act co-dominantly.
• Sequence signature (the “code”): Successful NC splicing requires specific CAG (near the 5′ side) and CTG (near the 3′ side) motifs, precise spacer lengths (3 nt before CAG; 5–6 nt after CTG), and a purine (A/G) as the first base of the downstream exon. Complementary bases around these motifs form an RNA hairpin that brings splice sites together. The code can be summarized as 5′-N₃CDG-/-CH′G N₅–₆|R(exon)-3′.
• Genetic proof using Euglena gracilis: By knocking nonconventional and synthetic 50-bp introns into the EgGSL2 gene (which controls paramylon granule formation), the team showed that mutating CAG/CTG or altering spacer length disrupts splicing and function, whereas code-compliant synthetic introns splice cleanly—proving the code works.
• Design variants: Swapped or derivative motifs (e.g., CGG–CCG, CGG–CTG) can also support splicing when they respect the complementary pattern, refining the allowable motif space.

How they did it

• De novo whole-genome and transcriptome assembly for E. agilis to map exon–intron boundaries at scale.
• CRISPR–Cas9 ssODN knock-in in E. gracilis to insert natural or synthetic introns into EgGSL2, with paramylon granules and RT-PCR/Sanger sequencing used as direct readouts of splicing success.

Potential applications

• Programmable splicing in Euglena: engineer exon skipping or inclusion to tailor protein functions, metabolic pathways, and productivity.
• Strain improvement for bio-manufacturing: boost useful metabolite production, and enhance traits linked to CO₂ fixation and biomass productivity.
• Evolutionary insights: reveals a coexisting dual splicing system in a eukaryote and a plausible role of inverted-repeat sequences and circular RNA formation in NC intron removal.

Reference

Nomura T., Kim J-S., Iwata O., Yamada K., Atsuji K., Uehara-Yamaguchi Y., Yoshida T., Inoue K., Takahagi K., Sakurai T., Shinozaki K., Ito T., Suzuki K., Goda K., Mochida K. “Genetic dissection of nonconventional introns reveals codominant noncanonical splicing code in Euglena.” Proceedings of the National Academy of Sciences (PNAS), 122(39): e2509937122. Published September 23, 2025.

Acknowledgements & funding

The project involved RIKEN CSRS (Mochida Lab), Yamagata University, the University of Tokyo (Goda Lab), Euglena Co., Ltd. (including Executive Fellow Kengo Suzuki), Kochi University, and Tsuruoka College, among others, with support from Japanese national programs including JST OPERA, GteX, SATREPS, and JSPS KAKENHI.