How microalgae and cyanobacteria rewrite their RNA to survive stress

Deep inside every cell, a quiet molecular editor decides which parts of a gene’s message survive. That editor, known as pre-mRNA splicing, cuts out non-coding introns and stitches together coding exons, and when it chooses different combinations for different transcripts, the process is called alternative splicing. A new review published in Advanced Biotechnology pulls together what scientists know about this editing machinery in two of the planet’s most industrially promising photosynthetic organisms: microalgae and cyanobacteria. The verdict is striking. In these organisms, splicing is not a background housekeeping chore but a front-line stress response system, one that could hold the key to coaxing more biofuel, omega-3 fatty acids, astaxanthin and other valuable products out of cells that already grow faster than almost any other biological feedstock.

Alternative splicing comes in a handful of recognizable flavors. Exon skipping drops an exon located between two others out of the mature message. Mutually exclusive exons keep one exon or group of exons while splicing out a rival, producing similarly shaped proteins with slightly different sequences. Intron retention leaves one or more introns in place, alternative 5′ splice sites and alternative 3′ terminal exons extend or trim exons at their respective ends. The frequencies of these events vary dramatically across species, and that variation tells an evolutionary story. In animals, exon skipping dominates, a pattern that intensified along the bilaterian lineage and is tied to growth, differentiation and movement. In plants, intron retention rules, a characteristic linked to their stationary lifestyle and their need to adapt stress responses in place. The logic is simple: animals can move away from stressors, so they use splicing to diversify their tissues, while plants must stay and endure, so they use splicing to rewire their stress machinery.

The numbers from land plants show just how responsive this system is. In Arabidopsis, roughly 22 percent of genes, or 42 to 61 percent of intron-containing genes, undergo alternative splicing under normal conditions. After treatment with abscisic acid, a compound that mimics a stress signal, that figure can climb to 83.4 percent of intron-containing genes. Wheat edits 11,141 genes in response to salt stress. Tea plants activate an alternatively spliced terpene synthase as a defense against pathogens. Lilies accumulate a spliced variant of the heat stress transcription factor LlHSFA3B, which in turn activates a protein governing tolerance to both high temperature and salinity. Maize and Arabidopsis rely on spliced isoforms of circadian clock-associated genes for drought response. In tea, splicing even processes the structural genes and MYB transcription factors needed for flavonoid synthesis, the chemistry behind tea’s taste.

Microalgae mirror this plant-like pattern, and the model species Chlamydomonas reinhardtii illustrates it in remarkable detail. Its genome is intron-rich, with approximately 8.5 introns per gene and only about 8 percent of genes lacking introns entirely. Between 50 and 55 percent of its genes show alternative site selection, and intron retention is the most prevalent event type, making up roughly 29 to 50 percent of total alternative splicing. Frame-preserving and frame-disrupting splicing occur at similar frequencies, 1412 and 1555 events respectively, but frame-disrupting events show lower percent-spliced-in values. That discrepancy points to two possibilities: either nonsense-mediated decay is removing transcripts with premature termination codons, or frame-disrupting splicing is simply rarer. Nonsense-mediated decay may itself contribute to transcriptional regulation in Chlamydomonas, achieved when alternative splicing introduces an in-frame premature termination codon that triggers the decay pathway.

Splicing in Chlamydomonas fluctuates through every phase of the cell cycle. About 29 percent of events overlap across the light G1, S-M and dark D1 phases, but some are unique to a single phase: roughly 19 percent occur only in S-M, about 17 percent exclusively in dark G1, and nearly 10 percent only in light G1. Most genes show no significant change in expression as they progress through the cycle, indicating that splicing events are regulated separately from transcription. Intron retention in Chlamydomonas can introduce premature termination codons that disrupt RNA recognition motif domains, directly influencing gene expression. Cold stress tells a similar story. Low temperatures induce three splicing factors, including two from the U1 snRNP family, while other factors such as SC35-like protein and H/ACA snoRNP are lost, suggesting the spliceosome machinery is physically rebuilt to adapt. Splicing events also precede structural ribosomal changes in the cytoplasm, mitochondria and plastid, meaning cold reshapes the proteome through translation and possible post-translational processes.

Nitrogen deprivation activates an even broader splicing program. Under these conditions, 3500 genes undergo alternative splicing, totaling 5806 events, heavily concentrated in spliceosome genes and transporter pathways. The events appear to dampen the citrate cycle and fatty acid degradation to help regulate triacylglycerol accumulation, modify membrane lipids through peroxisome-related genes, and induce amino acid and peptide metabolism for protein degradation. Intron-mediated enhancement adds another regulatory layer: the second intron of the RuBisCo small subunit gene boosts nuclear transgene expression 5.5-fold, and exogenous introns with splice sites resembling native ones achieve high enhancement, while foreign introns without recognizable boundaries actually reduce expression. A TFIIS-like protein, conserved from Arabidopsis where it is essential for heat stress adaptation and alternative splicing, may play a similar regulatory role in Chlamydomonas.

Dunaliella salina, a wall-less halotolerant green alga prized for beta-carotene production, takes a different genomic approach. Its genome is 343.7 megabase pairs, roughly three times larger than Chlamydomonas, with 53 percent introns compared to 30 percent. Its mitochondrial DNA carries about 1.5 introns per gene and its plastid DNA about 0.4, with 18 group I introns in the former and 43 introns in the latter, 35 of them group I and eight group II. Under hypersaline stress, the species increases spliceosome processing to manage genes critical for salt response. During palmella formation, a vegetative state induced by salinity changes, RNA processing proteins decline while G-patch domain-containing proteins and DEAD-box helicases shift in abundance, and phosphorylation of the RNA polymerase subunit RPB8 changes in ways that would affect polymerase assembly. The MEK1 gene produces two splice variants: the full-length DsMEK1-X2, preferred under salt stress, drives glycerol accumulation for osmotic regulation, while the truncated DsMEK1-X1, preferred under oxidative stress, features only a partial kinase domain and interacts weakly with partners. Four transcripts encoding the 19-kilodalton subunit of respiratory complex I have also been isolated, though their functions remain unreported.

Haematococcus pluvialis, famous for synthesizing astaxanthin, an antioxidant 10 times more potent than beta-carotene and 550 times more potent than vitamin E, shows intron retention as its dominant splicing mode across a 669-megabase draft genome containing 18,545 genes. Its delta-12 fatty acid desaturase gene HpFAD2, with a 1137-base-pair open reading frame coding for 378 amino acids and nine introns, increases transcription under light stress, possibly implying higher splicing rates. Beyond these models, sequencing advances have revealed 3137 alternative splicing events in the bloom-forming dinoflagellate Akashiwo sanguinea, 2030 new loci in the brown alga Ectocarpus, and significant splicing increases when Auxenochlorella protothecoides shifts from autotrophic to heterotrophic growth, with 705 differentially expressed genes and 660 differentially abundant sequences identified. In Haematococcus lacustris, splicing processes the channelrhodopsin gene into a protein unable to interact with light, while a fragment of the Bracteacoccus aggregatus gene yields a functional light-responsive protein, suggesting splicing may be crucial for light-regulated cellular functions across non-model algae.

Cyanobacteria, photosynthetic prokaryotes, play an entirely different splicing game based on self-splicing catalytic RNAs and proteins. Group I introns, conserved catalytic RNAs with paired segments P1 through P10, splice themselves out. One such intron sits in the ribonucleotide reductase gene of Nostoc punctiforme PCC73102, where splicing is essential to assemble the functional enzyme that catalyzes deoxyribonucleotide production for DNA replication. The Anabaena PCC7120 pre-tRNA-Leu intron becomes a ribozyme after self-splicing, and its shorter 3-base-pair internal guide sequence, compared to the 6-base-pair version in the well-studied Tetrahymena ribozyme, holds substrates more loosely, slowing single-turnover cleavage but enabling more ready splicing under multiple-turnover conditions. Group II introns, regarded as evolutionary ancestors of the eukaryotic spliceosome’s components, excise themselves as lariats with 2′-5′ phosphodiester bonds, and twintrons, introns within introns, require the inner intron to remove itself before the host can be properly spliced. Inteins, mobile protein elements that splice themselves out of flanking exteins, follow a tightly coordinated four-step mechanism: thioester formation at the N-terminal junction, branched intermediate creation, asparagine cyclization and intein release, then a spontaneous acyl shift converting the ester bond between exteins into an amide bond. Split inteins, which reassociate from separate polypeptide halves with high affinity, serve as powerful tools for protein engineering, though divalent copper ions can block their trans-splicing by oxidizing terminal cysteine residues, a block reversible only through chelation and reduction.

The practical implications are considerable. Microalgae can be cultivated at scale in photobioreactors, now increasingly designed with 3D printing flexibility to cater to specific species’ growth requirements, and stress conditions enhance the accumulation of lipids, carotenoids and other prized products. Understanding how alternative splicing mediates stress tolerance could therefore translate directly into productivity gains. CRISPR-Cas9 editing is already delivering results: knocking out the KJFTSY gene in the industrial alga Coccomyxa sp. strain KJ, which can synthesize triacylglycerols comprising more than 60 percent of dry weight under nitrogen deficiency, and editing transcription factors in Nannochloropsis gaditana to produce mutants with twice the lipid content of wild type without slowing growth. Detection methods are advancing too, with short-read and long-read RNA sequencing, percent-spliced-in calculations using tools like rMATS and SUPPA2, and specialized detectors such as IRFinder for intron retention and Whippet for complex events. A ΔPSI greater than 0.1 is typically regarded as a strict criterion for confirming significant splicing events. As sequencing and gene editing mature in tandem with bioreactor design, the molecular editing systems of microalgae and the self-splicing machinery of cyanobacteria may become levers for engineering the photosynthetic cell factories of the future.

Subject of Research: Pre-mRNA splicing regulation in microalgae and cyanobacteria

Article Title: Pre-mRNA splicing and its regulation in microalgae and cyanobacteria

Article References: Pre-mRNA splicing and its regulation in microalgae and cyanobacteria. (n.d.). https://doi.org/10.1007/s44307-025-00087-3

Image Credits: AI Generated

DOI: 10.1007/s44307-025-00087-3

Keywords: alternative splicing, microalgae, cyanobacteria, intron retention, Chlamydomonas reinhardtii, Dunaliella salina, Haematococcus pluvialis, group I introns, group II introns, inteins, spliceosome, stress response