Biology subjects

Stark, M. R.

Publications and source records attributed to Stark, M. R..

4 recordsLinked to original sources

bioRxiv2026

Dramatically reduced spliceosome, intronome, and splicing efficiency in Cyanidiococcus yangmingshanensis and Cyanidium caldarium

Eukaryotic pre-mRNA splicing is catalyzed by the spliceosome, whose ribonucleoprotein composition and the number of intron substrates it acts upon vary widely across eukaryotic lineages. The red alga Cyanidioschyzon merolae possesses a reduced spliceosome lacking the U1 snRNP, and an unusually small intron repertoire. We asked whether these traits are unique to C. merolae or shared across the related Cyanidiales and Cyanidioschyzonales lineages, as well as how they relate to splicing efficiency under light conditions relevant to photosynthetic growth. Genomic and transcriptomic analysis of C. merolae, Cyanidiococcus yangmingshanensis, and Cyanidium caldarium reveal that all three species harbour a reduced, but broadly conserved, set of splicing proteins. Strikingly, covariance model searches failed to detect U1 snRNA in either C. yangmingshanensis or C. caldarium, establishing U1 loss as a shared feature of all three lineages. We identified only 39 introns in C. merolae, 40 in C. yangmingshanensis, and 54 in C. caldarium. Splicing efficiencies were 42-50%, substantially lower than most organisms in which splicing has been measured, but low splicing is compensated by 2-4x higher expression of intron-containing genes than intron-lacking genes. Notably, light can enhance splicing efficiency in C. merolae and C. yangmingshanensis by up to 100%. Furthermore, the splice site and branch site consensus sequences are highly conserved and similar to those found in hemiascomycetous yeasts such as Saccharomyces cerevisiae. 85% of introns contain an in-frame stop codon with a strong bias towards the 5' end of the intron. These results indicate that dramatic streamlining of the spliceosome and intronome, together with inefficient splicing, predated the divergence of these lineages [~]320 million years ago, and is therefore a defining molecular trait of these extremophilic red algae.

molecular biology↗

bioRxiv2026

Epigenetic regulation of a heat-trainable sHSP locus controls thermomemory in a unicellular alga

A powerful way to enhance heat tolerance is to prime organisms with a moderate heat treatment to establish a molecular stress memory permitting the survival of the organism when exposed to subsequent heat shocks. While this has been extensively studied in multicellular organisms, we demonstrate that the unicellular red alga Cyanidioschyzon merolae exhibits heat stress memory. We show that, similarly to more complex organisms, thermomemory in this alga is underpinned by transcriptomic reprogramming, with the chloroplast emerging as the main site of gene trainability. Additionally, we find a conserved small heat shock protein (sHSP)-encoding locus in the nuclear genome to be heat-trainable, likely by histone depletion and sustained removal of the repressive mark histone H3 Lysine 27 trimethylation (H3K27me3). Of C. merolaes two sHSPs, only the nuclear-localizing CmsHSP2 is necessary for proper HS memory establishment. Finally, we reveal a role for the H3K27me3-transferase CmE(z) (Enhancer of zeste) in heat stress memory which shapes the transcriptome to recurring heat exposures, beyond regulating the trainable sHSP locus. Overall, our work provides a molecular framework for the regulation of heat stress memory in a unicellular eukaryote.

genetics↗

bioRxiv2026

Genome Position Does Not Impact Transgene Expression Efficiency in the Ancient Red Alga Cyanidioschyzon merolae

The thermoacidophilic red alga Cyanidioschyzon merolae represents one of the simplest photosynthetic eukaryotes and an ancient divergent group in the primary endosymbiotic Viridiplantae. Because of its [~]16 Mbp genome, containing few introns, and capacity for transgene integration by homologous recombination, it is an emerging chassis for synthetic biology. However, genomic integration sites and scalable transformation methods have not been established to systematically investigate the effect of genome position on transgene expression. Here, we combined bioinformatic genome analysis, liquid-handling robotics, and assays of heterologous protein and metabolite production to establish a reproducible framework for nuclear genome engineering in C. merolae. We mapped and annotated 40 intergenic loci as candidate neutral sites across 16 out of 20 chromosomes and could validate 38 of them through robotic-assisted transformation. Reporter gene expression analysis revealed highly uniform expression at all integration sites across broad populations of transformants, indicating surprising minimal positional effects and transcriptional neutrality. The functional equivalence of these genomic landing pads was determined by expression of a heterologous isoprene synthase, and coupling algal photobioreactors to headspace analysis to quantify isoprene production driven by transgene expression from different integration sites. Single copy transgene integrants, regardless of genome position, exhibited comparable reporter signals and consequent isoprene production. Together, these results provide the first experimentally validated set of neutral integration sites in C. merolae and establish a high-throughput transformation protocol for its genetic engineering in the context of synthetic genome biology.

synthetic biology↗

bioRxiv2024

A rapid CAT transformation protocol and nuclear transgene expression tools for metabolic engineering in Cyanidioschyzon merolae 10D

The eukaryotic red alga Cyanidioschyzon merolae 10D is an emerging algal host for synthetic biology and metabolic engineering. Its small nuclear genome (16.5 Mb; 4775 genes), low intron content (38), stable transgene expression, and capacity for homologous recombination into its nuclear genome make it ideal for genetic and metabolic engineering endeavors. Here, we present an optimized transformation and selection protocol, which yields single chloramphenicol-resistant transformants in under two weeks. Transformation dynamics and a synthetic modular plasmid toolkit are reported, including several new fluorescent reporters. Techniques for fluorescence reporter imaging and analysis at different scales are presented to facilitate high-throughput screening of C. merolae transformants. We use this plasmid toolkit to overexpress the Ipomoea batatas isoprene synthase and demonstrate the dynamics of engineered volatile isoprene production during different light regimes using multi-port headspace analysis coupled to parallel photobioreactors. This work seeks to promote C. merolae as an algal system for metabolic engineering and future sustainable biotechnological production.

bioengineering↗