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Selection for transcript diversity in the extremophile Galdieria sulphuraria results in retention of the splicing machinery

ABSTRACT: Purpose: Red algae show two putative phases of massive genome reduction. RNA-splicing represents an important form of post-transcriptional regulation in eukaryotes. This process generates increased transcriptome and proteome diversity via alternative splicing that can maximize the DNA-encoded genetic diversity embedded within genomes. Why would Spliceosomal Machinery genes and a relatively more complex intron-exon structure be preserved in the compact Galdieria genome? The answer may lie in the fact that RNA-splicing represents an important form of post-transcriptional regulation in eukaryotes. It is hypothesized that his process generates increased transcriptome and proteome diversity via alternative splicing that can maximize the DNA-encoded genetic diversity embedded within genomes. Methods: The experimental design followed a temperature shift timeline: after two weeks of cultivation at 42°C, constant illumination (90 μE) and constant shaking (160 rpm) in photoautotrophic conditions the first sampling took place (H-42) and the cultures of G.sulphuraria were swiftly moved to 28°C. After cold treatment at 28°C for 48 hours, a second sampling was performed (C-28.1). The G. sulphuraria was then switched to 46°C for 48 hours, at end of which a third sampling was retrieved (H-46). It again was followed by a cold treatment at 28°C for 48 hours when a fourth sampling was retrieved (C-28.2). Altogether, two 48h timepoints from high temperatures (42°C and 46oC) and two 48h timepoints from cold temperature (28°C) were targeted for sampling. Results: We show that RNA-splicing provides an avenue for generating transcriptome diversity under strong constraints on genome size and gene number. Conclusions: We report the relatively high retention of Spliceosomal Machinery genes and the enrichment of intron-exon structures in G. sulphuraria that might be associated with the exceptional transcriptome and proteome diversity of this species. These results suggest that the losses of genes and functions caused by genome reduction might be mitigated by sequence diversity generated by a robust RNA-splicing system. More generally, the strong constraints imposed by genome reduction appear to be ameliorated by processes that operate on the transcriptomic and proteomic levels.

ORGANISM(S): Galdieria sulphuraria

PROVIDER: GSE89169 | GEO | 2017/03/01

SECONDARY ACCESSION(S): PRJNA350520

REPOSITORIES: GEO

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Project description:Purpose: We aimed at i) obtaining insight into how a thermophile organism reacts to cold stress, and ii) evaluating the impact of HGT candidates on the acclimation process to temperature decrease. Methods: The experimental design followed a temperature shift timeline: after two weeks of cultivation at 42°C, constant illumination (90 μE) and constant shaking (160 rpm) in photoautotrophic conditions the first sampling took place (Hot_T48_1) and the cultures of G.sulphuraria were swiftly moved to 28°C ( = cold temperature). "Cold" samples were taken after 3h (Cold_T3_2), 12h (Cold_T12_3) and 48h (Cold_T48_4). After cold treatment at 28°C for 48 hours, the G. sulphuraria was then switched to 46°C for 48 hours (="Hot"). Again, samples were taken after 3h (Hot_T3_5), 12h (Hot_T12_6) and 48h (Hot_T48_7). Altogether, a 48h temperature timeshift at 28°C and successive recovery at 46°C were targeted for sampling. Results: Galdieria sulphuraria is a unicellular red alga that lives in hot, acidic, toxic metal-rich, volcanic environments, where few other organisms survive. Its genome harbours up to 5% of genes most likely acquired through horizontal gene transfer. These genes probably contributed to G. sulphuraria’s adaptation to its extreme habitats, resulting in today’s polyextremophilic traits. Here, we applied RNA-sequencing to obtain insights into the acclimation of a thermophilic organism towards temperatures below its growth optimum and to study how horizontally acquired genes contribute to cold acclimation. A decrease in growth temperature from 42 °C/46 °C to 28 °C resulted in an upregulation of ribosome biosynthesis, while excreted proteins, probably components of the cell wall, were downregulated. Photosynthesis was suppressed at cold temperatures, and transcript abundances indicated that C-metabolism switched from gluconeogenesis to glycogen degradation. Folate cycle and S-adenosylmethionine cycle (one-carbon metabolism) were transcriptionally upregulated, probably to drive the biosynthesis of betaine. All these cold-induced changes in gene expression were reversible upon temperature increase. Numerous genes acquired by horizontal gene transfer displayed pronounced temperature-dependent expression changes, corroborating the view that these genes contributed to adaptive evolution in G. sulphuraria.

Project description:Alternative splicing is prevalent in plants, but little is known about its regulation in the context of developmental and signaling pathways. We describe here a new factor that influences pre-mRNA splicing and is essential for embryonic development in Arabidopsis thaliana. This factor was retrieved in a genetic screen that identified mutants impaired in expression of an alternatively spliced GFP reporter gene. In addition to the known spliceosomal component PRP8, the screen retrieved a previously uncharacterized protein containing a Replication termination factor2 (Rtf2) domain defined by a C2HC2 zinc finger. The Rtf2 protein was discovered in fission yeast, where it stabilizes paused DNA replication forks by an unknown mechanism. When homozygous, a null mutation in Arabidopsis RTF2 (AtRTF2) is embryo-lethal, indicating that it encodes an essential protein. As revealed by quantitative RT-PCR, impaired expression of GFP in atrtf2 and prp8 mutants is attributable to inefficient splicing of the GFP pre-mRNA. A genome-wide analysis using RNA-seq demonstrated that 12% of total introns display a significant degree of retention in atrtf2 mutants. Intron-retaining transcripts are enriched from genes encoding proteins involved in signaling pathways and membrane transport. Affinity purification of AtRTF2 followed by mass spectrometry identified several known and predicted splicing proteins. In a yeast two-hybrid screen, AtRTF2 interacted with Exo70B1, a peripheral subunit of the exocyst, which is involved in vesicle trafficking. Considering these results and previous suggestions that Rtf2 constitutes an ubiquitin-related domain, we discuss possible roles of AtRTF2 in ubiquitin-based regulation of pre-mRNA splicing and membrane signaling to the spliceosome. Rtf2 is SDR1 (= AtRTF2) and was discovered in a genetic suppressor screen using the dms4 mutant. DMS4 was described in Kanno et al (2010) EMBO Rep. 11:65-71. Examination of whole-genome DNA methylation status in transgenic Arabidopsis plants

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Selection for transcript diversity in the extremophile Galdieria sulphuraria results in retention of the splicing machinery

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