Tiny Genomes, Tricky Introns, and Thermal Springs: How Intron-Encoded RNAs Allow Some Introns to Persist in a Dramatically Reduced Algal Genome

Abstract

C. merolae is a thermo-acidophilic, unicellular red alga that inhabits sulfuric hot springs (up to 55 C) as well as soil and endolithic environments with sufficient humidity and light, for instance around fumaroles. We have previously demonstrated a dramatic reduction in the complexity of the splicing machinery, including the wholesale elimination of the U1 snRNP. We have recently found that this reduction is also present in two related lineages, pushing back the timing of this genomic streamlining. Bioinformatic and biochemical searches have revealed only 49 splicing proteins in this organism. While its red algal ancestor is estimated to have had ~2000 introns, C. merolae has only 39. This raises the question of whether these 39 were preserved over evolution due to their crucial functions, or whether the stochastic and contingent process of intron removal has simply not yet reached the last few.

To address this question, we have taken a biochemical, transcriptomic, and genetic approach. We deleted 35 of the introns individually, and find that only one is essential: it turns out to encode the RNase MRP that has previously been shown to be essential in S. cerevisiae where it is involved in rRNA biogenesis. Half of the introns encode RNAs that are stably expressed at 42 C (the normal growth temperature), about half of which are predicted to be snoRNAs. We have experimentally confirmed the RNA modifications (2’ O-methylation) in the rRNA and snRNA targets, supporting the view that these are bona fide snoRNAs. Many of the remaining stable intron sequence RNAs (sisRNAs) are predicted to be complementary to protein-coding transcripts, suggesting a possible role in gene regulation. Notably, approximately half of the intron deletion strains grow more slowly than the WT control, demonstrating that the introns, or the ncRNAs they encode, contribute measurably to normal cellular function.

To further investigate the function of C. merolae sisRNAs, we subjected the cells to a variety of stress conditions to attempt to determine whether the sisRNAs are involved in stress responses. Under heat stress, we observed apparent accumulation of the sisRNAs in transcriptomic data. Further investigation by northern blotting and RT-qPCR showed that the response is actually poly-adenylation of the sisRNAs, which we measured at ~50-150 nucleotides. Chemical modification structure probing confirms that the sisRNAs are highly structured and largely resistant to heat denaturation. To seek further clues to their possible function, we are currently using antisense oligo pulldowns to isolate sisRNPs so that we can identify the associated proteins by mass spectrometry. Our results strongly suggest that at least half of C. merolae’s preserved introns encode stable RNAs that contribute substantially to normal cellular function and adaptation to stress.