Is splicing essential in Cyanidioschyzon merolae?

Protein-coding sequences of eukaryotic genes are often interrupted by non-coding sequences called introns. Introns must be removed from mRNA precursors, and retained segments known as exons are then ligated together to form mature messenger RNA. This essential process in eukaryotic gene expression is called mRNA splicing. Splicing is carried out by a large complex that contains small nuclear RNAs (snRNAs) and many proteins. Due to the complexity of the spliceosomal machinery in other eukaryotes, studying splicing has been very challenging. Cyanidioschyzon merolae is a unicellular red alga with only 39 introns in its genome and a much simpler set of splicing machinery than in humans. It has been estimated that the ancestral red alga contained ~1700 introns, from which we can infer that C. merolae has lost almost all of its introns. This raises the possibility that splicing is no longer essential for this organism. I addressed whether this vestigial splicing system is biologically important by inhibiting splicing in various ways. If splicing is not required for the survival of this organism, inhibiting this process should not impact cell survival. In contrast, if splicing is essential, a deleterious phenotype and cell death are expected upon inhibition. I attempted to inhibit splicing using antisense RNA and degron techniques. In the first approach, which seeks to silence a target by base pairing the transcript to inactivate it, I transiently expressed the antisense version of three essential splicing factors under the control of a nitrate inducible promoter by transforming an engineered plasmid with a selectable marker into the cells. Additionally, I integrated antisense versions of two splicing factors in the genome under the control of the same inducible promoter. The antisense RNA should bind the target RNAs in both cases, leading to their degradation or sequestration. The nitrogen source for C. merolae in rich media is ammonium, where the antisense promoter will be off. By shifting cells to nitrate media, I activated antisense expression, after which I expected splicing to be inhibited and cell death to occur. Control experiments showed that the inducible promoter works, but I could not demonstrate the antisense strand induction. In the second approach, I implemented an inducible degron system to degrade splicing proteins. Degrons are motifs that target proteins for degradation, and they can be fused to target genes to allow the corresponding protein to be degraded by adding rapamycin. This small molecule activates the degradation system. I targeted Prp8 and Clfl, both core spliceosomal proteins. The degron results were consistent with protein degradation and splicing inhibition, but for technical reasons, I cannot conclude whether splicing is essential for C. merolae . Despite numerous attempts with morpholino oligos, gene deletion, splicing inhibitors, etc., the only experiment in our lab to date consistent with Cm splicing being essential was our failure to delete the gene for the splicing protein Cefl.