The endoplasmic reticulum (ER) protein folding capacity is balanced with the protein folding burden to prevent accumulation of un- or misfolded proteins. The ER membrane-resident kinase/RNase Ire1 maintains ER protein homeostasis through two fundamentally distinct processes. First, Ire1 can initiate a transcriptional response through a non-conventional mRNA splicing reaction to increase the ER folding capacity. Second, Ire1 can decrease the ER folding burden through selective mRNA decay. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, the two Ire1 functions have been evolutionarily separated. Here, we show that the respective Ire1 orthologs have become specialized for their functional outputs by divergence of their RNase specificities. In addition, RNA structural features separate the splicing substrates from the decay substrates. Using these insights, we engineered an S. pombe Ire1 cleavage substrate into a splicing substrate, which confers S. pombe with both Ire1 functional outputs.

MegZ03_index13_AGTCAA_1MISS_lowerM_wildtype_polyA_PLUSstrand_COUNTSThis is .WIG file (processed from .fastq files) for DMS-seq data in S. pombe cells (plus strand) (generated by Meghan Zubradt)MegZ03_index13_AGTCAA_1MISS_lowerM_wildtype_polyA_MINUSstrand_COUNTSThis is .WIG file (processed from .fastq files) for DMS-seq data in S. pombe cells (minus strand) (generated by Meghan Zubradt)MegZ03_index13_AGTCAA_L005_R1_001.fastq.gzThis is the unprocessed sequencing .fastq file (compressed) for S. pombe DMS-seq experiment (generated by Meghan Zubradt)