Mucorales sealing-only Trl1s can perform HAC1 splicing during the UPR. (A) R. arrhizus appears to have an Ire-dependent intron in HAC1 mRNA (encoding the 149 amino acid hypothetical protein G6F37_009984). Sequence similarity to Hac1 is highest in the indicated bZIP domain. Depicted is the unspliced (RaHAC1u) and spliced (RaHAC1s) mRNA structure based on aligning RNA-seq data (SRR1013749) to the genome with RNA STAR, and the predicted function of Ire1 and sealing-only Trl1 in generating spliced HAC1 mRNA. (B) The M. circinelloides, R. arrhizus, and R. irregularis Trl1 were expressed in S. cerevisiae trl1Δ [trl1-K114A] [TRL1, URA3] strain background and subjected to plasmid shuffle assay in the presence or absence of 0.2 µg/mL tunicamycin. The wild-type, ire1Δhac1Δ, and ire1Δ strains were used as a control. (C) RT-PCR of HAC1 indicates that upon UPR induction, the Mucorales Trl1 gives rise to spliced HAC1 mRNA (HAC1S). The HAC1 splicing defective ire1Δ strain was used as a control, which only produced the unspliced HAC1 mRNA (HAC1U).
