Identification of an Exceptionally Long Intron in the HAC1 Gene of Candida parapsilosis

Abstract

The unfolded protein response (UPR) in the endoplasmic reticulum (ER) is well conserved in eukaryotes from metazoa to yeast. The transcription factor HAC1 is a major regulator of the UPR in many eukaryotes. Deleting HAC1 in the yeast Candida parapsilosis rendered cells more sensitive to DTT, a known inducer of the UPR. The deletion strain was also sensitive to Congo red, calcofluor white, and the antifungal drug ketoconazole, indicating that HAC1 has a role in cell wall maintenance. Transcriptomic analysis revealed that treatment of the wild type with DTT resulted in the increased expression of 368 genes. Comparison with mutant cells treated with DTT reveals that expression of 137 of these genes requires HAC1 Enriched GO term analysis includes response to ER stress, cell wall biogenesis and glycosylation. Orthologs of many of these are associated with UPR in Saccharomyces cerevisiae and Candida albicans Unconventional splicing of an intron from HAC1 mRNA is required to produce a functional transcription factor. The spliced intron varies in length from 19 bases in C. albicans to 379 bases in Candida glabrata, but has not been previously identified in Candida parapsilosis and related species. We used RNA-seq data and in silico analysis to identify the HAC1 intron in 12 species in the CTG-Ser1 clade. We show that the intron has undergone major contractions and expansions in this clade, reaching up to 848 bases. Exposure to DTT induced splicing of the long intron in C. parapsilosisHAC1, inducing the UPR.IMPORTANCE The unfolded protein response (UPR) responds to the build-up of misfolded proteins in the endoplasmic reticulum. The UPR has wide-ranging functions from fungal pathogenesis to applications in biotechnology. The UPR is regulated through the splicing of an unconventional intron in the HAC1 gene. This intron has been described in many fungal species and is of variable length. Until now it was believed that some members of the CTG-Ser1 clade such as C. parapsilosis did not contain an intron in HAC1, suggesting that the UPR was regulated in a different manner. Here we demonstrate that HAC1 plays an important role in regulating the UPR in C. parapsilosis We also identified an unusually long intron (626 bp) in C. parapsilosisHAC1 Further analysis showed that HAC1 orthologs in several species in the CTG-Ser1 clade contain long introns.

Keywords: Candida parapsilosis; Hac1; introns; unfolded protein response.