Functional characterisation of splicing factor Slu7 in Cryptococcus neoformans – roles in cell cycle, constitutive and alternate splicing

Abstract

Fungal genomes, including in pathogenic yeasts like Cryptococcus neoformans (Cnn) and Candida albicans are rich with short introns. 99% of Cryptococcus neoformans genes have introns with an average of 5 introns per gene and 52nts as average intron length. Such short introns, with degenerate cis splice-signals throws open questions on roles for conserved spliceosome factors in the precise splicing and the influence of splicing on processes such as the cell cycle. Here we report the functional characterization of CnnSlu7, a zinc finger motif-containing spliceosome protein whose budding yeast and fission yeast homologs have predominant roles for intron 3’splice site definition during constitutive and alternative splicing. Using a Cryptococcus neoformans H99 strain with CnnSlu7 conditional knockdown, we show it is an essential factor required for efficient cell cycle transitions and with a major role during mitosis. Significant abnormalities during nuclear migration from mother to the daughter cell, defects in nuclear position during division, and in spindle position were found when cells were depleted of Slu7. However, the temporal pattern for kinetochore clustering was unaffected, and the Slu7 knockdown-associated defects in mitosis were not under the surveillance of Spindle Assembly Checkpoint. Mining of the global transcriptome changes in the knockdown strain identified several transcripts with functions in DNA damage checkpoint were expressed at high levels. Further, transcripts encoding some important cell cycle transitions regulators factors (e.g., Cyclin1) and for nuclear migration during mitosis (e.g., PAC1, DAD4) have introns dependent on CnnSlu7 for their splicing. Analysis of the global transcriptome for splicing efficiency and isoforms uncovered a stringent dataset of 17,188 introns among the total 40,768 introns as being dependent on Slu7 for their splicing. We found that degeneracy in intronic cis-elements such as 5’ss, BrP, and 3’ss had only marginal contributions to dependency. Other features such as intron length, AU content, 5’ss to BrP distance, and BrP to 3’ss distance were examined. Slu7 dependent introns were found to be longer, with a greater distance between 5’ss to BrP and a greater distance between the BrP to 3’ss. Since earlier studies in our lab have reported the role of fission yeast Slu7 in alternate 3’ss choice, we carried out a bioinformatic analysis to examine any role of CnnSlu7 in alternate splicing. Using a bioinformatic pipeline (Ishigami et al., 2021), we discovered that Slu7 regulates alternate splicing in Cnn through its effects on the choice between alternate 3’ss sites in >15,000 introns. The alternate splice site chosen in conditions where Slu7 was depleted was with weak/sub-optimal cis-elements. These findings indicate that Slu7 depletion activated the use of a weak 3’ss during splicing catalysis. Similar results were obtained in the case of alternate events resulting from the use of an alternate 5’ss. This function for CnnSlu7 in influencing 5’ss choice contrasts with the splicing functions of its budding yeast and human counterparts, where its role has been reported mostly in the selection of 3’ss during the second step of splicing. We conclude that CnnSlu7 plays a role in suppressing the usage of weak 3’ss and 5’ss in wildtype cells during splicing catalysis.

Overall, our study indicates splicing dependent role of CnnSlu7 in regulating the nuclear migration from the mother cell to the daughter bud during mitosis in C. neoformans. Its critical role in positioning the mitotic spindle to facilitate timely nuclear migration is executed in part through efficient splicing of introns in cell cycle regulators and spindle positioning cytoskeletal elements. In addition, we propose that Slu7 is an evolutionary conserved splicing factor among the fungi group playing roles in constitutive and alternate splicing.