Cryptococcus neoformans Slu7 ensures nuclear positioning during mitotic progression through RNA splicing

TL;DR

This study shows that the splicing factor CnSlu7 in Cryptococcus neoformans is essential for proper nuclear positioning during mitosis by regulating gene expression.

Contribution

The study reveals a novel role of the conserved splicing factor Slu7 in ensuring nuclear positioning during mitosis in an intron-rich fungal pathogen.

Findings

CnSlu7 is essential for viability and cell cycle progression, particularly during mitosis.

Depletion of CnSlu7 causes nuclear and spindle positioning defects without activating the SAC.

CnSlu7-dependent splicing of specific genes is critical for nuclear migration and cell cycle regulation.

Abstract

The position of the nucleus before it divides during mitosis is variable in different budding yeasts. Studies in the pathogenic intron-rich fungus Cryptococcus neoformans reveal that the nucleus moves entirely into the daughter bud before its division. Here, we report functions of a zinc finger motif containing spliceosome protein C. neoformans Slu7 (CnSlu7) in cell cycle progression. The budding yeast and fission yeast homologs of Slu7 have predominant roles for intron 3’ splice site definition during pre-mRNA splicing. Using a conditional knockdown strategy, we show CnSlu7 is an essential factor for viability and is required for efficient cell cycle progression with major role during mitosis. Aberrant nuclear migration, including improper positioning of the nucleus as well as the spindle, were frequently observed in cells depleted of CnSlu7. However, cell cycle delays observed due to Slu7 depletion did not activate the Mad2-dependent spindle assembly checkpoint (SAC). Mining of the global transcriptome changes in the Slu7 knockdown strain identified downregulation of transcripts encoding several cell cycle regulators and cytoskeletal factors for nuclear migration, and the splicing of specific introns of these genes was CnSlu7 dependent. To test the importance of splicing activity of CnSlu7 on nuclear migration, we complemented Slu7 knockdown cells with an intron less PAC1 minigene and demonstrated that the nuclear migration defects were significantly rescued. These findings show that CnSlu7 regulates the functions of diverse cell cycle regulators and cytoskeletal components, ensuring timely cell cycle transitions and nuclear division during mitosis.

Nuclear position in eukaryotic cells is spatio-temporally regulated during the cell cycle. Unlike in Saccharomyces cerevisiae, where nuclear migration to mother-daughter bud neck precedes mitotic segregation, in Cryptococcus neoformans, the nucleus moves entirely into the daughter bud before division. Transcription dynamics during the C. neoformans cell cycle show periodic expression changes and since all nascent pre-mRNAs must be processed to mRNA, splicing is indispensable for gene expression. Yet, how evolutionarily conserved splicing factors modulate the complex intron-rich C. neoformans transcriptome and their impacts on atypical mitotic nuclear position is unexplored. Here, we show CnSlu7 a zinc finger motif spliceosome factor is essential for viability and for cell cycle progression with a major mitotic role. Live imaging of the nucleus, the spindle and other factors revealed significant abnormalities in nuclear migration from mother to daughter cells, as well as nuclear and spindle position defects upon CnSlu7 depletion. Depletion of CnSlu7 altered the transcript status of various cell cycle players, including those critical for nuclear migration, as their introns were CnSlu7 dependent for splicing. Together, these findings highlight the roles of C. neoformans Slu7 in fine-tuning gene expression levels of transcripts that ensures timely cell cycle progression and spatial control of nuclear position.

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