Deciphering canonical and non-canonical role of splicing factor Prp16 in pathogenic yeast C. neoformans

Abstract

RNA splicing is a ubiquitous process during eukaryotic gene expression, where non-coding introns are removed from primary transcripts to form functional mRNA. This vital process is carried out by the spliceosome, a large ribonucleoprotein complex consisting of 5 snRNAs and more than 150 proteins. The spliceosome functions to recognize conserved cis sequences, primarily those at the 5’ splice site (5’SS), branch site (BPS), and 3’ splice site (3’SS) of pre-mRNA introns, to assemble catalytic reaction centers by sequential remodeling of pre-mRNA bound spliceosomal complexes. These catalytic reactions are two transesterifications that excise introns and join exons in the primary transcript. The sequence variation in the conserved cis-elements and changes in their interactions with spliceosomal factors can influence splicing efficiency and thus provide mechanisms to modulate gene expression levels. The diversity in intronic elements and the vast variation in intron lengths (20nts to 2kb) prompts questions about how the evolutionarily conserved core spliceosomal factors act to provide efficient and accurate splicing in different genomes. While splicing factors and spliceosome assembly are extensively studied in yeast models S. cerevisiae and, to a lesser extent, in S. pombe, their roles in the basidiomycete Cryptococcus neoformans with a very intron-rich genome is relatively unexplored.

Objective I. Functional characterization of DEAH-box helicase Prp16 in short intron-rich yeast Cryptococcus neoformans

S. cerevisiae DExD/H-box protein Prp16 is required for recognition and proofreading of the branch site and aberrant 5’ splice site in precursor mRNA to ensure splicing accuracy and splice site choice. This function is ascribed to the helicase activity of Prp16, which utilizes ATP hydrolysis to enable the transition of spliceosome complexes. Previously our group investigated fission yeast Prp16 and reported this essential splicing factor has substrate-specific functions and interacts with factors typical of early pre-catalytic spliceosomal complexes. Interestingly, SpPrp16 facilitates G2/M cell cycle progression and promotes silencing at centromeres. Given the high prevalence of introns in > 99% transcripts of C. neoformans and the observation that these introns are majorly short to ultra-short in length, we aimed to examine roles for C. neoformans Prp16 in splice site selection. We first engineered a strain for conditional knockdown of CnPrp16 by replacing the native promoter with the GAL7 promoter that would allow for conditional expression of a tagged mCherry-Prp16 protein from the endogenous locus. Growth assessment of this strain (PGAL7:Prp16) in glucose-containing media (YPD) that repress the expression from PGAL7 shows that Prp16 is essential for the growth and viability of C. neoformans. We carried out deep transcriptomic sequencing of wild-type and PGAL7:Prp16 cells grown in YPD media to understand the impact of CnPrp16 depletion on genome-wide splicing. A Python pipeline to map reads to all splice junctions coupled with statistical analysis showed that CnPrp16 is required for efficient splicing of > 88% introns in the global transcriptome. We took up semi-quantitative RT-PCR validation on selected sets of candidates picked from the global splicing analysis. In these assays, we confirm an increase in unspliced pre-mRNAs across affected introns on CnPrp16 knockdown. On the other hand, we also confirmed that for certain introns splicing remains efficient under depleted CnPrp16 conditions. Interestingly, in two other strains that were engineered for expression of missense mutants of helicase motif I and motif II of Prp16 (K628A and D719A), splicing of the latter set of introns remained efficient. Hence, we confirm that for a small but significant set of introns, splicing occurs independent of CnPrp16 activity. Extensive bioinformatic analysis of intronic signatures in the majority of CnPrp16-dependent introns revealed a pattern of significant and differential enrichment of specific 5’ exonic sequences that immediately precede the splice site. We note that AAG at N-1,-2 and -3 positions preceding the 5'SS and the enrichment of G at the first nucleotide following the 3’SS (i.e., the first residue of the 3’exon) are signatures that indicate splicing can occur independent of Prp16. Notably, these exonic consensuses at 5'SS and 3'SS, which our study uncovered, would confer stronger base-pairing interactions with the spliceosomal U5snRNA in assembling spliceosome complexes. We generated strains to express a Pac1 E5I5E6 model mini-transcript and assessed its splicing when CnPrp16 is abundant or when it is depleted. We also assessed the consequences of changing the normally occurring UUC sequences at the 5’ splice site of this mini-transcript to AAG residues, which are the cis signature in introns spliced independent of Prp16. These experiments with mini-transcripts validated the predictions from global splicing analysis that the interaction of 5’exon-intron sequences in pre-mRNAs with U5snRNA dictates the pre-mRNA dependency on CnPrp16 for efficient splicing. Other extensive bioinformatic studies showed that CnPrp16 promotes efficient splicing of ultra-short (20-60nts) and short introns (60-100nts) in the intron-rich transcriptome of C. neoformans.

Objective II. Investigating the role of Prp16 during Cryptococcus neoformans cell cycle progression

Analysis of the global transcriptome for gene sets/pathways that are most impacted by depletion of CnPrp16 showed that transcripts with strongly dependent introns are enriched for genes encoding factors for cell cycle progression and meiosis. We followed this lead to examine cell cycle progression by detailed flow cytometry analysis of the C. neoformans PGAL7:Prp16 strain under conditions of Prp16 depletion. We uncovered mitotic defects reflected by the increased time taken for mitosis and also noted an increase in the population of aneuploidy-induced drug fluconazole-resistant cells. By live cell tracking GFP-Histone4 marked nuclei in PGAL7:Prp16 strain, we identify delayed mitotic progression on CnPrp16 knockdown and also instances of abnormal mitosis (with two or more nuclei in the mother cell and unequal nuclear segregation) underscoring defects in chromosome segregation. To study the underlying causes of delayed mitosis, we examined whether the spindle assembly checkpoint (SAC) had a role to play. We generated the PGAL7:Prp16 strain in the background of the MAD2 kinase null mutant (mad2Δ) and found exacerbated growth defect and hypersensitivity to mild spindle insult. This indicates active SAC in conditions of CnPrp16 knockdown, which contributes to delayed mitosis. Additionally, by imaging mitotic spindles using GFP-Tub1 reporter, we identify abnormal mitotic spindle positions in Prp16 knockdown cells. These data suggest that the spindle position checkpoint (SPOC) is another additional checkpoint that contributes to the mitotic delay created on the CnPrp16 knockdown. Consistent with reports of roles for S. pombe Prp16 in silencing and heterochromatinization at centromeres, we found that in the CnPrp16 knockdown cells, there is a notable decrease in repressive histone methylation mark H3K9me2 at Tcn1 locus in centromeres (cen1, 2, 3, 9 and 11). We also conducted a genetic test by combining mutants in factors of the RNAi pathway (ago1Δ or rdp1Δ) with conditional knockdown of Prp16 (PGAL7:Prp16). The double mutant rdp1Δ, PGAL7:Prp16, exhibits exacerbated growth defects compared to PGAL7:Prp16. However, the mutants do not show any sensitivity to spindle insult, indicating that defective centromeric silencing in the Prp16 knockdown of C. neoformans could be independent of the cell cycle phenotypes.

Overall, our study reports a very widespread requirement of Prp16 for the constitutive splicing of introns in the C. neoformans transcriptome, yet the roles for this helicase are intron-specific. We identified an important pre-mRNA cis feature that confers dependency on Prp16, which is the strength of interactions between pre-mRNA 5’exon terminal residues and the spliceosomal U5 snRNA loop1. We also report that ultra-short introns found commonly in this unusual genome are also dependent on Prp16 for their efficient splicing. We show that C. neoformans Prp16 contributes to faithful chromosome segregation and mitosis by its role in spindle assembly and position. Collectively, the data from our study proposes a mechanistic model for how Prp16 mediates efficient pre-mRNA spicing and underscores its role in maintaining genome integrity.