Intron retention-dependent gene regulation in Cryptococcus neoformans

Abstract

The biological impact of alternative splicing is poorly understood in fungi, although recent studies have shown that these microorganisms are usually intron-rich. In this study, we re-annotated the genome of C. neoformans var. neoformans using RNA-Seq data. Comparison with C. neoformans var. grubii revealed that more than 99% of ORF-introns are in the same exact position in the two varieties whereas UTR-introns are much less evolutionary conserved. We also confirmed that alternative splicing is very common in C. neoformans, affecting nearly all expressed genes. We also observed specific regulation of alternative splicing by environmental cues in this yeast. However, alternative splicing does not appear to be an efficient method to diversify the C. neoformans proteome. Instead, our data suggest the existence of an intron retention-dependent mechanism of gene expression regulation that is not dependent on NMD. This regulatory process represents an additional layer of gene expression regulation in fungi and provides a mechanism to tune gene expression levels in response to any environmental modification.

Figure 1. Genome comparison between C. neoformans var. neoformans and C. neoformans var. grubii.

(A) Position of orthologous genes in C. neoformans var. neoformans and C. neoformans var. grubii as visualized using SynTView. (B) Right panel. Conservation of intron position between C. neoformans var. neoformans and C. neoformans var. grubii according to the position of the introns within the genes. The blue bars represent the percentage of var. grubii introns conserved in the var. neoformans and the red bars represent the percentage of var. neoformans introns conserved in the var. grubii. Left panel. Size conservation of the ORF introns between the two varieties.

Figure 2. Regulation of AS by the environment.

(A) Repartition of the different classes of alternative splicing in C. neoformans. (B) Number of AS events identified in each growth condition (i.e. exponential phase 30 °C [Expo]; exponential phase 37 °C [37 °C], stationary phase glucose [Stat D]; stationary phase galactose [Stat G]; SDS 0.01% treatment [SDS]; 10 mg /L fluconazole treatment [Fluco]). (C) Examples of RT-PCR analyses of alternative splicing regulation by temperature and stage of growth in C. neoformans. Actin mRNA was used as a control. CS, A-SS, and IR stand for constitutive splicing, alternative splice site utilization, and intron retention, respectively. Full-length blots/gels are presented in Supplementary Figure S9. (D) Venn diagrams drawn using lists of AS events in each variety, revealing the specificity of AS regulation by environmental cues.

Figure 3. Alternative splicing contributes little to proteome diversity in C. neoformans.

(A) Relationship between the number of IR events identified and the threshold used for identification. (B) Relationship between the number of A-SS events identified and the threshold used for identification. (C) Relationship between the number of AS events identified in the UTRs and the threshold used for identification.

Figure 4. RNA-Seq profiles (left) and semi-quantitative RT-PCR validation (right) of representative alternative splicing events that are subject to or immune to NMD.

The green and black curves represent the transcription coverage in the upf1Δ and wild-type strains, respectively; the blue boxes represent the CDS; and the vertical black bars represent the stop codons found in each frame. Primers (arrows) were chosen on the exons flanking the alternatively spliced introns in order to amplify both the constitutively spliced (CS) and the alternatively spliced (AS) or the unspliced (IR) isoform of each transcript. (A) CAS3 (CNB01440) is an example of the absence of the IR regulation of upon UPF1 deletion in C. neoformans. The first and the last intron were partly retained (red arrows), and this retention was not altered in the upf1Δ strain. (B) YRA1 (CNG03240) is an example of an intron-retaining mRNA up-regulated by UPF1 deletion. (C) In URA4 (CNA07120) the use of an in-frame alternative 5′ splice site introduces a PTC on the mRNA, which is regulated in the upf1Δ mutant. (D) In the ELO3 mRNA (CNC00110), the use of an in-frame alternative 5′ splice site does not introduce a PTC on the mRNA, and it is equally expressed in the upf1Δ mutant as compared to the wild-type strain. Full-length blots/gels are presented in Supplementary Figure S9.

Figure 5. Intron retention and gene expression regulation is linked.

(A) The most highly expressed genes are less regulated by IR than moderately expressed genes. Genes were separated into deciles according to level of expression at 30 °C in the exponential phase. The percentage of genes regulated by IR in this condition (1% threshold blue; 5% threshold red) is reported. (B) Gene expression regulation in each comparison. (i.e. Exponential phase 30 °C [Expo]; Exponential phase 37 °C [37 °C], Stationary phase glucose [StatD]; Stationary phase galactose [StatG]; SDS 0.01% treatment [SDS]; 10 mg/L fluconazole treatment [Fluco]).(C) Proportion of genes up- or down-regulated when IR is up- or down-regulated. For each comparison, the gene lists for which IR down- or up-regulation was observed were compared with the gene lists for which up- or down-regulation of expression was observed. The percentages of overlap between these lists are reported.

Figure 6. IR-dependent regulation of gene expression in C. neoformans.

(A) Example of IR-dependent regulation of gene expression in gene CNM00240. Top panel. RNA-Seq profiles as visualized with Artemis. The blue and red curves represent transcription coverage at 30 °C and 37 °C, respectively; the blue boxes represent the CDS; and the vertical black bars represent the stop codons found in each frame. Bottom panel. RT-PCR analysis of intron-retention. Primers (arrows) were chosen on the exons flanking the alternatively spliced introns in order to amplify both the spliced (CS) and unspliced (IR) mRNAs. The experiment was performed on 3 independent replicates and the percentages of retention are given. Full-length blots/gels are presented in Supplementary Figure S9. (B) Model of alternative splicing-dependent regulation of gene expression. Top panel. In the case of A-SS, the mRNAs are exported to the cytoplasm. If it contains a PTC, it is degraded by the NMD pathway. In the absence of PTC an alternative protein is produced. Bottom panel. If an intron is retained, export from the nucleus is repressed.