Early splicing functions of fission yeast Prp16 and its unexpected requirement for gene Silencing is governed by intronic features

Abstract

Prp16 is a DEAH box pre-mRNA splicing factor that triggers a key spliceosome conformational switch to facilitate second step splicing in Saccharomyces cerevisiae. However, Prp16 functions are largely unexplored in Schizosaccharomyces pombe, an attractive model with exon-intron architecture more relevant to several other eukaryotes. Here, we generated mis-sense alleles in SpPrp16 whose consequences on genome-wide splicing uncover its nearly global splicing role with only a small subset of unaffected introns. Prp16 dependent and independent intron categories displayed a striking difference in the strength of intronic 5' splice site (5'SS)-U6 snRNA and branch site (BS)-U2 snRNA interactions. Selective weakening of these interactions could convert a Prp16 dependent intron into an independent one. These results point to the role of SpPrp16 in destabilizing 5'SS-U6snRNA and BS-U2snRNA interactions which plausibly trigger structural alterations in the spliceosome to facilitate first step catalysis. Our data suggest that SpPrp16 interactions with early acting factors, its enzymatic activities and association with intronic elements collectively account for efficient and accurate first step catalysis. In addition to splicing derangements in the spprp16F528S mutant, we show that SpPrp16 influences cell cycle progression and centromeric heterochromatinization. We propose that strong 5'SS-U6 snRNA and BS-U2 snRNA complementarity of intron-like elements in non-coding RNAs which lead to complete splicing arrest and impaired Seb1 functions at the pericentromeric loci may cumulatively account for the heterochromatin defects in spprp16F528S cells. These findings suggest that the diverse Prp16 functions within a genome are likely governed by its intronic features that influence splice site-snRNA interaction strength.

Keywords: SpPrp16; cell-cycle; heterochromatin; interaction; intron-like; ncRNA; snRNA; splice-site; transcriptome.

Figure 1.

spprp16F528S mutant affects splicing of introns with diverse features. (a) Growth kinetics of wild-type (WT) and mutants (F528S, F528H and G515A) on EMM complete media at 23°C, 30°C and 37°C analysed by spotting 10-fold serial dilutions of cultures grown to OD₅₉₅ of 0.7. Schematic representation of the wild-type (leu1:spprp16⁺) and mutant strains (leu1:spprp16) generated is shown. (b) Semi-quantitative RT-PCRs showing the splicing of tfIId⁺I1 in WT, spprp16G515A and spprp16F528S cells grown at 30°C. Comparison of tfIId⁺I1 splicing in the WT and spprp16F528S mutant at 30°C and 23°C is also shown. (C and D) Semi-quantitative RT-PCRs showing the splicing of mdm35⁺I1 (c) and nda3⁺I4 (d) in WT, spprp16G515A and spprp16F528S cells grown at 30°C. Reverse transcription for each transcript was done with the reverse primer (RP) corresponding to the downstream exon. PCR on the cDNA was performed with the same RP in combination with upstream exonic forward primer (FP). RT-PCR on the intronless actin transcript (act1⁺) served as normalization control. P- pre-mRNA, M – mRNA and g. DNA – genomic DNA control as indicated. Bar graphs represent data from three biological replicates. In this and other quantitative assays, the statistical significance was determined by unpaired student’s t-test . *** = p < 0.001, ** p < 0.005, * = p < 0.05 and ns = non-significant.

Figure 2.

SpPrp16 has nearly ubiquitous splicing functions. (a) Frequency histogram representing the number of introns plotted against their respective log₂ differential splice index (SI). Introns with log₂ differential SI ≥ 1 (i.e., ≥2 fold difference between the wild-type and mutant) are considered strongly dependent on SpPrp16 for splicing and those with log₂ differential SI < 0.6 (i.e. <1.5 fold difference between the wild-type and mutant) are considered unaffected in the spprp16F528S mutant. The introns with marginally affected differential SI values (log₂ differential splice index between 0.6 and 1) were excluded from this representation. (b) Semi-quantitative RT-PCR validation of the transcriptome-based splicing phenotype at 30°C for SpPrp16 dependent seb1⁺ intron1 (left panel), dga1⁺ intron2 (middle panel) and independent intron dga1⁺ intron3 (right panel). (c) Schematic of a S. pombe transcript with global sequence consensus for the intronic 5’SS, BS and 3’SS elements. Base-pairing of the 5’SS and the BS with U6 snRNA and U2 snRNA, respectively, are indicated by dotted lines. (d) Graph representing the number of introns from SpPrp16 dependent and independent classes categorized based on the complementarity of 5’SS +4, +5 and +6 nucleotides with the U6 snRNA sequence – ACA. Base pairing at each position is denoted by a ‘+’sign and no base pairing by ‘–’ sign. The number of introns with complete complementarity indicated as ‘+++’ is significantly different between the dependent and independent intron categories at p = 0.03. (e) Principal Component Analysis (PCA) showing variance between the number of dependent and independent introns (Y axis) with loss of BS-U2 snRNA base-pairing for each indicated BS residue (X axis). The lines (Z axis) represent data for intron subsets with varying 5’SS-U6 snRNA strength. Green (– – –), red (– – +) and blue (– + +) lines indicates three intron subsets categorized based on 5’SS-U6 complementarity at the 5’SS +4, +5 and +6 positions. The invariant branch residue A was positioned as ‘0’ and other BS nucleotides are numbered with respect to this residue.

Figure 3.

SpPrp16 destabilizes 5’SS-U6 snRNA and BS-U2 snRNA interactions. (a) Primer extension to assess the splicing of seb1⁺ E1-I1-E2 mini-transcripts having wild-type or mutant 5’SS (depicted as 5’SS and 5’SS* respectively) in the WT (leu1:spprp16⁺) and F528S (leu1:spprp16F528S) mutant cells. The mutations introduced in the 5’SS (highlighted in red) and positions which can base-pair with U6 snRNA are shown (black lines). Primer extension on snu2⁺ transcripts served as the loading control. Lane M- 100 nts to 1000 nts DNA size marker (b). Semi-quantitative RT-PCRs to analyse the splicing of tif313⁺ intron2 in mini-transcripts comprising exon2-intron2-exon3 with wild-type or mutant branch site (BS) in the WT (leu1:spprp16⁺) and F528S (leu1:spprp16F528S) mutant strains. The mutations introduced (highlighted in red) and the positions that can base-pair with U2 snRNA are shown (black lines).

Figure 4.

SpPrp16 facilitates first step splicing catalysis. (a and b) Genetic interaction of spprp16F528S and spprp16G515A with dbr1Δ. Comparative growth profile of double mutants with respective single mutants at the indicated temperatures. (c) Primer extensions to estimate the levels of pre-mRNA, mRNA and lariat-exon2 species from tfIId⁺I1 splicing in spprp16F528S and appropriate control strains grown at 30°C and 23°C. The schematic to the left marks the expected position of extension products from pre-mRNA (P), mRNA (M) and lariat exon-2 (L) species. RNA from WT (leu1:spprp16⁺), F528S (leu1:spprp16F528S), WTdbr1 (leu1:spprp16⁺ dbr1Δ) and dbr1Δ strains grown at 30°C were used in lanes 1 to 4. For lanes 5 and 6, RNA from WT and F528S cultures grown at 23°C was the input. The DNA sequencing ladder (lanes GATC) was prepared using the tfIId exon2 RP on pDBlet tfIId E1-I1-E2 plasmid as the template. ATTAG is the reverse complement of BS sequence and asterisk marks the position for invariant branch residue A. Lane M indicates DNA size markers in the range of 100–1000 nts. (d) Primer extension reactions to assess tfIId⁺intron1 splicing in the spprp16G515A dbr1Δ double mutant grown at 37°C as compared to the single mutant and wild-type control strains. RNA from WT (leu1:spprp16⁺), WT dbr1Δ (leu1:prp16⁺ dbr1Δ), G515A (leu1:spprp16G515A) and G515A dbr1Δ (leu1:spprp16G515A dbr1Δ) were assessed in lanes 1 to 4. (E and F) Genetic interaction of spprp16F528S with splicing factor mutants cwf10-1 (e) and spprp8-1 (f). Growth of respective double mutants was compared with single mutant strains at the indicated temperatures. Ten-fold serial dilutions of each culture from an initial inoculum grown to equal OD₅₉₅ were spotted. (g) Semi-quantitative RT-PCR assessment of tfIId⁺I1 and tim13⁺I2 splicing in spprp16F528S cells that overexpress spprp22⁺. (h) The proposed juncture of SpPrp16 function in the splicing pathway based on the genetic interaction of spprp16F528S with other splicing factor mutants.

Figure 5.

Spprp16F528S mutant exacerbates the splicing defect of substrates with branch-site mutations. (a) Schematic of tfIId E1-I1-E2eGFP mini-transcript. The 5′SS, BS with invariant branch nucleotide A or mutants C or G residues and the 3′SS of the mini-transcript are shown. The GFP-RP primer used for reverse transcription is marked by an arrow. (b) Primer extensions to detect the splicing of tfIId mini-transcripts with branch residue A or C in spprp16⁺, spprp16F528S and spprp16G515A strains. The expected positions of cDNA from pre-mRNA (P), lariat-exon2 (L) and mRNA (M) species are depicted. Primer extension on snu2⁺ transcripts served as the loading control. Lane M – 100 nts to 1000 nts DNA size marker (c) Primer extension on tfIID mini-transcripts with the invariant branch residue A or the mutant residue G in spprp16 wild-type and mutant strains. (d) Alignment of N-terminal 50 amino acids in S. cerevisiae Cwc25 and S. pombe Cwf25. The red outline marks residues detected in the cryo-electron microscopy structure of budding yeast spliceosome [52]. (e) The structure shows N-terminal region from Cwc25 (magenta) [52] and Cwf25 (modelled based on homology with Cwc25, cyan – magenta overlap) in proximity to the branch site consensus (blue labelled A70 is the invariant branch residue) of the UBC4 pre-mRNA substrate (orange). The close-up images to the right show interactions of the N-terminal most residue in budding yeast Cwc25 (serine, Ser3-orange) and Cwf25 (glycine, Gly3-orange) with the intronic branch residue A. The side chain interactions of serine and glycine (orange) with the -NH2 group (blue) of branch site adenosine are indicated by dotted red lines.

Figure 6.

Biochemical characterization of Spprp16 helicase domain mutants. (a) Schematic of SpPrp16 domain architecture. Arrow marks indicate primer positions used to amplify the helicase domain (501–862 amino acids) of SpPrp16 wild type and mutant proteins. (b) ATP hydrolysis by wild-type and Spprp16F528S helicase protein upon incubating 10 nM of each protein with 1mM ATP and tracer amounts of γ^P32ATP at 30°C for the different time points indicated. (c) dsRNA unwinding activity of the wild-type and Spprp16F528S helicase proteins on a 3′ overhang containing RNA duplex labelled at the 5′ end (schematic to the left). The reactions were arrested at the indicated time points (in minutes). The upper and middle panels represent the activity of 10 nM and 40 nM proteins, respectively. Δ duplex indicates heat denatured RNA duplex, a marker for ssRNA. Bottom panel shows helicase activity of the wild-type and Spprp16F528S helicase protein on a 5ʹ overhang containing RNA duplex; 40 nM of wild-type and mutant protein was used in this experiment.

Figure 7.

SpPrp16 is required for successful mitosis and for efficient transcriptional gene silencing at centromeres and telomeres. (a) Bright field and DAPI stained images of spprp16⁺, spprp16F528S and spprp16G515A cells. The ‘cut’ cells (white arrowheads at the site of cell separation) and lagging chromosome (fragmented nuclei) in spprp16F528S are shown. Scale bar is 10 µm in all panels. (b) Thiabendazole (TBZ) sensitivity of spprp16 mutants as compared to wild-type and spago1Δ strains at 30°C. Cultures were grown to equivalent OD₅₉₅ before spotting 10-fold serial dilutions on PMG complete media containing TBZ (at 15 µg/ml concentration). The growth proficiency on PMG complete media (left) served as the control. (c) Normalized transcript levels for centromeric ncRNAs obtained from RNA-seq of spprp16⁺ (WT) and spprp16F528S mutant cells (left graph). P value significance for upregulated ncRNAs in the spprp16F528S mutant is shown. The semi-quantitative RT-PCR for two centromeric transcripts ncRNA.232 and ncRNA.362 are shown in the right upper and lower panels respectively. For ncRNA.232, ‘*’ to the right side of the gel indicates the unspliced precursor. Control PCR reactions without reverse transcription are shown in the -RT panel. (d) Semi-quantitative RT-PCR to assess the splicing of ncRNA.232 mini-transcript in the WT (leu1:spprp16⁺) and F528S (leu1:spprp16F528S) mutant strain. (e) Transcriptional silencing of ade6⁺ centromeric reporter in the wild-type and spprp16F528S mutant cells assessed in media with limiting adenine. cwf10-1 and spago1Δ served as positive controls for defective centromeric heterochromatin.