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. 2013 Aug;33(16):3125-36.
doi: 10.1128/MCB.00007-13. Epub 2013 Jun 10.

Splicing functions and global dependency on fission yeast slu7 reveal diversity in spliceosome assembly

Shataparna Banerjee  1 , Piyush Khandelia, Geetha Melangath, Samirul Bashir, Vijaykrishna Nagampalli, Usha Vijayraghavan
Affiliations

Splicing functions and global dependency on fission yeast slu7 reveal diversity in spliceosome assembly

Shataparna Banerjee et al. Mol Cell Biol. 2013 Aug.

Abstract

The multiple short introns in Schizosaccharomyces pombe genes with degenerate cis sequences and atypically positioned polypyrimidine tracts make an interesting model to investigate canonical and alternative roles for conserved splicing factors. Here we report functions and interactions of the S. pombe slu7(+) (spslu7(+)) gene product, known from Saccharomyces cerevisiae and human in vitro reactions to assemble into spliceosomes after the first catalytic reaction and to dictate 3' splice site choice during the second reaction. By using a missense mutant of this essential S. pombe factor, we detected a range of global splicing derangements that were validated in assays for the splicing status of diverse candidate introns. We ascribe widespread, intron-specific SpSlu7 functions and have deduced several features, including the branch nucleotide-to-3' splice site distance, intron length, and the impact of its A/U content at the 5' end on the intron's dependence on SpSlu7. The data imply dynamic substrate-splicing factor relationships in multiintron transcripts. Interestingly, the unexpected early splicing arrest in spslu7-2 revealed a role before catalysis. We detected a salt-stable association with U5 snRNP and observed genetic interactions with spprp1(+), a homolog of human U5-102k factor. These observations together point to an altered recruitment and dependence on SpSlu7, suggesting its role in facilitating transitions that promote catalysis, and highlight the diversity in spliceosome assembly.

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Figures

Fig 1
Fig 1
The SpSlu7 C113A mutant protein is nuclear localized. (A) Diagram of the FY527 pREP42EGFPN-spslu7+ and FY527 pREP42EGFPN spslu7C113A strains. (B) Cellular localization of EGFP-tagged wild-type (left panel) and zinc knuckle mutant (C113A) (right panel) SpSlu7 proteins in live cells. A merge of differential interference contrast (DIC) and fluorescence images is shown. (C) Immunoblotting results showing stability of MH-tagged SpSlu7 wild-type or mutant (C113A) proteins in whole-cell extracts of FY527-pREP41MHN spslu7+ (lane 1), FY527-pREP41MHN spslu7C113A (lanes 3 and 4), FY527-pREP41MHN vector (lane 5), as a control, and spslu7Δ-pREP41MHN spslu7+ cells (lane 6). The Coomasie blue-stained gel served as a loading control.
Fig 2
Fig 2
A thiamine-repressible spslu7 missense mutant has intron-specific splicing roles. (A) Diagram of the spslu7+ Pnmt81:spslu7+ (WT) and Pnmt81:spslu7I374G (spslu7-2) strains. (B) Growth kinetics of WT or mutant cells at 30°C, the optimal temperature, in the absence (−T) or presence (+T) of 15 μM thiamine added to early-log-phase cultures. (C and D) Reverse transcription-PCR analyses of the splicing status of tfIId+ I1 (C) and ade2+ I2 (D) in RNA from WT and mutant cells grown in the absence (−T) or presence (+T) of thiamine for 28 h. RNA from the temperature-sensitive prp2-1 mutant grown at 25°C or at 37°C for 2 h (lanes 6 and 7) was a control for transcript isoforms. Genomic DNA PCR product served as a mobility marker for the pre-mRNA (lanes 5). Pre-mRNA and mRNA levels normalized to that of the intronless act1+ transcripts were plotted for the WT and mutant as found from multiple experiments (n = 3 or 4). P and M denote positions of pre-mRNA and mRNA in the gel, respectively.
Fig 3
Fig 3
Global splicing roles for SpSlu7. Schematic illustration of array probes: intronic (P), splice junction (M) for each exon-exon junction, intron-exon junction probe (IE), and the 3′ exon-specific gene expression (T) probes. Shown is a hierarchically clustered heat map of the splicing profile of 611 introns in WT and spslu7-2 mutant cells. Each horizontal row depicts the fold induction or repression of the normalized transcript isoform for an individual intron detected by the probes labeled below. Three classes of various splicing behaviors are magnified in panels A, B, and C on the right. The introns chosen for validation are indicated by arrows, as follows: black, unaffected; red, both pre-mRNA and message levels affected; green, only precursor levels affected.
Fig 4
Fig 4
Semiquantitative reverse transcription-PCR assays validated microarray data. Four introns dependent on spslu7+ showed pre-mRNA accumulation and a spliced mRNA decrease (A), two introns showed pre-mRNA accumulation with no reduction in mRNA levels (B), and two introns were spliced independent of spslu7+ (C). RNA samples are labeled as described for Fig. 2. Reverse transcription was done using a downstream exon reverse primer followed by limiting cycle PCR in combination with upstream exon forward primer. Pre-mRNA and mRNA levels were calculated by densitometric quantification of the PCR products. The values were normalized to intronless act1+ levels, to obtain the fold change of pre-mRNA and message levels in mutant versus the wild type (n = 3 for all except SPAC13G7.11+ I2 [n = 2]).
Fig 5
Fig 5
Differential dependence of introns on two splicing factors SpSlu7 and SpPrp2. RT-PCR analysis results are shown for the three introns in various cellular transcripts based on the total RNA isolated from WT cells, prp2-1 cells grown at 25°C or 37°C for 2 h, and spslu7-2 mutant cells. Bar graphs show the fold changes (n = 3) in unspliced and spliced products seen in WT and spslu7-2 mutant strains. P and M on the left indicate the positions of amplicons from precursor and message species, respectively. PCR for genomic DNA (lane 5) was provided as a mobility marker for the amplicon from pre-mRNA species. The table (right panel) shows the fold changes in mRNA and pre-mRNA species for multiple introns in dim1+, rhb1+, and naa25+ transcripts and in their gene expression levels in the WT, spslu7-2, and prp2-1 strains from the microarray data.
Fig 6
Fig 6
SpSlu7 inactivation arrests splicing before the catalytic steps. (A) Primer extension analysis results to detect the message, precursor, and lariat intermediate for naa10+ I1. The 5′-end-labeled E2 reverse primer (22 nt) used on RNA from WT without (−T) or with (+T) thiamine (lanes 3 and 4), spslu7-2 cells −T and +T (lanes 5 and 6), and in the prp2-1 control strain grown at 25°C or 37°C for 2 h (lanes 1 and 2) is shown. An intronless transcript, snu2+, was independently measured in the same RNA samples as a normalization control (lower panel). The schematic representation of the cDNAs from pre-mRNA, mRNA, and the expected position of cDNA from the lariat intermediate are indicated to the right. (B) Schematic representation of the RT-PCR results for lariat species. The lariat RP, depicted as an open arrow, was used for reverse transcription on naa10+ I1 and phospholipase I4. This was followed by limiting PCR cycles in combination with either the lariat FP to detect lariat RNA species (upper panel) or the 5′ exon FP in the upstream exon to detect pre-mRNA (lower panel) in independent PCRs. The cDNA amplicons from WT RNAs (lanes 1 and 2) and spslu7-2 cells (lanes 5 and 6) were compared with RNA from the negative-control prp2-1 mutant (lanes 3 and 4) and positive-control dbr1Δ mutant (lane 7). The intronless gene act1+ served as an internal control. White vertical lines in the gels in panels A and B separate sections of a gel that were assembled to appropriately position the relevant lanes of data.
Fig 7
Fig 7
cis features dictate intron-specific roles for SpSlu7. (A) Graphical representation of the intron length distribution for 90 unaffected and 422 affected introns. Indicated P values were calculated for intron classes by using χ2 analysis. (B and C) The overall intron percent AU (x axis), excluding the 5′ss and 3′ss residues (B), and the percent AU for the region between the 5′ss and BrP (C) for unaffected and affected introns are shown. P values were determined with unpaired Student's t test. (D) Intron distribution (y axis) for various BrP-3′ss distances in 90 unaffected and in 104 strongly affected introns. The P values from χ2 analyses for distances of ≥16 nt are indicated along the dashed line.
Fig 8
Fig 8
Splicing status of wild-type and modified rhb1 I1 and nab2 I2 minitranscripts. Representative semiquantitative RT-PCR analyses to ascertain the splicing status of (A) rhb1+ I1 wild-type (i), rhb1 I1Δ10 (ii), and rhb1 I1 with 10BrPΔ10 minitranscripts (iii) and (B) nab2+ I2 wild-type (i) and nab2 I2 with 11 (ii) minitranscripts are shown. cDNAs primed with a minitranscript-specific reverse primer (T7) were used with a 5′ exon forward primer in limiting PCR cycles. Total RNA from WT and mutant cells, transformed with the indicated minigene plasmid, grown in the absence (−T) or presence (+T) of thiamine for 28 h were used. PCR with the same primers on the plasmid DNA of the wild-type nab2+ I2 minigene plasmid construct served as a mobility marker for this mini-pre-mRNA (denoted Pl). Pre-mRNA and mRNA levels were normalized to that of the intronless act1+ transcripts and are plotted as bar graphs for the WT and mutant samples. The number of experiments for each construct is denoted (n).
Fig 9
Fig 9
Spliceosomal interactions of SpSlu7. (A) MH-SpSlu7 immunoprecipitated at 150 mM NaCl (lanes 3 and 6) or 300 mM NaCl (lane 9). The coprecipitated snRNAs were detected by solution hybridization with antisense oligonucleotide probes for U1, U2, U5, and U6 (lanes 3 and 9) and U4 (lane 6) followed by electrophoresis on native PAGE gels. Hybridization to detect U4 snRNA was done with a separate RNA aliquot (for both input and immunoprecipitate), since U4 comigrates with U5 snRNA on native gels. snRNAs in an aliquot of the input extract were detected in lanes 1, 4, and 7. Nonspecific association of snRNAs with the beads is shown in lanes 2, 5, and 8. (B) Tetrad spores showing parental ditypes (PD) and three tetratype spore patterns, I, II, and III, obtained upon dissecting spslu7-2 × prp1-4 (UR100) (top panel) and those showing parental ditypes, nonparental ditypes (NPD), and tetratype patterns upon dissecting WT × prp1-4 (bottom panel). The total number of tetrads dissected and the number of tetrads obtained for each genotype are indicated within brackets.
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