Splicing of branchpoint-distant exons is promoted by Cactin, Tls1 and the ubiquitin-fold-activated Sde2

Abstract

Intron diversity facilitates regulated gene expression and alternative splicing. Spliceosomes excise introns after recognizing their splicing signals: the 5'-splice site (5'ss), branchpoint (BP) and 3'-splice site (3'ss). The latter two signals are recognized by U2 small nuclear ribonucleoprotein (snRNP) and its accessory factors (U2AFs), but longer spacings between them result in weaker splicing. Here, we show that excision of introns with a BP-distant 3'ss (e.g. rap1 intron 2) requires the ubiquitin-fold-activated splicing regulator Sde2 in Schizosaccharomyces pombe. By monitoring splicing-specific ura4 reporters in a collection of S. pombe mutants, Cay1 and Tls1 were identified as additional regulators of this process. The role of Sde2, Cay1 and Tls1 was further confirmed by increasing BP-3'ss spacings in a canonical tho5 intron. We also examined BP-distant exons spliced independently of these factors and observed that RNA secondary structures possibly bridged the gap between the two signals. These proteins may guide the 3'ss towards the spliceosome's catalytic centre by folding the RNA between the BP and 3'ss. Orthologues of Sde2, Cay1 and Tls1, although missing in the intron-poor Saccharomyces cerevisiae, are present in intron-rich eukaryotes, including humans. This type of intron-specific pre-mRNA splicing appears to have evolved for regulated gene expression and alternative splicing of key heterochromatin factors.

Figure 1.

Intronic features define the role of Sde2 in pre-mRNA splicing. (A) Schematic of Sde2 activation and function for the splicing of rap1-i2. (B) Design of splicing reporters (ss, splice site; BP, branchpoint). The numbers on the reporter show the insertion site of introns in the ura4 gene. rap1-i1 and i2 were individually inserted at the designated insertion site mentioned in the schematic to generate rap1-i1 and rap1-i2 reporters, respectively (C) Table showing the expected result from the reporter in S. pombe. (D) The splicing-proficient strain grows on –URA plate but does not grow on +FOA plates, and vice versa (incubation time: –LEU, –URA, 4 days; +FOA, 5 days). (E) Ubiquitin-like processing of Sde2 is essential for its intron-specific splicing activity. The Sde2 processing-defective strains Δubp5 Δubp15 and sde2 (AAK) could not splice the rap1-i2 reporter, while the ubiquitin–^KSde2-C chimeric strain (Ubi–sde2-C) spliced the reporter (incubation time: –LEU, –URA, 4 days; +FOA, 5 days). (F) The N-terminal lysine of ^KSde2-C is crucial for the intron-specific splicing activity of Sde2. The sde2 (GGM) mutant strain was unable to splice rap1-i2 (incubation time: –LEU, –URA, 4 days; +FOA, 4 days).

Figure 2.

Sde2 target introns have longer spacing between the BP and 3′ss. (A) Distribution of distances (number of nucleotides) between the BP adenosine and the 5′- and 3′-splice sites (ss) of the Sde2-dependent and -independent introns (Sde2-dependent ≥0.5 log₂Δsde2/wt ratio at 30°C; Sde2-independent ≤log₂Δsde2/wt ratio at 30°C). Red peaks and histograms show 122 Sde2-dependent introns, and blue peaks and histograms show 4418 Sde2-independent introns. The numbers on the peaks show their maxima. (B) Nucleotide sequence of rap1-i2 with different spacing between the BP and 3′ss. (C and D) Growth assay and RT–PCR showing that rap1-i2 with 39 nt between the BP and 3′ss was Sde2 dependent (incubation time: –LEU, –URA 4 days; +FOA 5 days). The reduction of this spacing made its splicing Sde2 independent. RT–PCR was performed by two sets of primers as indicated in the schematic. ex1-ex2 indicates PCR performed with primers that bind to exon 1 and exon 2, thereby monitoring both spliced and unspliced ura4 transcripts, and ex1-Jn indicates PCR performed with primers that anneal to exon 1 and the exon 1–exon 2 junction, thus monitoring only the spliced ura4 transcripts. The arrows in the schematics depict the binding of primers in the reporter. (E) Nucleotide sequence of tho5-i1 with different spacing between the BP and 3′ss. (F and G) Growth assay and RT–PCR showing that an Sde2-independent intron, tho5-i1, required Sde2 for splicing after increasing the distance between the BP and 3′ss. Growth assay and RT–PCR were performed similarly to (C) and (D) (incubation time: –LEU, –URA 4 days; +FOA, 5 days). Arrows in the schematics depict primer binding to the reporter.

Figure 3.

Threshold distance between the BP and 3′ss for the Sde2 target introns. (A) Schematics of tho5-i1 reporters with increasing spacing between the BP and 3′ss. (B, C) Growth assay and immunoblot analysis show that an increase in the distance between the BP and 3′ss makes the intron Sde2 dependent (incubation time: –LEU, –URA, 3 days; +FOA, 5 days). × marks the stop codon that arises during the translation of intron-retained mRNA. * marks proteins possibly arising from aberrant splicing or proteolytic cleavage. (D) Semi-quantitative RT–PCR showing splicing of tho5-i1 reporters with different spacing between the BP and 3′ss. RT–PCR was performed in the same way as in Figure 2D. Small amounts of Ura4 protein appeared enough for the cells to grow on –URA, but the cDNA and protein's detection by RT–PCR and immunoblots seems to require more efficient splicing. Thus, growth assays discriminate the reporters’ activities better with lower splicing efficiency, whereas RT–PCR and immunoblots discriminate the reporters’ activities better with higher splicing efficiency. (E) qRT-PCR analysis to quantify spliced transcripts from the tho5-i1 reporter with different spacings between the BP and 3′ss in wt (grey bars) and Δsde2 (black bars). The quantitation was against leu2 transcripts arising from the same plasmid. The forward primer used in qRT-PCRs binds to exon 1, and the reverse primer binds to the exon 1–exon 2 junction, thus specifically amplifying the spliced transcripts. The y-axis denotes log₂spliced ura4 transcripts against leucine transcripts, and the x-axis depicts different spacings between the BP and 3′ss in the tho5-i1 reporter.

Figure 4.

The spliceosome prefers BP-near 3′ss over BP-distant 3′ss. (A) Splicing of tho5-i1 reporters with competing 3′ss (BP-near and BP-distant) in the wt was tested on –URA and +FOA plates (incubation time: –LEU, –URA, 3 days; +FOA, 4 days). (B) Semi-quantitative RT–PCR monitoring splicing of BP-near and BP-distant 3′ss in the tho5-i1 reporter. Primers are as shown in the block diagram (ex-exon). * marks the band that appears due to the use of BP-near 3′ss, which leads to the incorporation of nine additional nucleotides in the ura4 cDNA transcript. (C) Western blot analysis shows the translational product of mRNA spliced using BP-near 3′ss but not from BP-distant 3′ss. Ura4-N3 and -N4 indicate proteins from mRNAs with exon 1 up to the stop codon (labelled with x).

Figure 5.

RNA structures may bring the 3′ss closer to the BP. (A) Predicted secondary structures of the RNA between the BP and 3′ss in rap1-i2 variants. Arrows indicate nucleotides where mutations were made in the rap1-i2 construct. The bold letters in the table indicate the mutations, and the underlined letters indicate the BP and 3′ss. (B) S. pombe growth on the indicated plates with rap1-i2 reporter variants (incubation time: –LEU, –URA, 3 days; +FOA, 4 days). (C) Western blot analysis to check for splicing defects with different rap1-i2 reporter variants. (D–F) The assays with the atg20-i2 reporter variant are similar to (A–C). –LEU and –URA plates were scanned after 3 days, and +FOA plates after 4 days.

Figure 6.

Sde2 functions with Cactin/Cay1 and Tls1. (A and B) Growth assay and immunoblot analysis showing introns with a longer distance between the BP and 3′ss also require Cay1 and Tls1, similar to Sde2. Reducing the distance in rap1-i2 makes its splicing independent of these factors (incubation time: –LEU, –URA, 3 days; +FOA, 4 days). (C and D). The growth assay and immunoblot analysis show that increasing the distance in the tho5-i1 reporter makes its splicing dependent on Cay1 and Tls1, as Sde2. * marks proteins arising from aberrant splicing or proteolytic cleavage (incubation time: –LEU, –URA, 3 days; +FOA, 4 days). (E) Genetic interaction among intron-specific splicing factors. The double mutants Δsde2 Δtls1 and Δtls1 Δcay1 grow more slowly in comparison with the respective single mutants at all temperatures. We crossed Δsde2 and Δcay1, and the lack of Δsde2 Δcay1 mutants [expected at (A) in TT and at (B) and (D) in NPD] indicates the synthetic lethality of the double mutant. Plates at 30°C and 37°C were scanned after 3 days, and the plate at 20°C was scanned after 5 days. TT, tetratype; NPD, non-parental ditype.

Figure 7.

Sde2, Cay1 and Tls1 control the expression of heterochromatin factors through intron-specific pre-mRNA splicing. (A) Rap1, Hif2 and Dsh1 proteins are lower in deletion mutants of intron-specific splicing factors. Immunoblotting was performed for the chromosomally C-terminal 6HA-tagged strains using an anti-HA antibody. (B) Semi-quantitative RT–PCR was performed to assay the splicing of heterochromatin factors in the indicated strains. Arrows in the schematic indicate where the primer binds.