Mechanism of alternative splicing of yeast HEH1 through competing 5’ splice sites

Domain: biology
Published: 2025-12-16
Editorial outcome: minor_revision

Abstract
Alternative splicing of precursor-messenger RNAs (pre-mRNA) bearing introns with competing 5’ splice sites (5’SS) produces two mRNAs. We investigated the mechanism of alternative splicing of the yeast HEH1/SRC1 gene, which contains four nucleotide-apart competing 5’SS, by monitoring its RNA and protein products. HEH1 alternative splicing requires a sixteen-nucleotide pre-mRNA segment spanning its two 5’SS. The nucleotides are decoded by U5 and U6 small nuclear RNAs (snRNAs), supported by specific proteins of the spliceosomal B and Bact complexes, including Prp8. HEH1 alternative splicing became independent of the supporting proteins following recalibration of the pre-mRNA-snRNA base pairings through changes in the 5’SS or U5 and U6 snRNAs. Assisted by proteins that stabilize low-fidelity, high-efficiency spliceosome conformations, the competing HEH1 5’SS are marked for alternative splicing by U5 and U6 snRNAs during the B to Bact transition of the spliceosome.

Introduction
Splicing of precursor-messenger RNAs (pre-mRNA) occurs through a multi-step process involving large and dynamic ribonucleoprotein (RNP) complexes called spliceosomes. Stage-specific spliceosomes are assembled on a pre-mRNA following intricate rearrangements of five distinct snRNPs containing cognate small nuclear RNAs (snRNAs) and associated proteins1,2. Specific trans-acting snRNPs recognise cis-acting splicing signals in pre-mRNAs: the donor 5’ splice site (5’SS) ‘GU’, the acceptor 3’ splice site (3’SS) ‘AG’, and the branch point adenosine ‘A’. Two steps of transesterification reactions, catalysed by divalent metal ions and U6 snRNA in the spliceosomes, remove introns and join exons to form functional mRNA. The spliceosome machinery is essential for constitutive and alternative splicing.

Multiple mRNAs can be produced from the same gene through alternative splicing. The process amplifies protein diversity from limited gene pools in an organism3,4. Different classes of alternative splicing exist in eukaryotes. Key among them occurs through the alternative selection of competing 5’SS5 and 3’SS4, and alternative splicing through competing 5’SS is a prevalent mode. A majority of pre-mRNAs in the budding yeast Saccharomyces cerevisiae have the highly conserved GUAUGU hexanucleotide as 5’SS donors6, but the sequences are highly variable in intron-rich eukaryotes, where alternative 5’SS selection is widespread7. Two major classes of 5’SS (NN/GURAG and AG/GUNNN; N represents any nucleotide and R represents a purine, ‘/’ indicates exon-intron junction), distinguished by the presence of ‘G’ at −1 and +5 positions, have been reported8. Alternative 5’SS are often found 4 nucleotides across the dominant 5’SS, and such occurrences in the human genome are estimated to be large9,10. The canonical ‘GU’ dinucleotide deviates to ‘GC’ in 0.87% of 5’SS in humans11.

The HEH1 gene (also called SRC1) in S. cerevisiae is alternatively spliced12–14. Its pre-mRNA has alternative donors with ‘GC’ and ‘GU’ dinucleotides embedded into two non-canonical 5’SS, /GCAAGU and /GUGAGU, arranged as a partially overlapping sequence /GCAA/GUGAGU. /GUGAGU is the dominant 5’SS, and splicing through this site generates mRNA-encoding a longer full-length protein, Heh1L. Splicing through the upstream /GCAAGU is tightly regulated and produces mRNA-encoding a shorter protein, Heh1S (the removal of the GCAA tetranucleotide gains an in-frame stop codon early in HEH1S mRNA). The two proteins share a common N-terminus but differ in their C-termini, thus attaining different topologies within the inner nuclear membrane and having distinct functions15–18.

Competing 5’SS are selected through early recognition of pre-mRNAs by RBPs (RNA-binding proteins), U1 snRNA and associated proteins19–21. Specific RBPs, SR (serine/arginine-rich) proteins, and hnRNPs (heterogeneous nuclear ribonucleoproteins) have been reported to be crucial for 5’SS selection5,22–27. These proteins act as splicing enhancers and silencers, and regulate spliceosome assembly on target pre-mRNAs in association with the U1 snRNP. The yeast RPL22B gene possesses an alternative 5 ′ splice site (5’SS) decoded through U1 snRNP-associated proteins, including Nam8 and Mud128. β-globin, MAPT, and WT1 genes in humans utilise the core U1 snRNP proteins (U1-70K, U1A, U1C) and regulatory splicing factors such as SR proteins and hnRNPs to modulate alternative 5′SS choice29.

The role of successive spliceosomal complexes in 5’SS selection has been reported30. U5 and U6 snRNAs modulate 5’SS selection8,31. In the absence of an authentic 5’SS, U5 snRNA can promote splicing from cryptic sites32,33. U1 snRNA-induced selection of a nearby 5’SS by U6 snRNA during spliceosome assembly34. m6A modification (methyl group addition at N6 position of adenosine) of U6 snRNA in humans, Caenorhabditis elegans, and Schizosaccharomyces pombe altered 5’SS selection, particularly of +4A positions in introns, and caused a shift to an alternative nearby site35,36. Furthermore, RNA structures modulate cis motifs in pre-mRNAs at different stages of the splicing cycle. A large number of S. cerevisiae introns are structured37. Structured introns in fission yeast and human are also regulatory to splicing outcomes and alternative splicing38,39. Thus, 5’SS selection depends on both early recognition and late processing of target introns.

The HEH1 gene is a valuable tool for investigating the RNA and protein factors required for alternative splicing through competing and overlapping 5’ SS. Its alternative splicing requires non-covalent associations of the ubiquitin-like protein Hub1 (UBL5 in humans) with the U1 and U2 snRNP-bridging RNA helicase Prp540, and the U4/U6.U5 tri-snRNP protein Snu6613. Thus, Hub1 acts early in spliceosome assembly by activating the RNA helicase, as well as later with the tri-snRNP component41. The latter activity suggests a possible role of the spliceosome core in HEH1 5’SS selection. We find that HEH1 alternative splicing requires optimal pairing of U5 and U6 snRNAs across a 16-nucleotide segment of the pre-mRNA across the two 5’SS at the exon 1-intron boundary. The selection of the competing 5’SS by U5 and U6 snRNAs and alternative splicing was supported by specific proteins of the spliceosome core, including Prp8.

Results
Trans-acting proteins for HEH1 alternative splicing
Between two 5’SS of HEH1/SRC1 pre-mRNA /GCAA/GUGAGU, the usage of the upstream 5’SS /GCAAGU requires non-covalent binding of the ubiquitin-like protein Hub1 to Snu66. The downstream 5’SS /GUGAGU was dominant and selected independently of this complex13. We searched for additional HEH1 alternative splicing factors in S. cerevisiae through the following approaches.

(i) HEH1–LACZ alternative splicing reporters HEH1S–LACZ and HEH1L–LACZ were prepared (Supplementary Fig. 1a), on the line of RP51–LACZ splicing reporter42, by fusing a HEH1 gene segment containing parts of the exons across the intron upstream of the β-galactosidase-encoding LACZ gene. In-frame translatable reporter mRNA would be made only after precise 5’SS selection and excision of the intron. Reporter activities were monitored in yeast strains grown on solid (X-Gal overlay assay) and liquid (ONPG assay) media. Splicing from the alternative 5’SS was diminished in the deletion strains of the RES (Retention and Splicing complex) complex subunits Snu17, Bud13, Pml1, and Urn1. The deletion of the NTC (Nineteen Complex) factor Ecm2 showed similar defects (Supplementary Fig. 1b, c). By contrast, splicing from the dominant 5’SS remained unaltered in the mutants, suggesting a role of these proteins in the selection of the weaker alternative 5’SS.

(ii) Heh1 protein isoforms were monitored in deletion and point mutants of splicing factors by Western blot assays. The gene was expressed with N-terminal epitope tags from a plasmid or tagged chromosomally. Supporting the data with the reporter assays (Supplementary Fig. 1a–c), Heh1S protein was strongly diminished in the knockout mutants of Snu17, Bud13, Pml1, and Urn1, in addition to Hub1 (Supplementary Fig. 1d). The previously reported D22A and H63L mutants of Hub1 were defective in HEH1 alternative splicing. Potential RNA-binding mutant of Hub1, as deduced from cryo-EM studies of spliceosomes43,44, showed a similar lack of Heh1S protein (Supplementary Fig. 1h). Alternative splicing defect was also seen in the Hub1-binding-deficient Snu66-HIND mutant (Supplementary Fig. 1j). Prp8 alleles were also screened for HEH1 alternative splicing. Heh1S level was strongly reduced in the prp8-101 (E1960K) allele. No other Prp8 mutant tested showed similar defects. While Heh1S protein was diminished in the above mutants, they showed more Heh1L than wt (Fig. 1a), possibly because the lack of competition from HEH1S 5’SS led to increased usage of the HEH1L site.

(iii) HEH1 mRNA isoforms in the yeast mutants were monitored by reverse-transcription (RT) PCR assays, with primers binding to HEH1 exons, followed by sequencing of the cDNAs. The two mRNAs show a single band. Since they differ in GCAA nucleotides at the exon junction (HEHL gains GCAA due to the usage of downstream 5’SS), electropherograms show two mixed peaks after the common exon. Areas under the peak were integrated to estimate the relative abundance of the two mRNAs. Consistent with the findings from reporter assays and protein analysis, the above mutants showed diminished levels of HEH1S mRNA, confirming the 5’SS selection defects at the RNA level (Fig. 1c). The electropherograms also showed increased HEH1L mRNA level in the mutants, compared to the wt strain (Fig. 1d), consistent with higher protein levels seen in Fig. 1a. Thus, spliceosomes defective in using HEH1S 5’SS used the dominant HEH1L 5’SS more efficiently in the absence of competition.

(iv) Splicing factors essential for cell viability could not be explored with deletion strains. Hub1’s proximity to them was used to explore their potential role in HEH1 alternative splicing. Snu66-binding-deficient hub1(D22A) in the free form was defective in the usage of HEH1 alternative 5’SS, but its linear fusion to Snu66, Prp38 and Prp8 restored alternative splicing in cell lacking Hub113. Interestingly, Heh1S protein was restored after anchoring Hub1 to Prp3, Prp6, and Prp31, but not to other proteins or the components of U1 and U2 snRNPs (Fig. 1e). Thus, Hub1’s proximity to specific proteins of the spliceosome B and Bact complexes (Snu66, Prp38, Prp8, Prp3, Prp6, and Prp31) promoted HEH1 alternative splicing.

The data discussed in i-iv indicated the need for specific proteins of the spliceosome core for HEH1S formation. However, their excess did not dominate the 5’SS selection towards HEH1S. Overexpression of none of these proteins increased the Heh1S isoform or suppressed the Heh1L (Supplementary Fig. 1m, n). Even Hub1’s constitutive presence in the spliceosome through its chromosomal fusions to two HEH1S splicing-promoting factors, Snu66 and Prp38, could not alter the Heh1S/Heh1L ratio (Supplementary Fig. 1o). Since excess trans-acting proteins could not modulate the spliceosome towards HEH1S site, we next asked how were the competing 5’SS decoded?

U5 and U6 snRNAs decode pre-mRNA cis elements for alternative 5’SS selection
From the splicing reporters discussed above, and known binding sites of U1, U5, and U6 snRNAs at exon-intron boundaries, we narrowed a 16-nucleotide pre-mRNA segment (1913–1928 of the HEH1 gene) and studied its importance for alternative 5’SS selection by mutagenesis (Fig. 2a). Proteins originating from the variants were detected by western blots, and mRNAs by RT-PCR followed by cDNA sequencing (Fig. 2b, c). Almost any change in this segment lost alternative splicing (barring the G(−1)A variant of HEH1S) and produced either one of the Heh1 isoforms. Changes leading to stop codons were studied by cDNA sequencing. GCAA to GUAA change of the upstream 5’SS gains an in-frame stop codon of HEH1L mRNA, abolishing the Heh1L protein (this data explains the choice of the ‘GC’ donor for HEH1S splicing; a ‘GU’ would have abolished Heh1L). These results highlighted the invariability of nucleotides around the competing 5’SS.

The alternative splicing defects in the pre-mRNA variants, however, did not correlate with predicted U1 snRNA–5’SS interactions. Although U1 snRNA-HEH1S 5′SS pairing was weaker in HEH1 pre-mRNA variants #4, #7, #11, and #13, Heh1S protein remained unaffected. Similarly, Heh1L expression did not diminish with weakened U1 pairing of #9, #12, and #14. Strikingly, we noticed a perfect correlation in the 5’SS choice and their expected base-pairing strengths with U5 and U6 snRNAs. 5’SS that paired stronger to U5 or U6 snRNAs was the preferred donor, and the use of the second site was minimal (Fig. 2 and Supplementary Fig. 2).

U5 and U6 snRNAs can promote alternative 5’SS selection without the protein factors
From Fig. 2, U5 and U6 (but not U1) snRNA variants with modified base pairing to the competing sites were expected to bias the selection. Indeed, the respective 5’SS was used more efficiently upon overexpression of U5 and U6 snRNA variants with strengthened base pairing (Fig. 3b). Similar overexpression of the U1 snRNA variant did not alter 5’SS choice. U5 snRNA U(98)G and U6 snRNA A(45)G variants with stronger binding to HEH1L 5’SS increased Heh1L protein (Supplementary Fig. 3). Conversely, U5 snRNA U(98)C and U6 snRNA U(46)CA(45)U variants with stronger binding to HEH1S 5’SS increased Heh1S mRNA and protein (Fig. 3b, c and Supplementary Fig. 3a, b, e, f). Thus, the 46th and 45th nucleotides of U6 snRNA, beyond the ACAGA box, were also crucial for selecting the alternative 5’SS by base pairing at +7 and +8 nucleotides in the intron.

If the proteins discussed above act by stabilising U5 and U6 snRNAs on the pre-mRNA substrate, the snRNA variants pairing strongly to HEH1S 5’SS should bypass the need for the proteins. Indeed, overexpression of U(98)C variant of U5 snRNA and U(46)C, A(45)U variants of U6 snRNA restored alternative splicing defects in Δhub1, Δsnu17, Δecm2, and Δurn1 mutants (Fig. 3 and Supplementary Fig. 3). Notably, the defect in prp8-101 strain was partially restored by overexpression of the U5 and U6 snRNA variants, suggesting an additional role of the core spliceosome protein Prp8 in 5’SS selection.

The Prp8-101 surface is critical for processing the ‘GC’ donor
Prp8 is reported to exist in two distinct conformations to maintain equilibrium between the two catalytic steps of splicing; consequently, two opposing alleles of Prp8 rescue splicing defects in one another45. Among the three first-step alleles of Prp8 (101, syf77, and R1753K; Supplementary Fig. 4c), only prp8-101 showed defective HEH1 alternative splicing. Combining the second step Prp8 alleles, prp8-161 and prp8-162, with prp8-101 did not restore the defects (Supplementary Fig. 4d, e). Thus, the alternative splicing defects in the first-step Prp8 allele, prp8-101, could not be explained by the above mechanism.

In a pre-mRNA variant (#7), the upstream 5’SS /GCAUGU with ‘GC’ donor was preferred over the downstream /GUGAGU in wt, Δhub1, Δsnu17, Δecm2, and Δurn1 strains (Fig. 4b and Supplementary Fig. 4b), likely due to the stronger pairing of +4U with the ACAGA box of U6 snRNA. However, the prp8-101 allele could not use this site. The defect in prp8-101 was explored using the ACT1–CUP1 splicing reporter with a single 5’SS46. The 5’SS /GCAUGU was used efficiently in all mutants, except for Δhub1 and prp8-101. C(+2)U-exchanged /GUAUGU was efficiently used in all strains, including Δhub1 and prp8-101 (Fig. 4c). Thus, the donor with +2C was poorly used in prp8-101 and Δhub1, leading to the loss of alternative splicing. Similarly, splicing from a +3C 5’SS variant /GUCUGU was defective in Δhub1, Δsnu66, and prp8-101 strains (Supplementary Fig. 4f). Thus, the Prp8-101 surface promotes selection (and catalysis?) from non-canonical 5’SS containing +2C and +3C.

Specific competing 5’SS allow alternative splicing without the regulatory proteins
Optimal decoding of competing 5’SS by U5 and U6 snRNAs, together with the use of ‘GC’ donor by Prp8, is the primary determinant of alternative splicing. The process is facilitated by specific proteins of the spliceosome. Thus, competing 5’SS with modified binding to the snRNAs should allow alternative selection in the absence of the regulatory proteins. More pre-mRNA variants were assayed to identify such 5’SS.

Two 5’SS variants allowed alternative splicing not only in wt yeast (Supplementary Fig. 2) but also in strains mutated for the regulatory proteins (Fig. 4b and Supplementary Fig. 4b). U5 and U6 snRNAs’ base pairing was recalibrated in #15 with two non-canonical 5’SS /GCAA/GUAUAU. In contrast, 5’SS pairing with U6 snRNAs was strongest in #18, containing two canonical 5’SS /GUAU/GUAUGU. The sites being canonical were readily used without the need for the protein factors, including the Prp8-101 surface. The efficient use of canonical 5’SS in the absence of the protein factors was further supported by HEH1–CUP1 reporters (Supplementary Fig. 4g–i).

Ecm2, Urn1, and the RES proteins bring the weaker 5’SS into competition
HEH1S expression from /GCAA/GUAUAU 5’SS (#15) in cells lacking the protein factors was intriguing, since the changes in this variant were beyond the hexanucleotide /GCAAGU. The outcomes could be explained by two possibilities: (i) weakening of the HEH1L 5’SS /GUGAGU to /GUAUAU rebalanced the competition in favour of the HEH1S site, or (ii) improved binding of +7A and +8U in /GCAAGUAU to U6 snRNA nucleotides U46 and A45 favoured the HEH1S 5’SS. To study these possibilities, alternative splicing reporters, HEH1S–CUP1 and HEH1L–CUP1, were made (Fig. 5b).

HEH1L 5’SS was weakened into /GUGAGA (#10; it also lacks potential HEH1S-U6 pairing at +7 and +8 positions) or /GUAUAA (#20; with enhanced U6 pairing at +7 and +8 positions of HEH1S) (Fig. 5a, c). HEH1S 5’SS selection in Δhub1 improved for reporters with enhanced U6 pairing at +7 and +8 positions of the 5’SS, and the defect was partially restored in the prp8-101 allele. The data confirmed the importance of U6 snRNA’s U46 and A45 nucleotides for alternative splicing discussed earlier in Fig. 3. The 5’SS selection in Δsnu17, Δurn1, and Δecm2, on the other hand, was improved by both enhanced U6 snRNA pairing to HEH1S and weakening the competition from HEH1L (Fig. 5d). Thus, Hub1 and Prp8 promote selection of 5’SS with +2C by stabilising U5 and U6 snRNA on the target pre-mRNA. On the contrary, Snu17, Urn1, and Ecm2 promote competition for the weaker 5’SS by stabilising snRNA–pre-mRNA interactions.

The data presented support dominant roles of U5 and U6 snRNAs and regulatory roles of B and Bact proteins for the selection of competing 5’SS in HEH1 pre-mRNA (Fig. 6).

Methods
Reagents
All the reagents and software used in the study are listed in Supplementary Table 1.

Plasmids, S. cerevisiae strains, yeast transformation and growth assays
Plasmids and strains used in this study are listed in Supplementary Tables 2, 3, respectively. The yJU75 yeast strain, plasmids PRP8 and prp8-101, and ACT1–CUP1 reporter were kind gifts from C. Guthrie and M. Konarska’s lab. Prp8 mutant strains were made by shuffling out the Ura+ PRP8 wild-type plasmid with the Trp+ prp8 mutants on 5-fluoroorotic acid (FOA) plates. The S. cerevisiae deletion strains of splicing factors were obtained from the Euroscarf haploid deletion library. Competent cell preparation and transformation were performed following previously published protocols.68,69. Chromosomal tagging for western blot, double knockout strains for genetic interactions, and splicing factors deletion in the yJU75 background were made by using the reported protocol.68,69. Protein overexpression was achieved by expressing clones under a strong gal promoter. For growth/spot assays, fivefold serial dilutions of cells were spotted on the indicated agar plates, and plates were incubated at the temperatures indicated in the figure.

Chromosomal fusion of hub1–D22A to the C-termini of splicing factors
Splicing by overlap extension (SOE) PCR was done to make hub1(D22A) C-terminal fusion to essential splicing factors. This fusion was done based on the published protocol68. Three sets of primers were used to amplify three fragments. First set of primers amplified a 200–300 bp fragment of the C termini of the desired gene, excluding the stop codon. The reverse primer of this had an overhang of 20–25 bps that was complementary to the 20–25 bps of hub1(D22A) N-terminus. The second set of primers amplified the full-length hub1(D22A) with the Nat antibiotic selection marker cassette from the plasmid clones of pFA6a-hub1(D22A)-natNT2. The third set of primers amplified a fragment of 200–300 bp from the stop codon of the gene that was going to be tagged. The forward primer for this had an overhang of 20–25 bp with the end of the antibiotic-resistance marker. All three fragments were amplified using Vent DNA polymerase. These fragments were confirmed based on their size on the agarose gel, and the correct fragments were gel-purified. All three fragments were mixed and joined by SOE PCR using Vent DNA polymerase using the forward primer of the first fragment and the reverse primer of the third fragment. The joined fragment was confirmed by agarose gel electrophoresis and transformed into the S. cerevisiae strain. The transformants were selected on agar plates with an antibiotic. The chromosomal fusion was confirmed by colony PCR using a forward primer specific to the gene and a reverse primer specific to the antibiotic cassette.

Splicing reporters
The splicing reporters used in this study are listed in Supplementary Table 4. Splicing reporters are modified forms of the conventional ACT1–CUP146 and RP51–LACZ42. The HEH1S–CUP1 reporters have a 121 bp segment of the S. cerevisiae HEH1 gene (1874-2095 nucleotides), including the intron, using BamHI and KpnI restriction sites. The HEH1S mRNA is spliced using GCAAGU 5’SS, generating in-frame CUP1 mRNA for translation. The methionine initiation codon was introduced by inserting a point mutation at C1881G. Initiation codon and variants of the 5’SS were made by site-directed mutagenesis (SDM) using specific primers, and the change was confirmed using Sanger sequencing. For HEH1L–CUP1 reporter, a 120 bp segment of the HEH1 gene (1874-2094 nucleotides) was used to bring the HEH1L mRNA spliced using GUGAGU 5’SS in-frame with the CUP1 gene. For reporter assays, competent cells from the S. cerevisiae strain yJU75 were transformed with the reporters, and transformants were selected in media lacking leucine. 1 OD600 cells were spotted on solid media with different concentrations of CuSO4. The plates were incubated for 2–3 days at 30°C. Similarly, for HEH1–LACZ reporters, the RP51 part of the RP51–LACZ reporter (gift from M. Rosbash) was replaced with a HEH1 fragment of 399 bp (1792-2191 nucleotides) and 398 bp (1792-2190 nucleotides) to obtain HEH1S–LACZ and HEH1L–LACZ reporters, respectively.

β-galactosidase assay
β-galactosidase activity was observed on both solid and liquid media. The X-gal overlay and the ONPG assay were performed by following published protocols70.

RNA isolation and cDNA synthesis
RNA isolation and cDNA synthesis were done as described previously71. Briefly, five OD600 cells in the log phase were harvested. Total RNA was isolated by the hot acid phenol method using 15 mL phase lock tubes for phase separation. Residual DNA was removed by treating RNA with DNase I for 15 min at room temperature, followed by RNA clean-up using a Zymo-SpinTM II column. cDNA synthesis from 2 μg total RNA was done using reverse transcriptase (RT) and random-hexamer primers at 37°C for 2 h. Splicing defects were monitored by detecting intron-containing transcripts or post-splicing mature transcripts using exonic primers across the intron and analysed by agarose gel electrophoresis. For cDNA sequencing, five identical PCR reactions were performed. The PCR products were pooled and kept overnight for precipitation after adding 2.5 times the volume of isopropanol and 1/10th the volume of 3 M sodium acetate at −20°C. DNA was pelleted by centrifugation for 15 min at 4°C at maximum speed. The pellet was washed twice with 70% ethanol. Dried pellets were dissolved in 30 μl of nuclease-free water, and 10 μl of the DNA was sequenced by Sanger sequencing using a primer nested inside the forward primer used in amplification.

Quantification of HEH1 mRNA isoforms
HEH1 alternative splicing was analysed using RT-PCR followed by cDNA sequencing. We have two sets of primers. To check the splicing defect, we have used the F1 and R1 primers. We have cut the mRNA band (the lower one) and gel-purified it. We have used F2, which is nested in the F1 primer and R1, for reamplifying the mRNA band. We did ethanol precipitation of the PCR product and sent it for Sanger sequencing using the F2 primer. The primer sequences are listed in Supplementary Table 5. HEH1S and HEH1L mRNAs differ in GCAA nucleotides at their exon junctions (HEHL gains them due to the usage of downstream 5’SS), the electropherogram shows mixed peaks corresponding to the two isoforms after the exon-exon junction. The area under the peaks was integrated to find the relative abundance of the two mRNAs. Both common and isoform-specific peaks were analysed within the region corresponding to the 5’ exon/intron boundary. To understand alternative 5’SS usage, common nucleotides after the junction in the two isoforms were excluded from further analysis. For each site, we recorded the nucleotide proportions possible for both the cDNAs and calculated the ratio between the two nucleotides, normalising their combined total to 100%. The average value of both forms is directly related to their respective 5’SS usage.

Relative expression of HEH1L in wild-type and yeast mutants was obtained from the cDNA electropherogram. The areas under ten common and ten mixed peaks (before and after the junction, respectively), were integrated and averaged. Similarly, an area under the peaks specific to HEH1L was integrated and averaged. The ratio of common and HEHL peaks for different strains was obtained. The fold difference in HEH1L expression in the mutants was calculated against the wild-type strain by dividing the average of the area under HEH1L peaks (after the junction) by the average of the area under common peaks (before the junction).

Western blots
For protein western blots (immunoblotting), logarithmically growing cells equivalent to 2 OD600 were harvested. Whole protein was extracted using the trichloroacetic acid (TCA) method68. Total proteins used for Western blots were denatured by heating cells in a high-urea buffer at 65°C for 15 min and then centrifuged. The soluble protein was separated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) 8% gels, or NuPAGE™ 4–12% Bis-Tris gels (Invitrogen), transferred to a polyvinylidene difluoride (PVDF) membrane, and probed with specific primary and secondary antibodies. Protein bands were quantified using ImageJ following the published method72.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Banyal et al. describes an investigation into the factors affecting the selection of two alternative 5' splice sites in a yeast gene. These sites were separated by just 4 nucleotides, and the 5'-most site (S) has a GC rather than the more common GU at the 5' end of the intron. The authors show first that several RES and NTC proteins, components of the pre-catalytic spliceosome complexes, are required for use of the S site, and its use is also promoted by fusion of several other proteins to a protein known to be required for S site use. The major part of the manuscript, though, is concerned with the roles of U5 and U6 snRNAs. Here an extensive set of mutants was used to show that the balance of use of the two sites depended as expected on the strength of base-pairing between the 5' splice sites and the snRNAs. Moreover, stronger base-pairing reduced the dependence of site S on the proteins. Use of site S with a GC motif was found to depend on the protein Prp8, a major component of the spliceosome. Finally, some evidence was presented showing that the extent of U6 base-pairing to a pre-mRNA might extend further than is usually considered to be the case. The possible roles of the proteins were discussed.\nAlthough yeast does not exhibit extensive alternative splicing patterns, the dissection of factors influencing the use of two close potential sites might be broadly applicable. Thus, for example, if a higher eukaryotic U1 snRNP/5' splice site complex is brought into a complex formed on a 3' splice site by U2 snRNP and the tri-snRNP, then the selection of the right local site for splicing might involve similar interactions to those described here. That said, it is quite hard to argue that there is any definitive new knowledge here. The importance of U5 and U6 base-pairing has been known for many years, and establishing a list of proteins whose actions should now be investigated is not quite the same as unveiling a new mechanism.\nThe work is generally of a high standard. The quantitative assays in the main figures were all done as replicates, with error bars, but many of the supplementary figures do not contain error bars and were presumably single determinations. That said, there were usually assays of both ONPG production and protein expression, which supported each other, and I do not doubt the validity of the results. I do have minor comments about particular points, listed below.\nAlthough the interpretation of the mutant sequences given by the authors is supported by the use of compensating U snRNA mutations, the authors might have considered whether the effects of the mutations around the 5' splice sites could be connected with RNA secondary structures. The absence of any consideration of RNA structure in the entire text is regrettable. A similar point can be made about the alignments of the sequences with the complementary U5 and U6 snRNAs. Is it possible that alternative, slightly shifted alignments or bulged bases can be tolerated?\nComments for improvements\n1. The title should specify yeast and that the sites are 4 nts apart, i.e., within the 5' splice site consensus region.\n2. Line 84 and elsewhere: the L site is referred to as the dominant site, although the use of S is predominant in the WT. This is confusing.\n3. Line 150. The only evidence that there is no correlation with U1 snRNA interactions is in ED Fig3a and b.\n4. Lines 151-4. Mutant 8 does not fit the 'perfect correlation'. In Fig 2c, the S complementarity to U6 is improved, but the use of S vanished. Mutant 14 showed a greater improvement in S complementarity, but again the site was not used. In mutant 16, U6 base-pairs better to S than L, but the usage ration is as WT. The claim of a perfect correlation should be qualified by a closer look at the data.\n5.Re ED Fig. 3, can the authors explain the shift seen in U5-U98A to the L site?", "reviewer_2": "This is an interesting analysis of a case of alternative splicing of the HEH1 intron in yeast S. cerevisiae, in which one of the two overlapping 5’SS signals — weaker /GC and stronger /GU sites — is utilized. The careful, very well done genetic analyses show that the 5’SS selection is decided not at the stage of U1 recognition (as often described for mammalian alternative splicing) but later, in the pre-catalytic B/Bact complexes, and depends on U5 and U6 interactions, as well as spliceosomal proteins of RES and Hub1-Snu66 complexes. In addition, prp8-101 allele, known to improve the first step catalytic conformation, strongly inhibits the use of the weaker /GC 5’SS.\nThe authors present a very large (perhaps unnecessarily large?) number of experiments, supporting their claims. My only reservation is that the results and discussion describe the mechanism of 5’SS selection in a way that is difficult to follow. A simpler, clearer description of the mechanism and its relevance to alternative splicing in other organisms would greatly improve this manuscript.\nSpecific points:\n1. In discussing the importance of U6 pos. 45 and 46 contacting intron pos. +7, +8 at the 5’SS, the authors should reference the original Staley and Guthrie, 1999 Mol Cell paper that first described this interaction as stabilizing the 5’SS. Similarly, the importance of U5 loop pairing to the 5’ exon has been demonstrated by Newman and Norman, 1991.\n2. The authors claim that prp8-101 allele specifically inhibits the weaker /GC 5’SS selection. I would expect that all strong 1st step prp8 alleles would have similar effects (the authors tested only one other 1st step prp8 allele, syf77), as they stabilize the first step catalytic conformation of the spliceosome (B*) and thus would be expected to destabilize B/Bact. Surprisingly, the combination of two opposing prp8 alleles, the 1st step prp8-101 and the 2nd step prp8-161, did not cancel the prp8-101 effect alone. This prompted the authors to dismiss the possibility that the inhibition of the /GC 5’SS splicing by prp8-101 results from the previously described mechanism (the 1st step 8-101 allele improves the first step catalysis, and would thus be expected to inhibit the preceding conformation of B/Bact complexes). I checked the referenced Liu et al. paper and found that the prp8-101 + 8-161 combination does not fully restore wt splicing levels (suggesting that the effects of 8-101 allele are stronger than those of 8-161 allele). Instead, the authors should have used prp8-R1753K + 8-161 combination, as it would be a more appropriate (better balanced) pair to test. Thus, the authors’ interpretation may be incorrect.\n3. Fig. 1d. Why is the overall mRNA level improved in prp8-101 and strains with deleted for protein components of RES and Hub1-Snu66 complexes? I do not understand how reduced competition from the /GC site could increase splicing over the wt levels (in the case of prp8-101, the increase is 2-fold!). Is a similar increase in mRNA level observed for a reporter with a single 5’SS (mentioned in the paper)? The catalytic conformation of the prp8-101 spliceosome (B*) may be relatively more stable than B/Bact, improving catalysis of these 5’SS. This possibility should be at least discussed, if not tested.", "reviewer_3": "In this manuscript, the authors use the yeast HEH1 gene as a model system to study the mechanism of alternative 5’ ss selection. They demonstrated that this selection does not happen at the U1 snRNA recognition stage. Instead, by mutating nucleotides around the 5’ ss or on U5 or U6 snRNA and deleting spliceosomal proteins, they showed that the alternative 5’ ss compete with each other and is selected during B to Bact transition, by the strength of their interactions with U5/U6 snRNA facilitated by protein factors in the B or Bact complex. The manuscript provided important mechanistic details of an alternative splicing mechanism outside the U1 snRNA recognition stage. The experiments are well designed and convincing. A few issues need to be addressed to further strengthen the manuscript.\n1. The idea of 5’ ss selection by basepairing with U5 or U6 snRNA if not new (for example, citation 8 in the manuscript), although this is not clear from the manuscript. It will be beneficial if the authors can add some discussion on the status of the field and highlight the major new insight and contribution provided by this manuscript.\nRelated to this, some discussion on whether similar mechanism is likely to exist in other eukaryotic systems will be helpful.\n2. Page 2, line 60: “have” should be replaced with “having”\n3. Page 4, line 112, it will be helpful to specify where the primers bind to better understand these data.\n4. Page 5, line 140. The 16 nucleotides fragment that the authors mutated are referred to as 1913 to 1928 here, but they were labeled as 1912 to 1927 in Fig. 2a.\n5. In Fig. 2a, it will be useful to add a column that show the basepairing with U1 snRNA.\nFig. 2c, variant 7, last column, missing the underline that indicates the mutant\n6. Fig. 3c: orange triangle is not shown\n7. Fig. 4C: underline needs to be defined in figure legend\n8. Page 9, lines 293-299, it is not clear what this paragraph is trying to convey. Need to clarify.\n9. abstract: “Alternative splicing of pre-mRNA bearing introns with competing and overlapping 5’ ss produces more RNAs.”\n“more” here can be interpreted as higher level instead of more than one.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to comments from reviewers (reviewers’ comments are italicised, our response\nis shown in coloured text)\nReviewer 1:\nThe manuscript by Banyal et al. describes an investigation into the factors affecting the\nselection of two alternative 5' splice sites in a yeast gene. These sites were separated by just\n4 nucleotides, and the 5'-most site (S) has a GC rather than the more common GU at the 5'\nend of the intron. The authors show first that several RES and NTC proteins, components of\nthe pre-catalytic spliceosome complexes, are required for use of the S site, and its use is also\npromoted by fusion of several other proteins to a protein known to be required for S site use.\nThe major part of the manuscript, though, is concerned with the roles of U5 and U6 snRNAs.\nHere, an extensive set of mutants was used to show that the balance of use of the two sites\ndepended, as expected on the strength of base-pairing between the 5' splice sites and the\nsnRNAs. Moreover, stronger base-pairing reduced the dependence of site S on the proteins.\nUse of site S with a GC motif was found to depend on the protein Prp8, a major component of\nthe spliceosome. Finally, some evidence was presented showing that the extent of U6 base-\npairing to a pre-mRNA might extend further than is usually considered to be the case. The\npossible roles of the proteins were discussed. Although yeast does not exhibit extensive\nalternative splicing patterns, the dissection of factors influencing the use of two close potential\nsites might be broadly applicable. Thus, for example, if a higher eukaryotic U1 snRNP/5' splice\nsite complex is brought into a complex formed on a 3' splice site by U2 snRNP and the tri-\nsnRNP, then the selection of the right local site for splicing might involve similar interactions\nto those described here. That said, it is quite hard to argue that there is any definitive new\nknowledge here. The importance of U5 and U6 base-pairing has been known for many years,\nand establishing a list of proteins whose actions should now be investigated is not quite the\nsame as unveiling a new mechanism.\nResponse: We sincerely thank the referee for reviewing the manuscript, highlighting important\nfindings of the manuscript and constructive feedback and suggestions. Indeed, the roles of U5\nand U6 snRNAs for promoting 5’ splice sites usage are known. However, their roles in the\nalternative selection of 5’SS have not been explored. Previous studies on alternative splicing\nvia competing 5’SS have focused on the early recognition of the alternative 5 'splice site (5'SS)\nby U1 snRNA, with the help of SR proteins and hnRNPs. In our study, we have shown the\nroles of U5 and U6 snRNA in alternative splicing via competing 5 'splice sites (5’SS), which\nare regulated by trans-acting factors of the B and Bact complexes. U1 snRNA did not play a\nrole in the selection of closely spaced 5’SS. The protein factors identified in this study have\nnot been associated with alternative 5’SS selection (besides Hub1 and Snu66). To the best of\nour knowledge, HEH1 is the only known in vivo splicing target of these factors, described in\nthis study. Similarly, in vivo targets of the prp8-101 allele have not been reported. The\ndominant roles of U5 and U6 snRNAs in selecting the alternative sites over the trans-acting\nproteins, bring further novelty in the findings.\nThe work is generally of a high standard. The quantitative assays in the main figures were all\ndone as replicates, with error bars, but many of the supplementary figures do not contain error\nbars and were presumably single determinations. That said, there were usually assays of both\nONPG production and protein expression, which supported each other, and I do not doubt the\nvalidity of the results. I do have minor comments about particular points, listed below.\nimprovements.\nAlthough the interpretation of the mutant sequences given by the authors is supported by the\nuse of compensating U snRNA mutations, the authors might have considered whether the\neffects of the mutations around the 5' splice sites could be connected with RNA secondary\nstructures. The absence of any consideration of RNA structure in the entire text is regrettable.\nA similar point can be made about the alignments of the sequences with the complementary\nU5 and U6 snRNAs. Is it possible that alternative, slightly shifted alignments or bulged bases\ncan be tolerated?\nResponse: We thank the reviewer for highlighting the importance of RNA secondary structure\nin splicing. We did not wish to ignore the role of RNA structures in pre-mRNA splicing. In fact,\nwe published a number of splicing-promoting potential structures in S. pombe introns (Anil et\nal., Nucleic Acids Research 2022). We just did not have any concrete evidence of the role of\nRNA structures in HEH1 alternative splicing. The recently published comprehensive study on\nintronic structures in yeast did not indicate structures in the HEH1 intron (Rangan et al. NSMB\n2025).\nWe have incorporated potential roles of RNA structures in the discussion section (lines 250-\n253). The added lines are: “RNA structures modulate cis motifs in pre-mRNAs at different\nstages of the splicing cycle. A large number of S. cerevisiae introns are structured42. Structured\nintrons in fission yeast and human are also regulatory to splicing outcomes and alternative\nsplicing43,44”. We acknowledge this remains an open question which can be studied in a future\nwork. O’Keefe et al. (EMBO J.1998 and Cell 1996) showed that a single nucleotide change in\nU5 snRNA loop 1, especially a substitution, can be tolerated.\nComments for improvements:\n1. The title should specify yeast and that the sites are 4 nts apart, i.e., within the 5' splice site\nconsensus region.\nResponse: We have changed the title and made it more specific for yeast. The updated title\nis “Mechanism of alternative splicing of yeast HEH1 through competing 5’ splice sites”. We\nincorporated the words “four nucleotide-apart” 5’SS in the Abstract.\n2. Line 84 and elsewhere: the L site is referred to as the dominant site, although the use of S\nis predominant in the WT. This is confusing.\nResponse: The HEH1L site (GUGAGU) is referred to as the dominant one because of its\nbetter pairing with U5 and U6 snRNA as compared to the HEH1S site (GCAAGU), which also\ncontains the non-canonical ‘GC’. We have also shown that HEH1L production occurs\nindependently of the trans-acting factors that are required for the HEH1S selection. Thus,\nHEH1L appears to be a better splice site, which makes us use the term dominant over the\nadjacent HEH1S site. The term 'dominant' does not refer to protein level.\n3. Line 150. The only evidence that there is no correlation with U1 snRNA interactions is in ED\nFig. 3a and b.\nResponse: In Fig. 1e, we have checked the role of U1 proteins Mud1 and Nam8 by fusing\nhub1(D22A) at the C-termini of these factors. In Figs. ED1b and 1c, we have checked Nam8\ndeletion for alternative splicing by X-gal overlay and by β-galactosidase assay. Mud1 deletion\nis checked for alternative splicing by Western blot in Supplementary Fig.1d. We have further\nchecked the possible role of Nam8, which is important for the splicing via suboptimal 5’SS by\nsnRNA with strengthened base pairing to the GC site in HEH1S. These experiments did not\nsupport the role of U1 snRNA and associated proteins in alternative splicing via 4nts apart 5’\nsplice sites. It is likely that the competing sites are recognised in a single step by U1 snRNA\nand then handed over separately to U5 and U6 snRNAs. We have added two sentences on\nthis hypothesis.\n4. Lines 151-4. Mutant 8 does not fit the 'perfect correlation'. In Fig. 2c, the S complementarity\nto U6 is improved, but the use of S vanished. Mutant 14 showed a greater improvement in S\ncomplementarity, but again, the site was not used. In mutant 16, U6 base-pairs better to S\nthan L, but the usage ratio is the same as that of WT. The claim of a perfect correlation should\nbe qualified by a closer look at the data.\nResponse: In Mutant 8 (GCAAGUAAGU) improves U6 snRNA pairing with the HEH1S 5’SS,\nbut this mutation converts the HEH1L 5’SS into a canonical hexanucleotide (GUAAGU). As a\nresult, the HEH1L site dominates spliceosome recognition. The dominance of canonical sites\nGUAAGU and GUAUGU, irrespective of the position, could not be correlated with the base-\npairing efficiency of a single snRNA. Similarly, in Mutant 14 (GCAUGUAUGU), U6 pairing with\nHEH1S is enhanced, but the adjacent HEH1L 5’SS sequence becomes the canonical\nGUAUGU, again becoming the sole site for splicing. This strong HEH1L site suppresses usage\nof the HEH1S 5’SS, preventing detection of the HEH1S form.\nIn mutant 16 (GCAAGUAUUA), it improves U6 snRNA pairing to the HEH1S site, and\ndecreases the pairing for the HEH1L site. Although we are seeing both the forms in mt 16 but\nthe proportion of Heh1S protein (0.8815) in mt 16 is more compared to Heh1S (0.6411) in wt.\n5. ED Fig. 3, can the authors explain the shift seen in U5-U98A to the L site?\nResponse: Comparing U5-WT, U5-U98G, and U5-U98A has different effects on splice site\npairing and splice form abundance. When comparing U5-WT to U5-U98G, U5 pairing with the\nL site increases, while pairing to the S site decreases. As a result, the L form predominates in\nthe wild type, and only the L form is observed in the U98G mutant. In contrast, when U5-U98G\nis compared with U5-U98A, U5 pairing to the L site decreases in U98A, while pairing to the S\nsite remains unchanged. This reduction in L site pairing in U5-U98A makes the S site more\naccessible, resulting in more of the S form in the U98A mutant compared to U98G.\nWe thank the reviewer for their time, valuable comments, and suggestions for improving the\nmanuscript.\nThis is an interesting analysis of a case of alternative splicing of the HEH1 intron in yeast S.\ncerevisiae, in which one of the two overlapping 5’SS signals — weaker /GC and stronger /GU\nsites — is utilized. The careful, very well done genetic analyses show that the 5’SS selection\nis decided not at the stage of U1 recognition (as often described for mammalian alternative\nsplicing) but later, in the pre-catalytic B/Bact complexes, and depends on U5 and U6\ninteractions, as well as spliceosomal proteins of RES and Hub1-Snu66 complexes. In addition,\nprp8-101 allele, known to improve the first step catalytic conformation, strongly inhibits the use\nof the weaker /GC 5’SS.\nThe authors present a very large (perhaps unnecessarily large?) number of experiments,\nsupporting their claims. My only reservation is that the results and discussion describe the\nmechanism of 5’SS selection in a way that is difficult to follow. A simpler, clearer description\nof the mechanism and its relevance to alternative splicing in other organisms would greatly\nimprove this manuscript.\nResponse: We thank the reviewer for reviewing the manuscript. We also appreciate the\npositive feedback from the reviewer on our work. We agree with the reviewer’s concern\nregarding the complexity of the manuscript and have addressed this concern.We simplified\nthe discussion by adding important details and reshuffling the text. The key information has\nnot been alteredSpecific points:\n1. In discussing the importance of U6 pos. 45 and 46 contacting intron pos. +7, +8 at the 5’SS,\nthe authors should reference the original Staley and Guthrie, 1999 Mol Cell paper that first\ndescribed this interaction as stabilizing the 5’SS. Similarly, the importance of U5 loop pairing\nto the 5’ exon has been demonstrated by Newman and Norman, 1991.\nResponse: We thank the reviewer for the suggestion to include these important references.\nWe have cited these papers in the discussion (lines 240 and 244).\n2. The authors claim that prp8-101 allele specifically inhibits the weaker /GC 5’SS selection. I\nwould expect that all strong 1st step prp8 alleles would have similar effects (the authors tested\nonly one other 1st step prp8 allele, syf77), as they stabilize the first step catalytic conformation\nof the spliceosome (B*) and thus would be expected to destabilize B/Bact.\nResponse: We thank the reviewer for this point. We have tested the three first step alleles,\nR1753K in Supplementary Fig. 1l, prp8101, and syf77 in Supplementary Fig. 1k. For further\nclarity, we have again tested the three alleles together in Supplementary Fig. 4c. We have not\nseen any strong alternative splicing defect in Prp8 alleles other than prp8-101. There is a\nsubtle defect in the R1753K allele, but the defect is not strong enough to monitor a rescue by\nsecond-step alleles (Supplementary Fig.4c ).\nSurprisingly, the combination of two opposing prp8 alleles, the 1st step prp8-101 and the 2nd\nstep prp8-161, did not cancel the prp8-101 effect alone. This prompted the authors to dismiss\nthe possibility that the inhibition of the /GC 5’SS splicing by prp8-101 results from the\npreviously described mechanism (the 1st step 8-101 allele improves the first step catalysis,\nand would thus be expected to inhibit the preceding conformation of B/Bact complexes). I\nchecked the referenced Liu et al. paper and found that the prp8-101 + 8-161 combination does\nnot fully restore wt splicing levels (suggesting that the effects of 8-101 allele are stronger than\nthose of 8-161 allele). Instead, the authors should have used prp8-R1753K + 8-161\ncombination, as it would be a more appropriate (better balanced) pair to test. Thus, the\nauthors’ interpretation may be incorrect.\nResponse: We agree with the reviewer that, in reference Liu et al., the double mutant prp8-\n101 and 161 does not fully rescue the defect. In the same reference, the double mutant prp8-\n4\n101 and 162 has better splicing efficiency over the prp8-101 and 161 combinations. We tested\nthe HEH1 alternative splicing defect in prp8-101, prp8-162 double mutant (Supplementary\nFig.4e), but did not observe a significant gain in HEH1S. Following the reviewer's suggestion,\nwe combined prp8-R1753K with prp8-161 and checked the rescue of the alternative splicing\ndefect. The defect in prp8-R1753K was not strong enough to monitor a rescue by second-step\nalleles. Indeed, we have not seen any significant gain in HEH1S splicing (Supplementary\nFig.4e). It cannot be ruled out that prp8-101, in addition to being a first-step allele, plays an\nadditional role in HEH1 alternative splicing. Indeed, the usage of splice sites containing ‘C’ at\n+2 and +3 positions of the intron was strongly diminished in this mutant (Supplementary\nFig.4f).\n3. Fig. 1d. Why is the overall mRNA level improved in prp8-101 and strains with deleted for\nprotein components of RES and Hub1-Snu66 complexes? I do not understand how reduced\ncompetition from the /GC site could increase splicing over the wt levels (in the case of prp8-\n101, the increase is 2-fold!). Is a similar increase in mRNA level observed for a reporter with\na single 5’SS (mentioned in the paper)? The catalytic conformation of the prp8-101\nspliceosome (B*) may be relatively more stable than B/Bact, improving catalysis of these 5’SS.\nThis possibility should be at least discussed, if not tested.\nResponse: In Fig. 1d, we present the levels of the constitutive HEH1L mRNA across all strains\n(wild type and mutants), rather than total HEH1 mRNA, based on the electropherogram data\nshown in Fig. 1c. In wild type, both isoforms are present in nearly equal proportions, indicating\nthat the spliceosome recognizes S and L sites on nearly half of total pre-mRNA populations.\nIn the mutants, recognition of the HEH1S 5′SS is markedly reduced compared to wild type.\nBut when HEH1 alternative splicing factors are absent or defective, the spliceosome gets more\npre-mRNA substrates for the L site. As a result, in prp8-101 (and other mutants), where S-site\nrecognition is minimal, there is an increased accumulation of HEH1L mRNA.\nWe thank the reviewer for their time, valuable comments, and suggestions for improving the\nmanuscript.\nIn this manuscript, the authors use the yeast HEH1 gene as a model system to study the\nmechanism of alternative 5’ ss selection. They demonstrated that this selection does not\nhappen at the U1 snRNA recognition stage. Instead, by mutating nucleotides around the 5’ ss\nor on U5 or U6 snRNA and deleting spliceosomal proteins, they showed that the alternative 5’\nss compete with each other and is selected during B to Bact transition, by the strength of their\ninteractions with U5/U6 snRNA facilitated by protein factors in the B or Bact complex. The\nmanuscript provided important mechanistic details of an alternative splicing mechanism\noutside the U1 snRNA recognition stage. The experiments are well-designed and convincing.\nA few issues need to be addressed to further strengthen the manuscript.\nResponse: We appreciate the reviewer’s positive feedback on our work and valuable\nsuggestions to make the work more understandable.\n1. The idea of 5’ ss selection by basepairing with U5 or U6 snRNA if not new (for example,\ncitation 8 in the manuscript), although this is not clear from the manuscript. It will be beneficial\nif the authors can add some discussion on the status of the field and highlight the major new\ninsights and contributions provided by this manuscript. Related to this, some discussion on\nwhether a similar mechanism is likely to exist in other eukaryotic systems will be helpful\nResponse: We have added the following text to the discussion (lines 246-247). “Roles of U5\nand U6 snRNAs are also reported for 5’SS selection”.\nResponse: We have added these points to the discussion (lines 235-238). The mechanism is\nlikely to be conserved for closely-placed 5’SS in other eukaryotes. The ‘GC’ dinucleotide donor\nis found in nearly 1% introns in humans11, and the transacting proteins supporting its selection\nin yeast are conserved across eukaryotes.\n2. Page 2, line 60: “have” should be replaced with “having”\nResponse: We have rectified this error. Thank you for pointing that out.\n3. Page 4, line 112, it will be helpful to specify where the primers bind to better understand\nthese data.\nResponse: We agree with the reviewer and are thankful for pointing this out. We have added\na schematic in Fig1b and c showing the primers used. We have updated “Quantification of\nHEH1 mRNA isoforms” in the methods section.\n4. Page 5, line 140. The 16-nucleotide fragment that the authors mutated is referred to as\n1913 to 1928 here, but they were labelled as 1912 to 1927 in Fig. 2a.\nResponse: This was an error. We have corrected the figures. The first nucleotide is G, which\nis at 1913 nucleotide, and the last nucleotide is C, which is at 1928 position, are part of the\n16-nucleotide fragment.\n5. In Fig. 2a, it will be useful to add a column that show the basepairing with U1 snRNA. Fig.\n2c, variant 7, last column, missing the underline that indicates the mutant.\nResponse: We have modified Fig. 2a by adding extra rows showing pairings with U1 snRNA.\nWe have corrected the error in Fig. 2c.\n6\n6. Fig. 3c: orange triangle is not shown.\nResponse: We have corrected this error.\n7. Fig. 4C: underline needs to be defined in figure legend\nResponse: We have defined the underline in the figure legend.\n8. Page 9, lines 293-299, it is not clear what this paragraph is trying to convey. Need to clarify.\nResponse: We have rewritten the paragraph for better understanding.\n9. abstract: “Alternative splicing of pre-mRNA bearing introns with competing and overlapping\n5’ ss produces more RNAs.” “more” here can be interpreted as a higher level instead of more\nthan one.\nResponse: We have replaced more with two mRNAs in the abstract.\nWe thank the referee for their time, valuable comments, and suggestions for improving the\nmanuscript. The revised manuscript has improved significantly.\n7"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The rebuttal from Banyal et al. has addressed many of my points. However, two remain outstanding.\n1. The response to my first comment states the following:\n'Indeed, the roles of U5 and U6 snRNAs for promoting 5’ splice sites usage are known. However, their roles in the\nalternative selection of 5’SS have not been explored. Previous studies on alternative splicing via competing 5’SS have\nfocused on the early recognition of the alternative 5 'splice site (5'SS) by U1 snRNA, with the help of SR proteins and\nhnRNPs.'\nThis is simply not correct. The first evidence for a role of U5 snRNA in determining the usage of alternative sites was\npublished by Newman and Norman (1991), working with yeast. A cryptic site just 12 nts form the normal site was activated\nby a U5 mutation (Cell 65, 115-123). in a mammalian system, Cortes, Sontheimer, Siewert and Steitz (EMBO J. 12, 5181-9)\nshowed in 1993 that different exogenous U5 mutants could alter the usage of a set of cryptic 5' splice sites. Regarding U6, a\nvery comprehensive series of experiments by Hwang and Cohen in 1996 showed that the choice between two alternative\nsites, both close to a U1 binding site, was made by U6 snRNA (Genes Dev.10, 338-50). It is not difficult to find these\nseminal references. The authors might also be interested in the recent work from Simpson and colleagues, in which 5' splice sites\nare classified according to their dependence primarily on U5 or U6 base-pairing (Shen et al., 2024, Nuc. Acids Res. 52,\n9139-60; Parker et al., 2025, Open Biol. 15, 240293). The Shen et al. paper describes how the loss of methylation of U6\nsnRNA affects base-pairing with 5' splice sites containing a +4A and causes a shift to alternative nearby sites (Figure 2F).\nI repeat, therefore, that it is quite hard to argue that there is any definitive new knowledge in the current manuscript.\nEstablishing a list of proteins whose actions should now be investigated is not quite the same as unveiling a new\nmechanism.\n2. The response to point 5 was rather misleading. The authors compared the effects of U98A to those of U98G, whereas a\ncomparison with the WT U5 shows that the U98A mutant reduces potential base-pairing to both the L and S sites by one G-\nU pair (Supp. Fig 3a). Nonetheless, there appears to be a relative increase in the L isoform (Supp. Fig. 3b).", "reviewer_2": "The revised version corrects a number of previous issues, significantly improving the manuscript. The authors’ response to\nreviewers’ comments is very thorough, clarifying several previous questions.", "reviewer_3": "The authors have adequately addressed my concerns."}, "author_response": "NCOMMS-25-52483-T (Banyal et al.)\nResponse to comments from reviewers (reviewers’ comments are italicised, our response\nis shown in coloured text)\nReviewer 1\nThe rebuttal from Banyal et al. has addressed many of my points. However, two remain\noutstanding.\n1. The response to my first comment states the following: 'Indeed, the roles of U5 and U6\nsnRNAs for promoting 5’ splice sites usage are known. However, their roles in the alternative\nselection of 5’SS have not been explored. Previous studies on alternative splicing via\ncompeting 5’SS have focused on the early recognition of the alternative 5 'splice site (5'SS)\nby U1 snRNA, with the help of SR proteins and hnRNPs.'\nThis is simply not correct. The first evidence for a role of U5 snRNA in determining the usage\nof alternative sites was published by Newman and Norman (1991), working with yeast. A\ncryptic site just 12 nts form the normal site was activated by a U5 mutation (Cell 65, 115-123).\nin a mammalian system, Cortes, Sontheimer, Siewert and Steitz (EMBO J. 12, 5181-9)\nshowed in 1993 that different exogenous U5 mutants could alter the usage of a set of cryptic\n5' splice sites. Regarding U6, a very comprehensive series of experiments by Hwang and\nCohen in 1996 showed that the choice between two alternative sites, both close to a U1\nbinding site, was made by U6 snRNA (Genes Dev.10, 338-50). It is not difficult to find these\nseminal references. The authors might also be interested in the recent work from Simpson\nand colleagues, in which 5' splice sites are classified according to their dependence primarily\non U5 or U6 base-pairing (Shen et al., 2024, Nuc. Acids Res. 52, 9139-60; Parker et al., 2025,\nOpen Biol. 15, 240293). The Shen et al. paper describes how the loss of methylation of U6\nsnRNA affects base-pairing with 5' splice sites containing a +4A and causes a shift to\nalternative nearby sites (Figure 2F).\nI repeat, therefore, that it is quite hard to argue that there is any definitive new knowledge in\nthe current manuscript. Establishing a list of proteins whose actions should now be\ninvestigated is not quite the same as unveiling a new mechanism.\nResponse: We thank the reviewer for their encouraging comments and suggestions of\nimportant references for further improvements.\nNewman and Norman (Cell, 1991) demonstrated the importance of U5 snRNA in 5’SS\nselection. The authors showed that U5 snRNA mutants altered the specificity of 5’SS cleavage\nin the CYH2-lacZ and ADC1-CYH2 reporters. However, this was possible only when the\nauthentic 5’SS was abolished by mutating G1-A. Similarly, in Cortes, Sontheimer, Siewert, and\nSteitz (EMBO J, 1993), the U5 snRNA mutant promoted the use of cryptic 5’SS in the absence\nof the authentic 5'SS. In Hwang and Cohen (Genes Dev., 1996), U1 snRNA facilitated the\nselection of an adjacent 5’SS by U6 snRNA. These fundamental discoveries indeed\ndemonstrated the roles of U5 and U6 snRNA in 5’SS selection. However, the example of 5’SS\nselection in HEH1 is distinct, where relative strengths of U5/U6 pairing determine the choice\nof competing sites naturally present in the same pre-mRNA, and which simultaneously\nproduces two different mRNAs in the cell. In any case, we should have introduced the\nreferences suggested by the reviewer. We have summarised previous literature in the\nintroduction (lines 69-75). We did not discuss m6A modification in early versions since the\npresence of this modification has not been reported in S. cerevisiae. We have also mentioned\nthe role of m6A modification of U6 snRNA in alternative splicing by Shen et al. (Nuc Acids\nRes., 2024) in the revised manuscript.\nselection8,31. In the absence of an authentic 5’SS, U5 snRNA can promote splicing from cryptic\nsites32,33. U1 snRNA-induced selection of a nearby 5’SS by U6 snRNA during spliceosome\nassembly34. m6A modification (methyl group addition at N6 position of adenosine) of U6\nsnRNA in humans. Caenorhabditis elegans and Schizosaccharomyces pombe altered 5’SS\nselection, particularly of +4A positions in introns, and caused a shift to an alternative nearby\nsite35,36”.\n2. The response to point 5 was rather misleading. The authors compared the effects of U98A\nto those of U98G, whereas a comparison with the WT U5 shows that the U98A mutant reduces\npotential base-pairing to both the L and S sites by one G-U pair (Supp. Fig. 3a). Nonetheless,\nthere appears to be a relative increase in the L isoform (Supp. Fig. 3b).\nResponse: We thank the reviewer for pointing out this point. The expression of both isoforms\ndecreased upon expression of the U5 U98A variant (compare U5 WT and U98A lanes), due\nto weaker pairings resulting in poor selection of both sites. However, the negative effects of\nU98A appeared less pronounced on Heh1L, possibly because this site was the dominant one.\nTo clarify this point to readers, we have added the following line in the Supp. Fig.3b legend\n“U5•U98A variant paired weekly to both HEH1 5’SS, resulting in lower amounts of both proteins\n(compare lanes for WT U5 with the U5•U98A)”.\nReviewer 2: The revised version corrects a number of previous issues, significantly improving\nthe manuscript. The authors’ response to reviewers’ comments is very thorough, clarifying\nseveral previous questions.\nResponse: We sincerely thank the reviewer for their time in reviewing the manuscript and for\ngiving expert comments. The reviews have helped us improve the manuscript.\nReviewer 3: The authors have adequately addressed my concerns.\nResponse: We sincerely thank the reviewer for their time in reviewing the manuscript and for\ngiving expert comments. The reviews have helped us improve the manuscript.\n2"}]

Decision letter