An ultraconserved pseudo 5’ splice site fine-tunes development by regulating alternative splicing within TOR-related pathways

Domain: chemistry
Published: 2026-03-09
Editorial outcome: minor_revision

Abstract
Introns have expanded dramatically during evolution, and while their internal sequences have greatly diverged, the potential function of ultraconserved RNA motifs remains an important, unanswered question. Sharing the sequence with the utilized 5' splice sites (5'SSs), pseudo-5'SSs are widespread in introns but are never spliced. We searched homologous introns and identified eight ultraconserved pseudo-5'SSs (UCP-5'SSs). The most conserved one resides in the animal ENOX1/Enox genes that are involved in plasma membrane electron transport and cell enlargement. In vivo deletion of this 9-nt UCP-5'SS in Drosophila results in a significantly enlarged ovary and increased fecundity. We demonstrate that this UCP-5'SS is a silencer for alternative splicing (AS) regulation of an upstream ultraconserved essential exon through interaction with the U1 snRNP-core proteins. The AS changes are observed in all the tested Drosophila mutants from the dTOR and Insulin-like pathways. Remarkably, loss of this UCP-5'SS significantly mitigates the changes. Multiple-source human cells treated with the mTOR/Insulin pathway inhibitors also change the AS and specifically increase the translation of U1-70K, suggesting remarkable conservation of this mechanism. This study reveals an ultraconserved regulatory network in which a short intronic RNA element functions as a sensor of TOR-related pathways during ovarian development.

Introduction
Intronic sequences have expanded dramatically during the evolution of eukaryotes. From budding yeast to humans, the number of introns in a genome has increased from hundreds to hundreds of thousands. The average length of introns per gene has also extended substantially. For example, in the 10% of longest genes of a genome, the average intron length per gene has increased from ~200 nt to ~200K nt over the course of evolution, whereas the average length of exons (including UTRs) per gene has remained relatively constant at around 3K nt 1.

Pre-mRNA splicing removes the intronic sequences from nascent transcripts and ligates exonic sequences together; this is catalyzed by the spliceosome, a large and dynamic RNA-protein complex 2, 3. To define an intron, various spliceosomal subcomplexes recognize the four conserved regions, such as the 5' splice site (5'SS) by the U1 small nuclear ribonucleoprotein particle (snRNP), the branch site by U2 snRNP, the polypyrimidine tract and the 3' splice site (3'SS) by the U2 snRNP Auxiliary Factors (U2AF1/2) 4, 5.

Due to the selection of different splice sites, alternative splicing (AS) creates multiple mRNA isoforms from a single gene, playing crucial regulatory roles in development, differentiation, and metabolism 6, 7, 8. Aberrant AS is frequently observed in hereditary diseases and cancers, often resulting from mutations in spliceosomal components and intronic sequences 9, 10, 11. Changes in AS are also significant in response to environmental variations in nutrition, energy, and stress 12, 13, 14, which facilitates the adaptability of organisms during evolution 15. Interestingly, conserved AS events in homologous genes have been observed across organisms, representing conserved vital regulatory functions during development, cell cycle, and signal transduction 16, 17, 18, 19. There are five kinds of basic AS types, and the skipped exon (SE) is the most common type for the identified conserved AS events 20, 21.

In general, the AS is regulated by the interplay between the pre-mRNA cis-elements and trans-acting protein factors 6, 22. The interplay could be affected by the sequences, structures, and modifications of the RNA cis-elements, post-translational modifications of the trans-acting factors, and the chromatin structures and transcription process, resulting in changes of AS 23, 24, 25, 26, 27. The pre-mRNA cis-elements include enhancers and silencers in both exons and introns 28, and more cis-elements have been found in introns than in exons in humans 29. It has been reported that the flanking intronic sequences near the splice sites of the conserved skipped exon events are significantly more conserved than those of constitutive exons in primates 30, and the conserved intronic cis-elements play critical roles in the selection of splice sites in mammals 31.

Pseudo 5' splice sites (Pseudo-5'SSs) are widespread in higher eukaryotic introns 32. These sites are not spliced but have the same sequences as the utilized 5'SSs in a consensus of AG│GUAAGU (│, the exon-intron border). Pseudo-5'SSs have been frequently found in the regulation of alternative splicing, trans-splicing, transcription termination, and polyadenylation 33, 34. As a key spliceosomal subcomplex in recognition of the 5'SSs, U1 snRNP contains a 164-nt U1 snRNA that is associated with seven Sm proteins and three U1-core proteins, U1-70K, U1A, and U1C 35, 36. It has been shown that the components of U1 snRNP have critical regulatory functions in the selection of the 5'SS 37, 38, 39, 40. Recently, mutations of U1 snRNA have been found in multiple cancers, resulting in the usage of cryptic 5'SSs 41, 42. In addition to splicing, U1 snRNP is strongly coupled with transcription and mRNA polyadenylation, playing important roles in co-transcriptional RNA processing 43, 44, 45.

Compared to the exonic sequences, the intronic sequences are much less conserved during evolution. Due to the significant diversities in length and sequence, it is a challenge to find conserved cis-elements in homologous introns across a wide range of organisms. Here, we focus on the pseudo-5'SS and develop a pipeline to identify ultraconserved pseudo-5'SSs (UCP-5'SSs) in the animal kingdom. These short RNA elements exhibit ultraconserved sequence identity across species, a feature reflecting their functional significance that distinguishes them from genomic ultraconserved elements, which are defined based on length criteria. We thoroughly investigate the regulatory function of a UCP-5'SS in the ENOX1/Enox homologous introns, which is downstream to a conserved and alternatively spliced essential exon (EE). ENOX, the Ecto-NOX disulfide-thiol exchanger, is a terminal oxidase of plasma membrane electron transport and controls physical membrane displacements associated with cell enlargement 46, 47, 48. We find that the UCP-5'SS in ENOX1/Enox serves as a TOR pathway sensor in both Drosophila and humans, functioning in a regulatory network that includes the TOR-regulated translation of U1-70K, the U1 snRNP─UCP-5'SS interaction, AS of the ENOX1/Enox-EE, and the ovarian development in Drosophila.

Results
Identification of conserved pseudo-5'SSs in homologous introns

We first generated position weight matrices (PWMs) of the human and Drosophila utilized 5'SSs by retrieving splice sites from public databases and used them for searching the transcriptome-wide pseudo-5'SSs. In total, we obtained 578,872 and 9,027 pseudo-5'SSs from the human and Drosophila introns, respectively (Fig. 1a left). Further analyses revealed that 3,712 human and 922 Drosophila pseudo-5'SSs are conserved in homologous introns in vertebrates and insects, respectively, of which 2,835 and 856 pseudo-5'SSs are located in retained introns or flanking introns of SE events (Fig. 1a right, Supplementary Data 1). Analysis of locations found that the majority of the conserved pseudo-5'SSs in AS events are generally positioned within 1.5 Kb of either 5' or 3'SSs. In Drosophila, conserved pseudo-5'SSs are more frequently closer to utilized splice sites than non-conserved ones, whereas their distributions are similar in humans (Fig. 1b). In addition, most of the conserved pseudo-5'SSs are located in genes with neural functions (Fig. S1a) and regions associated with skipped exon (SE) or retained intron (RI) events (Fig. 1c).

To find functionally conserved pseudo-5'SSs across animals, we combined the above two groups of data and obtained eight pseudo-5'SSs that are conserved in homologous animal introns and located in a conserved AS event in at least seven representative model organisms, including Homo sapiens, Bos taurus, Gallus gallus, Mus musculus, Xenopus tropicalis, Drosophila melanogaster and Tribolium castaneum (Fig. 1d). Based on their high conservation and potential regulatory functions, these pseudo-5'SSs are named ultraconserved pseudo-5'SSs (UCP-5'SSs) (Fig. 1e & Supplementary Data 2). Among them, the UCP-5'SS in ENOX1/Enox genes (intron 10 in human ENOX1 and intron 4 in Drosophila Enox) shows the highest score in conservation and is associated with a conserved AS event that varies significantly between tissues from both human and the fly (Fig. S1b & Supplementary Data 2). Therefore, we chose this UCP-5'SS as a model and investigated its regulatory function and mechanism.

The ENOX1/Enox intronic UCP-5'SS is always downstream to an alternatively spliced essential exon

The ENOX1/Enox UCP-5'SS has a 7-nt consensus sequence (AGGUAA/GG) in the animal kingdom and expands to 11-nt (CGAGGUAAGUA) in Diptera (Fig. 1f & Fig. S1c). ENOX proteins act as a terminal oxidase of plasma membrane electron transport from cytosolic NAD(P)H via hydroquinones to acceptors at the cell surface 46, 47, and they are believed to control physical membrane displacements associated with vesicle budding or cell enlargement 48, 49. There are two homologous introns in the animal ENOX1/Enox genes, and the UCP-5'SS is in the homologous intron II. Interestingly, its upstream exon is a conserved 107-nt essential exon (EE) in more than 300 animal species with only one exception in the hood coral Stylophora pistillata, where the EE is 110-nt long (Fig. 1g & Fig. S1e). mRNA with this EE ensures the in-frame coding for the full-length ENOX, while skipping will result in a premature termination codon (PTC). Usually, two copies of ENOX genes are present in vertebrates (HsENOX1 and HsENOX2 in humans), one in invertebrates (DmEnox in Drosophila), and none in plants or fungi (Fig. S1d). The EE in ENOX1 and Enox are alternatively spliced; however, the EE in ENOX2 is always constitutively spliced. To confirm that UCP-5'SS is never used as a utilized 5'SS, we further analyzed hundreds of transcriptome data from multiple organisms, including human, mouse, zebrafish, and fly, and found no junction-reads between this UCP-5'SS and the 3'SS of the downstream exon (Fig. S1f).

Deletion of the Enox UCP-5'SS in Drosophila results in a significantly enlarged ovary

To study its physiological function, we used the CRISPR/Cas9 knock-in system and constructed a precise DmEnox UCP-5'SS-deleted strain (UCPΔ), as well as a DmEnox out-of-frame knockout strain (DmEnox-KO, KO). Both were confirmed by genomic DNA PCR and sequencing (Fig. 2a). We observed that the UCPΔ females were significantly larger than the WT females. We then monitored their weight after eclosion and found that the average weight of the UCPΔ females was ~12% above the WT females, but the average weight of males was not changed (Fig. 2b). Meanwhile, the average weight of both females and males of the control DmEnox-KO mutant was ~8% below the WT. After dissection, the UCPΔ females exhibited significantly enlarged ovaries (Fig. 2c). In contrast, the size of the testis from the UCPΔ males was similar to that of the WT males (Fig. 2d). Likewise, gonads from both the female and male KO mutants were much smaller. After crossing with either the UCPΔ or WT males, the UCPΔ females laid more eggs than the WT females, showing a significantly enhanced fecundity (Fig. 2e). Crossed with the UCPΔ males, the WT females laid fewer eggs, implying an unknown defect of the UCPΔ males. We conclude that the 9-nt intronic UCP-5'SS in DmEnox has a specific physiological function in ovarian development.

We further performed RNA-seq and identified hundreds of differentially expressed genes (DEGs) in the DmEnox-KO and -UCPΔ mutants (Fig. S2a & Supplementary Data 3), which were grouped into six clusters in females and five clusters in males (Figs. S2b-c). In the female clusters 3 and 4, 34 genes are involved in the eggshell chorion assembly (Fig. 2f & Supplementary Data 3) and critical for the eggshell formation in the ovary 50. Among them, 25 genes were significantly up-regulated in the KO females but dramatically down-regulated in the UCPΔ females. To validate, we performed RT-qPCR and confirmed that all five genes tested were down-regulated in the UCPΔ females but up-regulated in the KO females (Fig. 2g). These results imply that the enlarged ovary phenotype caused by deletion of the UCP-5'SS could be due to the changes in expression of the eggshell chorionic layer genes. Chorion genes are exclusively expressed in ovarian follicle cells, with transcriptional activity strictly restricted to stages 10-14 of oogenesis 51, 52. Additionally, perturbations in ENOX1 activity have been shown to disrupt cellular ultradian rhythms 53. We propose that increased DmENOX protein levels would disrupt the metabolic and circadian rhythms of follicle cells; in turn, this disruption would lead to altered expression of chorion genes, given their strict temporal regulation during oogenesis.

The UCP-5'SS is a silencer for splicing the ENOX1/Enox-EE

The conserved upstream EEs in DmEnox and HsENOX1 are exons 4 and 10, respectively; both of them are alternatively spliced (Fig. 3a). To address the function of ENOX1/Enox-EE’s UCP-5'SS in splicing, we constructed DmEnox and HsENOX1 mini-genes and transfected them into Drosophila S2 and human Huh7 cells, respectively (Figs. 3b-c). Each set of mini-genes includes the WT and the deletion, the mutation, and a mock-deletion of the UCP-5'SS. RT-PCR analyses showed that only 17% of the DmEnox-EE (E4) was spliced into the mRNA from the Drosophila WT mini-gene, whereas 32% and 34% were spliced from the UCP-5'SS deletion and mutation mini-genes, respectively (Fig. 3b). Meanwhile, retention of intron 4 from the two mutant mini-genes was significantly decreased. Similarly, splicing of the HsENOX1-EE (E10) was enhanced, with both decreased E10 skipping and reduced intron 10 retention observed in the deletion and mutation mini-genes in human Huh7 cells (Fig. 3c). As controls, splicing of the mock-deletion mini-genes did not show obvious changes in the two systems. These data demonstrate that this UCP-5'SS is an intronic silencer for splicing of the ENOX1/Enox-EE, dysfunction or loss of this UCP-5'SS leads to an enhanced splicing of the EE in both Drosophila and human cells.

Analyses of RNA-seq data from the FlyBase and our previous results 54 indicate that the DmEnox-EE is differentially spliced between tissues (Fig. S3a). Therefore, we then investigated the splicing changes of DmEnox-EE in the UCPΔ animal tissues. We found that splicing of the DmEnox-EE was significantly enhanced in the UCPΔ mutant for all the tested Drosophila samples from two lines of the mutant, in which the mutant ovary exhibits the most enhancement, ~2.5-fold changes of the EE inclusion (Fig. 3d). Being consistent with this individual RT-PCR analysis, significant AS changes of the EE were also observed in the RNA-seq data of the UCPΔ females and males (Figs. S3b-c). To detect the in vivo protein changes, we developed a polyclonal antibody against the N-terminus of DmENOX, which gives a clean and strong signal at the 110-KD position for the WT animal but not for the DmEnox-KO animal (Fig. S3d). We found that the DmENOX protein was significantly increased in the whole adult (~ 2-fold), ovary (~ 4.5-fold), and testis (~3-fold) in the UCPΔ mutant (Fig. 3e), consistent with changes of their mRNAs. These data demonstrate that the UCP-5'SS differentially regulates splicing of the DmEnox-EE, and its deletion increases the level of DmENOX in most tissues, especially in the ovary.

AS of DmEnox-EE is regulated by U1 snRNP through interaction with the UCP-5'SS

The utilized 5'SS is recognized by U1 snRNP during the pre-spliceosome assembly; therefore, we asked whether UCP-5'SS could be recognized and regulated by U1 proteins. Knockdown of the three U1 snRNP-core proteins (U1-70K, U1A, and U1C) in S2 cells using the dsRNA-induced RNAi significantly enhanced splicing of the endogenous DmEnox-EE and thereby increased DmENOX proteins. In contrast, the knockdown of PSI, encoding a protein that has been found closely associated with the U1-binding motifs in Drosophila 55, or two U2-specific proteins (Sf3a3 and Sf3b4) did not change splicing of the EE (Fig. 4a). Since LUC7Ls were reported as U1 snRNP-associated proteins and regulate the 5'SS selection in humans 56, we further tested knockdown of Drosophila LUC7L homologs in S2 cells. However, knockdown of Alsin2 or CG3198 (homologs of human LUC7L and LUC7L2/3, respectively) alone, or both simultaneously, did not exhibit obvious AS changes of the DmEnox-EE (Fig. 4b). These results suggest that AS of the DmEnox-EE is specifically regulated by the U1 snRNP-core proteins, not by the U1-associated proteins. We found that knockdown of U1-70K, U1A, and SF3B4 resulted in a moderate decrease in U1 snRNA, whereas knockdown of U1C and SF3A3 had no significant effect on U1 snRNA abundance (Fig. S3e). Additionally, RNA-seq analysis confirmed that U1-70K knockdown in S2 cells causes global splicing defects, while Enox exon 4 shows increased inclusion (ΔPSI = 0.159) and its intron 4 shows decreased retention (ΔPSI = -0.198) (Fig. S3f-gs).

We then checked the splicing of DmEnox mini-genes under the U1-70K knockdown condition. Compared to the WT and mock-deletion mini-genes, the enhanced splicing of DmEnox-EE from the UCP-5'SS-deleted or -mutated mini-genes was significantly mitigated when U1-70K was knocked down (Fig. 4c), down from ~1.9-fold enhancement to ~1.2-fold enhancement (Fig. 4d). These results demonstrate that AS regulation of the DmEnox-EE by U1-core proteins is dependent on the intronic UCP-5'SS. We conclude that the UCP-5'SS is recognized by the U1 snRNP, and their interaction would impair the intron definition between the utilized 5'SS and 3'SS, i.e. the DmEnox intron 4.

AS of DmEnox-EE is changed in the Drosophila dTOR and Insulin-like pathway mutants

It has been reported that dTOR and Insulin-like pathways regulate the development of the Drosophila ovary 57. Therefore, we collected or generated mutants including dTOR, Tsc1, and Rheb from the dTOR pathway, and dInR (insulin receptor), Pten, and Chico from the Insulin-like pathway 58, 59. These strains have different genetic backgrounds (Supplementary Table 1). These mutants were either knocked down (KD), activated (act) or overexpressed (Ex) using a GAL4/UAS system (Fig. 5a). Firstly, we observed that the ovary development is significantly changed in the two pathway mutants, such as in the dInR-KD, Pten-KD, Tsc1-KD, and RhebEx strains (Fig. 5b). Secondly, in comparison to controls, splicing of the DmEnox-EE was significantly impaired in the dTOR-KD larvae but enhanced in the Tsc1-KD and RhebEx larvae (Fig. 5c), resulting in consistent expression changes of the DmENOX protein (Fig. 5d). Since dTOR and Rheb positively and Tsc1 negatively contribute to the dTOR pathway, these results indicate that the dTOR pathway positively regulates the splicing of DmEnox-EE. Similarly, splicing of the DmEnox-EE was also changed in the Insulin-like pathway mutants, significantly enhanced in the dInR-KD and Chico-KD mutants but impaired in the Pten-KD and dInR activated (dInRact) mutants (Figs. 5e and Fig. S4a), which were further confirmed by changes of the DmENOX protein (Figs. 5f and Fig. S4b). Since dInR and Chico positively and Pten negatively contribute to the insulin-like pathway, these results indicate that the Insulin-like pathway negatively regulates the splicing of DmEnox-EE. We also tested the mRNA levels of U1 snRNP-core proteins and found that none of the U1-70K, U1A, or U1C mRNAs were detectably changed in these pathway mutants (Figs. S4c-e). Unfortunately, the available antibodies against human U1-core proteins are insufficient to give reliable signals for Drosophila samples, and we do not know whether the translation of these proteins was changed in these mutants. Taken together, we conclude that AS of the DmEnox-EE is regulated by both the dTOR and Insulin-like pathways.

The UCP-5'SS is a sensor of the Drosophila dTOR and Insulin-like pathways

To further address whether AS changes of the DmEnox-EE in the two pathway mutants rely on the UCP-5'SS, we crossed the pathway mutants with the UCPΔ mutant to obtain double mutants (Fig. 6a). Firstly, the dTOR-KD flies died at an early stage of the 3rd instar larva, which survived less than 72 hours after hatching. However, additional deletion of the UCP-5'SS significantly extended the life span of the dTOR-KD mutant, up to 168 hours (Fig. 6b), providing evidence that the UCP-5'SS in the pre-mRNA of DmEnox is genetically downstream to the dTOR pathway. Secondly, we found that the knockdown of dTOR resulted in a 0.21 decrease in the PSI value for EE inclusion; this decrease was reduced to 0.14 when the UCP-5'SS was additionally deleted (Figs. 6c-d left). Similarly, the Rheb-OE resulted in a 0.11 increase in the PSI value for EE inclusion; this PSI change was no longer significant in the absence of the UCP-5'SS (Figs. 6c-d right). We also observed mitigation of the AS changes in the Insulin-like pathway mutants when the UCP-5’SS was additionally deleted (Figs. 6e-f and Figs. S5a-b). The deletion of UCP-5'SS significantly mitigated the DmEnox-EE splicing changes caused by the two pathway mutants, strongly suggesting that the Enox UCP-5'SS serves as a sensor of the two pathways.

Human Insulin and mTOR pathways regulate AS of the HsENOX1-EE through the translational control of U1-70K

Since the UCP-5'SS is evolutionarily conserved, we then asked whether interference of the two pathways in humans would also change the AS of HsENOX1-EE. We addressed this question in three tissue-differentiated human cells: Huh7 from the liver, HEK293T from the kidney, and KGN from the ovary. These cells were treated with three pathway-specific inhibitors (Fig. 7a), including wortmannin for PI3K 60, Akti-1/2 for AKT 61, and rapamycin for mTOR 62, 63. Consistent with the previous reports 64, 65, the treatments of wortmannin and Akti-1/2 significantly inhibited the phosphorylation of AKT (p-AKT), production of mTOR, and p85-S6K in all three cells, while the treatment of rapamycin had no effect on AKT but strongly inhibited the production of mTOR, p85-S6K, and p70-S6K (Fig. S6). Interestingly, AS of the HsENOX1-EE (E10) was changed in the inhibitor-treated cells, showing a decreased exon 10 skipping and reduced intron 10 retention (Figs. 7b-c and Fig. S7a). Consistently, the HsENOX1 protein was significantly decreased in all the inhibitor-treated cells (Figs. 7d-e and Fig. S7b). These results demonstrate that the human mTOR and Insulin pathways also regulate the AS of HsENOX1-EE.

Similar to the results in Drosophila, there were no obvious mRNA changes in human U1-70K, U1A, or U1C mRNAs upon the treatment of pathway inhibitors (Figs. 7b-c and Fig. S7a, c-e). Using human antibodies, we found that the U1-70K protein was significantly increased in all the inhibitor-treated cells. Meanwhile, the U1A and U1C proteins were either not changed or slightly increased (Figs. 7d-e and Fig. S7b). We then performed luciferase assays and found the expression of luciferase-ORF with the 5'-UTR from human U1-70K was significantly increased at a medium level upon the treatment of rapamycin in Huh7 cells, while with the 5'-UTR from human U1A, U1C or Enox1 was not (Fig. S7f), indicating that the 5'-UTRs from U1-70K might contain cis-elements for translation in response to the regulation by mTOR pathway. Taken together, we conclude that the translation of U1-specific proteins, especially U1-70K, is controlled by the human mTOR and Insulin pathways, and their increased translation upon the treatment of pathway inhibitors decreases the AS of HsENOX1-EE.

Additionally, we performed RNA immunoprecipitation (RIP) assays using U1-70K antibodies in HuH7 cells transfected with plasmids encoding 250-nt RNA sequences containing either the WT, mutated, or deleted UCP-5'SS. Notably, only the RNA harboring the WT UCP-5'SS was co-immunoprecipitated by the U1-70K antibody (Fig. 7f). We also analyzed published CLIP-seq datasets 66 and found that the U1A protein binds precisely to the UCP-5'SS on Enox1 mRNA. Taken together, these results provide direct support for the conclusion that the UCP-5'SS is recognized by U1 snRNP.

Methods
Bioinformatic analyses

Conserved pseudo-5'SSs: Genome sequences and gene annotations of Homo sapiens and Drosophila melanogaster were downloaded from UCSC (https://genome-asia.ucsc.edu/) and FlyBase (http://flybase.org/), respectively. The upstream and downstream sequences (2 + 5 nts) of all the annotated 5'SSs were extracted to calculate the position weight matrixes (PWMs). Sequences closely resembling the 5'SS motif, log odds score > 6, were identified using the scanMotifGenomeWide.pl program from homer2 69. To obtain pseudo-5'SSs, sequences that overlapped with known splice junctions were excluded according to the GTEx database 70 and FlyBase. To identify conserved pseudo-5'SSs, human and Drosophila pseudo-5'SSs were filtered by phyloP > 2 from 100 vertebrates and 124 insects on the UCSC, respectively 71. Information about the AS-related human and Drosophila introns was retrieved from VastDB 72.

Ultraconserved pseudo-5'SSs: Conserved AS events were obtained based on VastDB and the NCBI database information. The above vertebrate and insect conserved pseudo-5'SSs were kept if they were located in conserved AS events in seven model animals, including three mammals (Homo sapiens, Bos taurus, and Mus musculus), a bird (Gallus gallus), an amphibian (Xenopus tropicalis), and two insects (Tribolium castaneum and Drosophila melanogaster). Allowing up to one mismatch, sequences of the pseudo-5'SSs were then checked in the seven organisms. The remaining pseudo-5'SSs are defined as ultraconserved pseudo-5'SSs (UCP-5'SSs). For each UCP 5'SS, ΔPSImax, the maximum difference of PSI values (Percent Spliced In) of the related AS event between tissues is calculated using PSI values in VastDB, which contains 84 human tissues and 72 Drosophila tissues.

RNA-seq analysis: Raw reads from RNA-seq were quality-filtered and trimmed, mapped to the Drosophila melanogaster genome (dm6) by STAR v2.6.1a, and counted by featureCounts 73. Analyses of the differentially expressed genes were performed by DEseq2_v1.22.2 74, and genes with |log2Fold Change| > 0.5 and FDR < 0.05 were screened as significant. Alternative splicing analysis was performed using rMATS v4.1.0.

Fly strains and culture

The wild-type D. melanogaster used in this study is a w1118 isogenic strain (BDSC 5905). Two Enox mutant strains (UCPΔ and KO) were constructed using a CRISPR/Cas9-mediated knock-in technique 75. Briefly, the target sequences of guide RNAs (sgRNAs) were selected, and the donor plasmids (pMD18-T) with deletion regions and the adjacent 1 kb sequences as homologous arms were constructed. The gRNAs and donor plasmids were co-injected into embryos of a transgenic line nanos-Cas9 by UniHuaii Technology Company. Specific primers were used for genomic PCRs to screen for the desired alleles, which were further validated by sequencing. The obtained strains were crossed for at least 5 generations with the WT to eliminate potential off-target events.

Strains with the dTOR or Insulin-like pathway mutation were purchased from BDSC or offered by other labs. Those strains were crossed with the da-GAL4 strain to induce mutation. Double mutants containing the deletion of UCP-5'SS (UCPΔ) and dTOR/Insulin-like pathway mutations were generated by two steps of crosses. The UCPΔ mutant strain was first crossed with the UAS-pathway mutants and da-GAL4, respectively, due to limitation of chromosome location, and the obtained strains were crossed with each other to get the double mutants. All the Drosophila strains used in this study are listed in Supplementary Table 1.

Cell cultures, drug treatment, and RNAi

Drosophila S2 cells were cultured in 10% of the Schneider's Drosophila medium at 28°C. Human cells were cultured in DMEM (Gibco) supplemented with 10% FBS and 1% penicillin and streptomycin at 37°C. Wortmannin, Akti-1/2, and Rapamycin were purchased from Selleck Chemicals, and their stock solutions were prepared in DMSO and stored at -20°C before use. Plated at a density of 1 x 105 cells/ml, human cells were treated with inhibitors in final concentrations of 50 nM Wortmannin, 10 µM Akti-1/2, and 100 nM Rapamycin for 5 min, respectively 76. For quantitation, all the treatments were performed at least in triplicate.

To knock down genes, dsRNAs were designed and transcribed using the T7 Ribo-MAX Express RNAi system (Promega) and transfected into S2 cells for Drosophila genes 54. DmEnox mini-genes containing a 3.4-kb fragment (exons 3 to 6) of WT or mutated sequences were cloned into a pIZT-V5 vector (Invitrogen) and transfected. HsENOX1 mini-genes containing a 2.4-kb fragment (exons 9 to 12) of WT or mutated sequences were cloned into a pcDNA3.0 vector (Addgene) and transfected into Huh7 cells.

Western blotting and antibodies

Cells and Drosophila tissues were collected and lysed with the RIPA buffer for Western blotting 65, their protein concentrations were determined by an Enhanced BCA Protein Assay Kit (Promega). Rabbit polyclonal antibodies against human proteins include anti-ENOX1 (Abclonal, A14282), anti-U1-70K (Abcam, ab83306), anti-U1A/SNRPA (Abclonal, A3686), anti-U1C/SNRPC (Santa Cruz, sc-101549), anti-mTOR, anti-p70 S6 Kinase and anti-4E-BP1 (Proteintech, 20657, 14485, 60246 respectively), anti-phosphoSer2448-mTOR, anti-phosphoThr37/46-4E-BP1, anti-phosphoThr389-p70 S6 Kinase, anti-AKT and anti-phosphoSer473-AKT (Cell Signaling Technology, 5536, 2855, 97596, 4691 and 4060, respectively). A rabbit polyclonal antibody against the Drosophila ENOX was developed using the 1-240 aa polypeptide as the antigen by Shanghai Ango Biotechnology.

RT-PCR and RNA-seq

Total RNAs from the human and Drosophila cells and tissues were isolated by TRIzol (Sigma) and treated with RNase-free DNase I (Invitrogen). For RT-PCR, reverse transcription was performed using PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa), and the obtained cDNA was amplified by 2x Hieff PCR Master Mix (Yeasen). RT-qPCR was performed using TB Green Premix Ex Taq II FAST qPCR (TaKaRa) in 96-well reaction plates with the Kubo Technology Real-Time PCR System, following the manufacturer’s instructions. Gene expression profiles were analyzed using the –ΔΔCt method. All experiments were performed in triplets. The statistical analyses were performed using GraphPad Prism 7 (San Diego, CA), and statistical differences were determined by t-test.

RNAs from Drosophila adults (the 5th day after eclosion) were isolated for mRNA-seq, and the construction of cDNA libraries and sequencing were performed using Illumina HiseqXten-PE150 by Novogene.

Drosophila assays

Fecundity: The number of eggs laid per female fly was measured. Briefly, ten individual mated female adults (24-28 hrs post-eclosion) from each strain were passed to new plates, and their laid eggs per plate were counted every day for ten days.

Weight: Female and male virgin flies, including the WT and mutants, were collected within 2 hrs post-eclosion. Fifty female or male flies were placed into one vial for culture. After feeding with fresh food for 24 hrs, the weights of each gender of fly were recorded daily for two weeks. All the tests were in triplets and at regular intervals. The statistical analyses were performed using GraphPad Prism 7 (San Diego), and t-tests were used for the statistical differences.

Gonads: Anesthetized adults were placed on a Sylgard 184 black silica dissection plate (Sigma-Aldrich) and soaked in a drop of ice-cold 1x PBS buffer. To obtain intact and clean samples of ovaries and testes, the internal fly organs were carefully pulled out after dissection, and other excess organs and fat were removed. The gonads were then transferred to a new black dissection plate with 1x PBS buffer for photographing under an OLYMPUS microscope. The sizes of the gonads were detected using ImageJ.

Survival of larva: the flies of different strains were cultured in egg-laying cages respectively. After 24 hours post-egg-laying, three parallel repeats of 100 larvae from each strain were transferred onto a new plate with yeast paste, and the survivals were checked and recorded every 12 hours as described 77.

Luciferase assay

The 5'-UTR sequences of human U1A, U1C, U1-70K, ENOX1, and RPL23 were amplified and inserted into the pmiGLO vector, upstream to the luciferase start codon. Huh7 cells were cultured in 12-well plates until 70% density and then transfected with 1 µg of plasmid DNA per well using Neofect transfection reagent (ALSTEM). Six hours post-transfection, the cells were treated with 0.1 µM rapamycin for 15 min. Fluorescence was measured by a microplate reader using the Dual-Luciferase Reporter Assay System (Beyotime) and processed using GraphPad software. Each sample was assayed in triplicates.

RNA immunoprecipitation (RIP) assays

Approximately 5×10⁶ Huh7 cells were used per RIP sample. Cells were lysed for 1h at 4°C in non-denaturing lysis buffer (Solarbio, R0030) containing protease/phosphatase/RNase inhibitors. Lysates were centrifuged to clarify, and 10% of the supernatant was retained as input. The remaining lysate was incubated with 5 µg anti-U1-70K antibody (Abcam, ab83306) or Rabbit IgG (CST, 2729). Pre-blocked Protein A/G Agarose beads were added, and the mixture was rotated overnight at 4°C. After washing with WB150 buffer (20 mM Tris-HCl pH 7.9, 150 mM NaCl, 0.1% NP-40), a fraction of the bead-bound protein was analyzed for Western blotting. RNA was isolated from the remaining beads and the input samples using RNAiso Blood (Takara, 9113), with glycogen added as a carrier during precipitation. For RT-qPCR, input and immunoprecipitated RNAs were reverse-transcribed using random primers.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Ding, Fang, Li et al search for evidence of conservation in pseudo-5’SS (sequences that resemble the 5’ splice site consensus but that they are not used as donor splice sites) across multiple species and identify such sequences in the neighbourhood of alternatively spliced exons in homologous genes of multiple species (ultraconserved pseudo-5’SS or UCP-5’SSs). They chose to focus on one such UCP-5’SSs conserved in humans and Drosophila, located in the ENOX1 /Enox gene. Precise deletion of the pseudosplice site in Drosophila resulted in enlarged ovaries and increased female fertility, while decreased male fertility. RNA-seq identified multiple changes in expression, including downregulation of the expression of various eggshell chorionic layer genes. Experiments in both Drosophila and human cell lines using minigenes, as well as in vivo analysis in Drosophila of DmENOX protein levels, indicate that the UCP-5’SS element reduces expression of the mRNA encoding ENOX protein and additional knock down experiments suggest that core protein components of U1 snRNP contribute to UCP-5’SS recognition and its regulatory function on mRNA expression. In an interesting twist, the authors then link this regulatory event with the TOR and insulin pathways, showing through a nice combination of genetic tools that the UCP-5’SS can serve as a sensor of the activity of these pathways. Remarkably, relevant aspects of this interplay were observed in mammalian cells treated with inhibitors of mTOR, p-AKT or PI3K and the authors mechanistically interpret these effects as the result of increased levels of the U1 snRNP protein U170K under these conditions.\nThis is a very interesting and thorough study that links, in unexpected ways, the still proorly understood mechanisms of recognition of pseudo splice sites with a remarkably conserved regulatory interplay with important signalling pathways and ovarian function in flies. The study opens a number of fascinating questions at various levels for the authors and others to follow in future studies.\nIn my opinion the following revisions could help to improve the manuscript:\n1) Figures 1b, c, and e. The results are clear but it is not clear what is the significance of these observations, i.e. what are these distances or association with alternative splicing events compared with. For example, are these localizations / associations different for conserved pseudo 5’SS compared to non-conserved pseudo 5’SS, or to other control sequences (e.g. pseudo 5’SS-like sequences matching the PWM but lacking G at position +1, which should be inactive in splicing)? In this regard “near” (line 104) should be defined more precisely.\n2) Figure 3. The quantification of IR events shows % values above 100%, which is confusing. It would be better to refer to values relative to those observed under WT condition, these values being referred to as 1. Related to this, while increased inclusion of exon 4 in the Drosophila minigenes correlates with decreased levels of the exon skipping isoform, the decrease in intron 10 retention in the human minigene does not seem to have an impact on the levels of spliced exon 10-11 transcripts. On the other hand, effects of mTOR/insulin inhibition in mammalian cells in Figure 7 show skipping of both exons 10 and 11 as a consequence of the inactivation. Has skipping of exons 11 and 12 been assessed in the minigene experiments such as those of Figure 3C?\n3) Related to the previous point: the mechanism by which recognition of the pseudo-5’SS by U1 snRNP leads to intron retention rather than to activation of the sequence as a donor splice site (e.g. splicing to the next available 3’SS) remains unclear. It would be too much to ask to decipher the mechanism in this first report, but the authors should comment on efforts to detect such products. For example, should these products be detectable using the primer pairs used to detect IR events? Are such shorter products indeed detected or not below the IR bands in (uncropped) full gel electrophoresis images?\n4) Figure 4. The results of the effects of knock down of U1 snRNP core proteins are clear. They are in fact remarkable because one could expect that such knock downs would lead to decreased recognition of 5’SS in general. Selectivity could be explained by these pseudo-5’SS being particularly sensitive to decreased levels/activity of U1 snRNP, a hypothesis that the authors may want to discuss. However the key question is whether the different knock downs (also those serving as controls) have an impact on the levels of U1 snRNP. I understand that the lack of good antibodies for the Drosophila factors make it difficult to assess this, but the authors could check the levels of U1 snRNA as a surrogate for U1 snRNP particles, assuming that U1 snRNA will not have the same half-life if not assembled in full particles. This control should be feasible and could add to the mechanistic interpretation of the results. Along similar lines, do the increases in U1-70K levels observed in Figure 7 correlate with increased levels of U1 snRNA, again as a surrogate for U1 snRNP particles?\n5) Figures 4d and 6d, 6e, 6f: are the differences statistically significant?\n6) Text:\na. Abstract: “in which ultraconserved RNA motifs may play essential regulatory roles”: I would suggest “Introns have expanded dramatically during evolution, and while their internal sequences have greatly diverged, the potential function of ultraconserved RNA motifs remains an important, unanswered question”.\nb. Abstract, line 31: “Conservatively, …” I would suggest removing and fisnishing the sentence with “…suggesting remarkable conservation of this mechanism”.\nc. Line 42: “while the length of exons per gene has remained relatively constant around 3K nt” may be confusing. Better to refer to the length of mature mRNAs, as exons are much shorter than 3K, particularly in vertebrates.\nd. Line 48: U2 snRNP auxiliary factors should be “U2 snRNP Auxiliary Factors (U2AF1/2).\ne. Line 67: clarify if this refers to the conservation of splice site signals or to other intronic sequences near the splice sites.\nf. Line 96 and throughout the text “actual 5’SS” is not a commonly used term to designate actively used (utilized) 5’SS.\ng. Line 131: “pre-termination codon (PTC)” should be “premature termination codon (PTC)”.\nh. Lines 219-220: relevant to point #3 above, it may not be entirely clear that recognition of the psedo-5’SS by U1 snRNP impairs intron definition.\ni. Lines 329-330: strictly speaking, the interaction between U1 snRNP and the pseudo-5’SS has not been directly tested in this manuscript (also relevant to the discussion in point #4).", "reviewer_2": "Ding et al. present a comprehensive and compelling study on the evolutionary conservation and functional mechanism of an ultraconserved pseudo 5’ splice site (UCP-5’SS) in regulating the alternative splicing of the ENOX1/Enox gene across the animal kingdom, and further explores its biological significance in ovarian development. Through integrative bioinformatic analyses, the authors identified UCP-5’SS within homologous introns, focusing on a particularly conserved UCP-5’SS located in the ENOX1/Enox genes. The authors effectively integrate bioinformatic analyses with functional assays from both Drosophila and human cell lines, to elucidate how UCP-5’SS acts as a splicing silencer responsive to TOR and insulin signaling pathways. The study reveals a molecular mechanism linking translational regulation of U1-70K to alternative splicing control, ultimately affecting physiological functions like ovary development. Importantly, this study provides valuable insights into the biological significance and underlying mechanism by which the UCP-5’SS function as cis-regulatory elements in alternative splicing. Overall, this manuscript is well-structured, with clear data presentation and substantial experimental design. Accordingly, I recommend this paper for publication, below are a few issues that need to be addressed before publication.\n1. The authors claimed that this short intronic RNA motif functions as a sensor of TOR-related pathways. How to define a TOR pathway sensor? What conditions does RNA motif possess to become a development sensor? Please clarify a TOR pathway sensor and whether it holds general significance?\n2. The authors showed that deletion of the Enox UCP-5'SS in Drosophila results in a significantly enlarged ovary, and that the enlarged ovary phenotype caused by deletion of the UCP-5'SS could be due to the changes in expression of the eggshell chorionic layer genes. However, the authors did not explain how deletion of the UCP-5'SS affect expression of the eggshell chorionic layer genes.\n3. Indeed, the AS changes are observed in all the tested Drosophila mutants from the dTOR and Insulin-like pathways, and loss of this UCP-5'SS significantly mitigates these changes. In addition, multiple-source human cells treated with the mTOR/Insulin pathway inhibitors also change the AS and specifically increase the translation of U1-70K. Are all these evidences sufficient to indicate that pseudo 5' splice site fine-tunes development by regulating alternative splicing within TOR-related pathways? In other word, does pseudo 5' splice site fine-tunes development mainly through regulating alternative splicing within TOR-related pathways? Are there other mechanisms for pseudo 5' splice site?\n4. What are the differences between pseudo-5'SS and alternative 5' splice sites? Is there evolutionary conversion between pseudo-5'SS and alternative 5' splice sites?\n5. In the Method section, the upstream and downstream sequences (2 + 5 nts) of all the annotated 5'SSs were extracted to calculate the position weight matrixes (PWMs). What is based on the upstream and downstream sequences 2 + 5 nts parameters? Are there some pseudo-5'SS not identified? Please clarify.\n6. In Discussion section, I suggest a deeper discussion of the evolutionary significance of conserved pseudo-5'SS?\n7. In Fig. 2e, there appears to be a misalignment of the group name.\n8. In Fig. 2g, authors choose RT-PCR to validate RNA-seq results, which may not be reliable enough. I suggest switching to RT-qPCR, which provides more accurate quantification.\n9. In Fig. 3b and 3c, there are some spacing gaps between the letters, such as \"mini gene\" and \"mock-del\". A carefully reviewing of all figures should be done.\n10. The quantification results in Fig. 3, 4, and 5 are missing group labels, which hinders the clear interpretation of the data. To improve clarity, I suggest adding the appropriate group labels in these figures.\n11. The layout of Fig. 4 appears somewhat disorganized and visually unbalanced. I recommend improving the alignment and overall formatting to enhance clarity and visual appeal. Similarly, the alignment of panels in Fig. 3d and 3e could also be refined for better consistency.\n12. Please ensure that the size of the significance asterisks in the quantification results of Fig. 7 is consistent. In particular, the asterisks in panels 7d and 7e appear to be too small.\n13. I suggest authors to use the full-term Western blot or the abbreviation WB to refer this experimental technique, rather than simply using “Western” in formal scientific figures.", "reviewer_3": "In their manuscript ‘An ultraconserved pseudo 5' splice site fine-tunes development by regulating alternative splicing within TOR-related pathways’, Xu and colleagues describe a role for an intronic sequence resembling a 5’ splice site in controlling gene expression and ovary development. The manuscript suggests a that the pseud 5’ splice site is downstream of mTOR signaling, interacts with U1 snRNP and thereby controls alternative splicing of an upstream alternative exon, which then controls ovary development.\nSeveral points make the manuscript difficult to read and parts of the conclusion are not justified by the data presented.\nThe sentence in the introduction: ‘length of introns per gene has expanded from ~200 nt to ~200K nt, while the length of exons per gene has remained relatively constant around 3K nt’ is wrong and the citation not correctly used.\nThis statement: ‘the conserved pseudo-5'SSs are involved in the events of skipped exon (SE) or retained intron (RI) (Fig. 1c) and located in genes with neural functions’ is wrong. Pseudo splice sites may be located within such genes, but that does not mean that they are involved in any splicing events.\nThroughout the entire manuscript, the function of ENOX1/Enox genes is described as ‘involved in plasma electron transport’. I think this should mean ‘plasma membrane electron transport’. Please correct and carefully proof read the entire manuscript.\nPhenotypes in figure 2 have to be shown in 2 independently generated lines per genotype (in fig 3d two UCPD lines are used – show the phenotype of the second line, also for ko). Fig 2f and 2g colors are confusing. In 2f ko shows mostly red (up) but this is blue in 2g. Same for UCPD. Please use consistent colors. Also, why are these genes down in the UCPD which are more fertile. Shouldn’t this be the other way?\nPlease use RT-qPCR instead of the standard RT-PCR in 2g bottom (an upregulation in ko is not visible in most cases in the data shown).\nIn all splicing quantifications, please indicate % inclusion +/- Standard deviation below the gel. It would also be better to plot % inclusion +/- SD rather than the normalization that is then shown +/- SEM.\nAll gel, especially the ones that quantify intron retention, need a -RT control.\nFig 3C show three gels that all investigate intron 10 retention. It would be much more informative to quantify real IR and not something normalized, which makes the effect look stronger than it is. This also needs a -RT control. Skipping of exon 10 has to be analyzed as well and a quantification as in Fig 3b has to be shown.\nIn the scheme in Fig. 3a, remove the introns after splicing\nThe splicing phenotype of the endogenous fly mutants in Fig. 3d has to be confirmed at the endogenous level in a human cell line as well.\nIn Fig. 4 it is surprising that knock down of U1 components that are required for all splicing events lead to increased exon inclusion. Increased IR could have been an expected outcome. To validate this, RNA-Seq should be performed to see what the global effect is and to confirm the finding for Enox splicing using a different read out. If this is confirmed, these experiments should be repeated in human cells.\nComparing splicing in Fig. 4a and 4c, there is a strong discrepancy in the control. In 4a there is 61% inclusion, in 4c in the control it is only 25%. This questions all splicing analyses.\nFig. 5c has also inconsistent controls. Splicing in the ‘-‘ controls differs from 55 to 36 % inclusion. This is too much variation.\nIn 5e there is hardly a change in splicing in the Pten mutant but a strong change in protein expression. The change in the protein can’t then be mediated by the change in splicing.\nPlease show that the pseudo-splice site is really by the U1 snRNP, e.g. using CLIP. To make the claim that ‘UCP-5'SS is recognized by the U1 snRNP and their interaction impairs the intron definition…’ a spliceosome assembly gel should be shown comparing RNA with and without the pseudo splice site.\n‘Secondly, we found that the knockdown of dTOR resulted in a 38% decrease in the EE-inclusion, and the decrease was reduced to 21% when the…’ Please use PSI change.\n‘Consistently, the HsENOX1 protein was dramatically decreased in all the inhibitor-treated cells (Figs. 7d-e and Fig. S7b).’ This is not a ‘dramatic’ change.\n‘short ultraconserved RNA motif’ – Ultraconserved has been used before and is defines as conserved elements of at least 100 nts. I would call this short element ultraconserved.\nA direct connection of TOR and Insulin pathways with the translation of U1-70k is not shown. It is also not clear how the level of U1-70k regulates binding of U1 snRNP to the pseudo splice site."}, "author_response": "Response: We appreciate the reviewer's insightful comments and valuable suggestions. Following the reviewer’s recommendation, we have conducted an additional round of analysis to compare conserved and non-conserved pseudo-5'SSs. In Drosophila, the locations of conserved pseudo-5'SSs in AS events are indeed more concentrated near the utilized splice sites than their non-conserved counterparts. In humans, however, we do not observe a similar pattern; instead, both conserved and non-conserved pseudo-5'SSs exhibit similar distributions in the vicinity of the utilized splice sites. Based on these findings, we have revised Fig. 1b and the corresponding text in the revised manuscript (see Page 6).\nRegarding the reviewer’s second suggestion, we have modified the text in line 104 to “…pseudo-5'SSs in AS events are generally positioned within 1.5 Kb of either 5' or 3'SSs. In Drosophila, conserved pseudo-5'SSs are more frequently closer to utilized splice sites than non-conserved ones, whereas their distribution are similar in humans.”\n2) Figure 3. The quantification of IR events shows % values above 100%, which is confusing. It would be better to refer to values relative to those observed under WT condition, these values being referred to as 1. Related to this, while increased inclusion of exon 4 in the Drosophila minigenes correlates with decreased levels of the exon skipping isoform, the decrease in intron 10 retention in the human minigene does not seem to have an impact on the levels of spliced exon 10-11 transcripts. On the other hand, effects of mTOR/insulin inhibition in mammalian cells in Figure 7 show skipping of both exons 10 and 11 as a consequence of the inactivation. Has skipping of exons 11 and 12 been assessed in the minigene experiments such as those of Figure 3C?\nResponse: We appreciate the reviewer for bringing these issues to our attention.\nFirst, we have adjusted the relative value of WTs to “1” in Figure 3 and all other relevant Figures.\nSecond, in response to the reviewer’s comment and to improve the comparability between human minigenes data in Figure 3 and the endogenous mTOR/insulin inhibition data in mammalian cells presented in Figure 7, we re-performed RT-PCR experiments. These revised experiments included analyses of both intron 10 retention of exon 10. The results are now consistently presented across Figures 3 and 7, revealing a clear correlation and similar trend in both Drosophila and human systems. Corresponding updates to these results have also been incorporated into the relevant sections of the manuscript text (Pages 9 and 14).\n3) Related to the previous point: the mechanism by which recognition of the pseudo-5’SS by U1 snRNP leads to intron retention rather than to activation of the sequence as a donor splice site (e.g. splicing to the next available 3’SS) remains unclear. It would be too much to ask to decipher the mechanism in this first report, but the authors should comment on efforts to detect such products. For example, should these products be detectable using the primer pairs used to detect IR events? Are such shorter products indeed detected or not below the IR bands in (uncropped) full gel electrophoresis images?\nResponse: The reviewer raises an intriguing question: why does the recognition of the pseudo-5'SS by U1 snRNP result in intron retention rather than activating the sequence as a donor splice site? This question also aligns with our own research interests. Recently, we have identified three potential explanations: 1) location (distance relative to the utilized splice sites), 2) flanking sequences, and 3) the presence of a nearby functional branch site. We plan to dissect these possible mechanisms in a forthcoming study.\nRegarding the reviewer’s concern about shorter products: 1) We did not observe such bands when using different intronic primers; 2) no shorter products were observed in the full gel electrophoresis images. Please refer to the two uncropped full-gel images provided below.\n4) Figure 4. The results of the effects of knock down of U1 snRNP core proteins are clear. They are in fact remarkable because one could expect that such knock downs would lead to decreased recognition of 5’SS in general. Selectivity could be explained by these pseudo-5’SS being particularly sensitive to decreased levels/activity of U1 snRNP, a hypothesis that the authors may want to discuss. However the key question is whether the different knock downs (also those serving as controls) have an impact on the levels of U1 snRNP. I understand that the lack of good antibodies for the Drosophila factors make it difficult to assess this, but the authors could check the levels of U1 snRNA as a surrogate for U1 snRNP particles, assuming that U1 snRNA will not have the same half-life if not assembled in full particles. This control should be feasible and could add to the mechanistic interpretation of the results. Along similar lines, do the increases in U1-70K levels observed in Figure 7 correlate with increased levels of U1 snRNA, again as a surrogate for U1 snRNP particles?\nResponse: To address the reviewer’s concern and follow the suggestion, we performed RT-qPCR analysis of U1 snRNA in the relevant samples.\nIn Drosophila S2 cells, our new data suggest that knockdown of U1-70K, U1A, and SF3B4 resulted in a moderate decrease in U1 snRNA levels, whereas knockdown of U1C and SF3A3 had no significant impact on U1 snRNA abundance. These observations indicate that the assembly and/or stability of U1 snRNP depends on U1 components in a component-specific manner. Consistent with this, previous studies in zebrafish (Röse et al., 2011, EMBO J) and yeast (Tang et al., 1997, EMBO J) demonstrated that depletion of U1C does not disrupt the core assembly of U1 snRNP particles. This suggests that while U1C may play regulatory roles in splice site selection and splicing efficiency, it is not required for maintaining the structural integrity of the U1 snRNP complex itself. In human 293T, Huh7, and KGN cells, our new data reveal that inhibition of mTOR and Insulin pathways led to an increase in U1 snRNA levels, accompanied by upregulation of the U1-70K protein. This finding establishes a positive correlation between the abundance of U1-70K and U1 snRNA. These new data have been added to the revised Figs. S3e and S8a.\nCollectively, these results strongly support our model that the availability and stability of U1 snRNP directly regulate pseudo-5'SS recognition and alternative splicing. Compared to the utilized 5'SSs, the selective sensitivity of pseudo-5'SS to U1 snRNP levels likely reflects its weaker binding affinity for U1 snRNP and greater reliance on optimal U1 snRNP concentrations for efficient recognition. This provides a mechanistic basis for the splicing regulation observed in our study. Corresponding updates to these results have also been incorporated in the relevant section of the manuscript text on pages 11 and 17.\n5) Figures 4d and 6d, 6e, 6f: are the differences statistically significant?\nResponse: To address the reviewer’s question, we performed comprehensive two-way ANOVA analyses for each gene independently, followed by planned contrasts to examine compensation effects.\nIn Figure 4d, both the Del and Mut samples exhibited highly significant compensation effects (p < 0.001, Cohen's d > 15).\nIn Figures 6d, 6e, and 6f, the significance test results are more complex. For the Rheb gene, the effect was statistically significant (p = 0.019, Cohen's d = 3.36), whereas other genes showed no significant effects (pTOR = 0.088, pInR = 0.076, pInRact = 0.085, pChico = 0.509, pPten = 0.610). This pattern of significance likely reflects the distinct roles of these genes within the insulin/TOR signaling network. As a direct TOR activator, Rheb may be more sensitive to compensatory regulation, while upstream effectors (InR, InRact) and pathway modulators (Chico, Pten) would have more variable responses depending on their specific regulatory contexts and feedback mechanisms.\nTo avoid confusion regarding these findings, we have not included this new analysis In the revised manuscript.\n6) Text:\na. Abstract: “in which ultraconserved RNA motifs may play essential regulatory roles”: I would suggest “Introns have expanded dramatically during evolution, and while their internal sequences have greatly diverged, the potential function of ultraconserved RNA motifs remains an important, unanswered question”.\nResponse: We appreciate the reviewer’s suggestion and have modified this sentence in the revised abstract as the reviewer suggested.\nb. Abstract, line 31: “Conservatively, …” I would suggest removing and finishing the sentence with “…suggesting remarkable conservation of this mechanism”.\nResponse: We have modified this sentence as the reviewer suggested.\nc. Line 42: “while the length of exons per gene has remained relatively constant around 3K nt” may be confusing. Better to refer to the length of mature mRNAs, as exons are much shorter than 3K, particularly in vertebrates.\nResponse: We apologize for this confusing statement. Given that there are many introns in UTRs, we have revised it to “while the length of mRNA (sum of exons) per gene has remained relatively constant around 3K nt”.\nd. Line 48: U2 snRNP auxiliary factors should be “U2 snRNP Auxiliary Factors (U2AF1/2).\nResponse: We corrected this as the reviewer suggested.\ne. Line 67: clarify if this refers to the conservation of splice site signals or to other intronic sequences near the splice sites.\nResponse: We clarified this as the reviewer suggested in the revised manuscript.\nf. Line 96 and throughout the text “actual 5’SS” is not a commonly used term to designate actively used (utilized) 5’SS.\nResponse: According to the reviewer’s suggestion, we modified “actual 5'SS\" to “utilized 5'SS” throughout the text.\ng. Line 131: “pre-termination codon (PTC)” should be “premature termination codon (PTC)”.\nResponse: We apologize for this mistake and have corrected it.\nh. Lines 219-220: relevant to point #3 above, it may not be entirely clear that recognition of the psedo-5’SS by U1 snRNP impairs intron definition.\nResponse: Based on our updated experimental data, we confirm that the U1 snRNP does interact with this pseudo-5'SS (see details in the response below and the revised manuscript). However, we acknowledge the reviewer’s concern that the extent to which U1 snRNP recognition of the pseudo-5'SS impairs intron definition remains insufficiently elucidated. Therefore, we have revised this sentence to “….would impair…”.\ni. Lines 329-330: strictly speaking, the interaction between U1 snRNP and the pseudo-5’SS has not been directly tested in this manuscript (also relevant to the discussion in point #4).\nResponse: We agree with this concern, which was also raised by another reviewer. To address it, we have performed an RNA immunoprecipitation (RIP) assay using three newly constructed plasmids and the U1-70K antibody in human Huh7 cells. Each plasmid transcribes a 250-nt RNA sequence containing either the WT UCP-5'SS, a mutated UCP-5'SS, or the deleted UCP-5'SS. Notably, only the RNA harboring the WT UCP-5'SS was co-immunoprecipitated by the U1-70K antibody. These new data have been incorporated into the revised Fig. 7f and the revised text on page 15.\nIn addition, we analyzed published CLIP-seq datasets and found that the U1A protein binds to Enox1 mRNA precisely at the UCP-5'SS site (Deng et al., 2020, Biochem Biophys Res Commun, A potential mechanism underlying U1 snRNP inhibition of the cleavage step of mRNA 3' processing). This finding is presented in a new supplementary figure (Fig. S7g) and the revised text on page 15.\nTaken together, these results provide direct support for the conclusion that the UCP-5'SS is recognized by U1 snRNP.\nReviewer #2 (Remarks to the Author):\nDing et al. present a comprehensive and compelling study on the evolutionary conservation and functional mechanism of an ultraconserved pseudo 5’ splice site (UCP-5’SS) in regulating the alternative splicing of the ENOX1/Enox gene across the animal kingdom, and further explores its biological significance in ovarian development. Through integrative bioinformatic analyses, the authors identified UCP-5’SS within homologous introns, focusing on a particularly conserved UCP-5’SS located in the ENOX1/Enox genes. The authors effectively integrate bioinformatic analyses with functional assays from both Drosophila and human cell lines, to elucidate how UCP-5’SS acts as a splicing silencer responsive to TOR and insulin signaling pathways. The study reveals a molecular mechanism linking translational regulation of U1-70K to alternative splicing control, ultimately affecting physiological functions like ovary development. Importantly, this study provides valuable insights into the biological significance and underlying mechanism by which the UCP-5’SS function as cis-regulatory elements in alternative splicing. Overall, this manuscript is well-structured, with clear data presentation and substantial experimental design. Accordingly, I recommend this paper for publication, below are a few issues that need to be addressed before publication.\n1. The authors claimed that this short intronic RNA motif functions as a sensor of TOR-related pathways. How to define a TOR pathway sensor? What conditions does RNA motif possess to become a development sensor? Please clarify a TOR pathway sensor and whether it holds general significance?\nResponse: We appreciate the thoughtful questions raised by the reviewer. The TOR/Insulin pathways orchestrate cell growth, metabolism, and autophagy by integrating signals from nutrient availability, growth factors, and other relevant cues. A TOR/Insulin pathway sensor can thus be defined as a molecule or structural unit that detects changes in these pathways and subsequently transduces signals or elicits corresponding cellular responses.\nIn this manuscript, we provide three key lines of evidence to support our conclusion. First, we present evidence that deletion of this 9-nt RNA motif leads to significant changes in Drosophila ovary development. Second, we demonstrate that the expression and AS of DmEnox and HsEnox1 are regulated by the TOR and Insulin pathways. Third, we find that this regulatory effect of the TOR/Insulin pathways is significantly attenuated when this RNA motif is absent. Based on these collective findings, we conclude that this 9-nt RNA motif functions as a sensor of the TOR/Insulin pathways. Corresponding revisions to the relevant text in the Discussion part (Pages 17-18) have been made.\n2. The authors showed that deletion of the Enox UCP-5'SS in Drosophila results in a significantly enlarged ovary, and that the enlarged ovary phenotype caused by deletion of the UCP-5'SS could be due to the changes in expression of the eggshell chorionic layer genes. However, the authors did not explain how deletion of the UCP-5'SS affect expression of the eggshell chorionic layer genes.\nResponse: This is an interesting question. So far, we don’t know the exact mechanism by which the deletion of this UCP-5'SS affects the expression of the eggshell chorionic layer genes. However, chorion genes are well-documented to exhibit highly tissue- and stage-specific expression profiles. They are exclusively expressed in ovarian follicle cells, with transcriptional activity strictly restricted to stages 10-14 of oogenesis (Orr-Weaver, 1991, Bioessays; Calvi et al., 1998, Gene & Dev). By contrast, ENOX proteins serve as key regulators of intracellular NAD(H) levels and redox homeostasis (Zhang et al., 2024, Redox Biology), and perturbations in ENOX1 activity have been shown to disrupt cellular ultradian rhythms (Morré & Morré 2012, ECTO-NOX proteins: growth, cancer, and aging).\nBuilding on these reports and our observations, we propose that deletion of this UCP-5'SS leads to increased ENOX1 protein levels, which in turn disrupts the metabolic and circadian rhythms of follicle cells. Given that chorion genes are tightly temporally regulated during late oogenesis, this rhythm disruption results in aberrant expression timing and levels of these genes, ultimately contributing to the enlarged ovary phenotype. Thus, the observed downregulation of chorion genes reflects a perturbation in developmental timing, rather than an intrinsic impairment of the genes’ own functional capacity.\nTherefore, we have added a paragraph describing this possibility in the revised manuscript on page 9: “Chorion genes are exclusively expressed in ovarian follicle cells, with transcriptional activity strictly restricted to stages 10-14 of oogenesis 51, 52. Additionally, perturbations in ENOX1 activity have been shown to disrupt cellular ultradian rhythms 53. We propose that increased DmENOX protein levels would disrupt the metabolic and circadian rhythms of follicle cells; in turn, this disruption would lead to altered expression of chorion genes, given their strict temporal regulation during oogenesis.”\n3. Indeed, the AS changes are observed in all the tested Drosophila mutants from the dTOR and Insulin-like pathways, and loss of this UCP-5'SS significantly mitigates these changes. In addition, multiple-source human cells treated with the mTOR/Insulin pathway inhibitors also change the AS and specifically increase the translation of U1-70K. Are all these evidences sufficient to indicate that pseudo 5' splice site fine-tunes development by regulating alternative splicing within TOR-related pathways? In other word, does pseudo 5' splice site fine-tunes development mainly through regulating alternative splicing within TOR-related pathways? Are there other mechanisms for pseudo 5' splice site?\nResponse: We thank the reviewer for this question. Based on the evidence in this manuscript, the main conclusion is that this pseudo-5’SS fine-tunes ovarian development by regulating the alternative splicing of the Enox1 gene within TOR-related pathways. While other mechanisms, such as epigenetic regulation, transcriptional control, or transcription-coupled splicing regulation, cannot be completely ruled out, they are considered less likely. In addition, the regulatory effect of this pseudo-5’SS may be tissue-specific. It is also possible that other, as-yet-unidentified developmental defects exist, which could arise from distinct mechanisms.\n4. What are the differences between pseudo-5'SS and alternative 5' splice sites? Is there evolutionary conversion between pseudo-5'SS and alternative 5' splice sites?\nResponse: The key difference between pseudo-5'SSs and alternative 5'SSs is that pseudo-5'SSs are never utilized as 5'SSs, whereas alternative 5'SSs are employed under specific conditions, such as in a tissue- or cell-specific manner, in response to stress induction, or even ubiquitously.\nRegarding the evolutionary interconversion between these two types of splice sites, this is an intriguing question. Although we have not yet performed bioinformatic analyses to explore this phenomenon, it represents a valuable direction for future investigation.\n5. In the Method section, the upstream and downstream sequences (2 + 5 nts) of all the annotated 5'SSs were extracted to calculate the position weight matrixes (PWMs). What is based on the upstream and downstream sequences 2 + 5 nts parameters? Are there some pseudo-5'SS not identified? Please clarify.\nResponse: The consensus sequence of 5'SSs is typically calculated based on the last 3 nts of exons plus the first 6 nts of introns (i.e., 3 + 6 nts) (Roca, et al., 2008, Genome Research, Features of 5′-splice-site efficiency derived from disease-causing mutations and comparative genomics; Wang, et al., 2019, BMC Bioinformatics, SpliceFinder: ab initio prediction of splice sites using convolutional neural network). Therefore, our use of a 2 + 5 nts internal window for identifying pseudo-5'SS is unlikely to result in the omission of such sites. However, pseudo-5'SS could potentially be missed if new defining characteristics of 5'SSs are identified.\n6. In Discussion section, I suggest a deeper discussion of the evolutionary significance of conserved pseudo-5'SS?\nResponse: As requested by the reviewer, we have added the following sentences to the Discussion section of the revised manuscript (Page 16):\nThe evolutionary conservation of this UCP-5'SS reflects a regulatory mechanism with multiple roles: it enables organisms to flexibly and rapidly adjust protein structure and abundance without genomic modifications; acts as an evolutionary capacitor; and functions as a splicing efficiency-modulated quantitative rheostat for gene expression (responsive to metabolic shifts, developmental cues, or cellular stress); and buffers environmental variation while preserving adaptive potential.\n7. In Fig. 2e, there appears to be a misalignment of the group name.\nResponse: We have aligned the group name in the revised Figure 2.\n8. In Fig. 2g, authors choose RT-PCR to validate RNA-seq results, which may not be reliable enough. I suggest switching to RT-qPCR, which provides more accurate quantification.\nResponse: According to the reviewer’s suggestion, we have performed RT-qPCR and added the new data to Fig. 2g.\n9. In Fig. 3b and 3c, there are some spacing gaps between the letters, such as \"mini gene\" and \"mock-del\". A carefully reviewing of all figures should be done.\nResponse: We have checked all the Figures and removed all the spacing gaps.\n10. The quantification results in Fig. 3, 4, and 5 are missing group labels, which hinders the clear interpretation of the data. To improve clarity, I suggest adding the appropriate group labels in these figures.\nResponse: We thank the reviewer for pointing this out. To optimize space, the quantification results share group labels with the gels presented above; these gels already include labels at the top of each panel.\n11. The layout of Fig. 4 appears somewhat disorganized and visually unbalanced. I recommend improving the alignment and overall formatting to enhance clarity and visual appeal. Similarly, the alignment of panels in Fig. 3d and 3e could also be refined for better consistency.\nResponse: According to the reviewer’s suggestion, we have reorganized Figs 3 and 4.\n12. Please ensure that the size of the significance asterisks in the quantification results of Fig. 7 is consistent. In particular, the asterisks in panels 7d and 7e appear to be too small.\nResponse: We have adjusted the size of asterisks to ensure consistency throughout.\n13. I suggest authors to use the full-term Western blot or the abbreviation WB to refer this experimental technique, rather than simply using “Western” in formal scientific figures.\nResponse: Thanks, we have revised this following the reviewer’s suggestions.\nReviewer #3 (Remarks to the Author):\nIn their manuscript ‘An ultraconserved pseudo 5' splice site fine-tunes development by regulating alternative splicing within TOR-related pathways’, Xu and colleagues describe a role for an intronic sequence resembling a 5’ splice site in controlling gene expression and ovary development. The manuscript suggests a that the pseud 5’ splice site is downstream of mTOR signaling, interacts with U1 snRNP and thereby controls alternative splicing of an upstream alternative exon, which then controls ovary development.\nSeveral points make the manuscript difficult to read and parts of the conclusion are not justified by the data presented.\nThe sentence in the introduction: ‘length of introns per gene has expanded from ~200 nt to ~200K nt, while the length of exons per gene has remained relatively constant around 3K nt’ is wrong and the citation not correctly used.\nResponse: We apologize for the confusing description and thank the reviewer for this important clarification. We have revised this sentence to: \"The total intron length per gene has expanded from ~200 nt to ~200K nt, while the length of exons (including UTRs) per gene has remained relatively constant at around 3K nt\".\nOur statement is based on a Figure from “McCoy, et al., 2020, BMC Genomics” (see below), which specifically analyzes and illustrates the cumulative length of introns and exons within the genes across evolutionary time. Regarding the length of exons per gene, the ~3K nt transcript length is well-supported by multiple independent studies (Piovesan et al., 2019, BMC Res Notes) and (Leung et al., 2021, Cell Rep). These values are consistent with our statement.\nThis statement: ‘the conserved pseudo-5'SSs are involved in the events of skipped exon (SE) or retained intron (RI) (Fig. 1c) and located in genes with neural functions’ is wrong. Pseudo splice sites may be located within such genes, but that does not mean that they are involved in any splicing events.\nResponse: We appreciate the reviewer’s comment. This statement is modified as “In addition, most of the conserved pseudo-5'SSs are located in genes with neural functions (Fig. S1a) and regions associated with skipped exon (SE) or retained intron (RI) events (Fig. 1c).\nThroughout the entire manuscript, the function of ENOX1/Enox genes is described as ‘involved in plasma electron transport’. I think this should mean ‘plasma membrane electron transport’. Please correct and carefully proof read the entire manuscript.\nResponse: We have made corrections in accordance with the reviewer’s suggestion.\nPhenotypes in figure 2 have to be shown in 2 independently generated lines per genotype (in fig 3d two UCPD lines are used – show the phenotype of the second line, also for ko). Fig 2f and 2g colors are confusing. In 2f ko shows mostly red (up) but this is blue in 2g. Same for UCPD. Please use consistent colors. Also, why are these genes down in the UCPD which are more fertile. Shouldn’t this be the other way?\nResponse: We thank the reviewer for these insightful questions and suggestions.\nFirst, we have revised Figure 2 by adding “line 1” to the UCPΔ strain (UCPΔ-L1) and adjusting the colors in Figs. 2f and 2g.\nSecond, we have corrected the color inconsistencies in Figure 2 to ensure consistent representation across panels.\nThird, the precise mechanism by which deletion of the UCP-5'SS affects the expression of the eggshell chorionic layer genes remains unclear. However, chorion genes are well-documented to exhibit highly tissue- and stage-specific expression profiles. They are exclusively expressed in ovarian follicle cells, with transcriptional activity strictly restricted to stages 10-14 of oogenesis (Orr-Weaver, 1991, Bioessays; Calvi et al., 1998, Gene & Dev). By contrast, ENOX proteins serve as key regulators of intracellular NAD(H) levels and redox homeostasis (Zhang et al., 2024, Redox Biology), and perturbations in ENOX1 activity have been shown to disrupt cellular ultradian rhythms (Morré & Morré 2012, ECTO-NOX proteins: growth, cancer, and aging). Building on these reports and our observations, we propose that deletion of this UCP-5'SS leads to increased ENOX1 protein levels, which in turn disrupts the metabolic and circadian rhythms of follicle cells. Given that chorion genes are tightly temporally regulated during late oogenesis, this rhythm disruption results in aberrant expression timing and levels of these genes, ultimately contributing to the enlarged ovary phenotype. Thus, the observed downregulation of chorion genes reflects a perturbation in developmental timing, rather than an intrinsic impairment of the genes’ own functional capacity.\nTherefore, we have added a paragraph describing this possibility in the revised manuscript on page 9: “Chorion genes are exclusively expressed in ovarian follicle cells, with transcriptional activity strictly restricted to stages 10-14 of oogenesis 51, 52. Additionally, perturbations in ENOX1 activity have been shown to disrupt cellular ultradian rhythms 53. We propose that increased DmENOX protein levels would disrupt the metabolic and circadian rhythms of follicle cells; in turn, this disruption would lead to altered expression of chorion genes, given their strict temporal regulation during oogenesis.”\nPlease use RT-qPCR instead of the standard RT-PCR in 2g bottom (an upregulation in ko is not visible in most cases in the data shown).\nResponse: According to the reviewer’s suggestion, we have performed RT-qPCR and added the new data to Fig. 2g.\nIn all splicing quantifications, please indicate % inclusion +/- Standard deviation below the gel. It would also be better to plot % inclusion +/- SD rather than the normalization that is then shown +/- SEM.\nResponse: We thank the reviewer for pointing out this issue. Actually, for the splicing quantifications, we used +/- SD for standard deviation; however, this was mislabeled in the original manuscript. We have indicated all splicing quantifications in the legends and below the gel of the relevant Figures.\nAll gel, especially the ones that quantify intron retention, need a -RT control.\nResponse: We agree with the reviewer that a “-RT” control is necessary to detect residual genomic DNA in samples. In recent years, we have been using the PrimeScript-RT reagent Kit with gDNA Eraser (TAKARA, Cat. No. RR047A), which contains a highly efficient DNase. Under -RT conditions, this kit consistently produce no detectable bands. To validate for the current study, we tested several representative samples under -RT conditions, and no PCR products were observed in any of these tests. Two relevant results are shown in the figures below. Based on these comprehensive findings, we consider it unnecessary to include this specific set of control data in the manuscript.\nFig 3C show three gels that all investigate intron 10 retention. It would be much more informative to quantify real IR and not something normalized, which makes the effect look stronger than it is. This also needs a -RT control. Skipping of exon 10 has to be analyzed as well and a quantification as in Fig 3b has to be shown.\nResponse: We thank the reviewer for these valuable suggestions. To enhance the comparability between human minigenes data in Figure 3 and the endogenous mTOR/insulin inhibition data in mammalian cells presented in Figure 7, we re-performed RT-PCR experiments. These revised experiments included analyses of both intron 10 retention and exon 10 skipping, with corresponding quantifications provided below the gels. The results reveal a clear correlation and similar trend in both Drosophila and human systems. Corresponding updates to these results have also been incorporated into the relevant sections of the manuscript text (Pages 9 and 14).\nRegarding the -RT control, please see the above response.\nIn the scheme in Fig. 3a, remove the introns after splicing\nResponse: We have removed introns after splicing in Fig. 3a.\nThe splicing phenotype of the endogenous fly mutants in Fig. 3d has to be confirmed at the endogenous level in a human cell line as well.\nResponse: Thank you for your suggestion regarding cross-species validation. We have already tried to delete this short RNA motif in human cells multiple times using the CRISPR/Cas9 system. Unfortunately, these attempts were unsuccessful, and we have not yet established a cell line with this pseudo-5'SS deletion. This failure may be due to technical challenges or the possibility that the deletion negatively affects human cell growth. Currently, we are collaborating with another lab to create conditional knockout mice. We hope to know whether this RNA motif influences the development of the mouse ovary or other organs soon.\nIn Fig. 4 it is surprising that knock down of U1 components that are required for all splicing events lead to increased exon inclusion. Increased IR could have been an expected outcome. To validate this, RNA-Seq should be performed to see what the global effect is and to confirm the finding for Enox splicing using a different read out. If this is confirmed, these experiments should be repeated in human cells.\nResponse: We agree that the reviewer had raised an important question. Following the reviewer’s suggestion, we performed RNA-seq on U1-70K knockdown S2 cells to investigate global splicing changes, including exon skipping and intron retention. Transcriptome-wide analysis confirmed the expected outcomes noted by the reviewer: knockdown of U1-70K results in hundreds of increased exon skipping events and elevated intron retention (IR) events.\nNotably, this RNA-seq data validated our experimental findings: the PSI value for Enox exon 4 skipping increased by 0.159 (corresponding to a 15.9% increase in the inclusion of this exon), and the PSI value for Enox intron 4 retention (IR) decreased by 0.198. These results confirm the validity of our other experimental data and proposed model, in which this pseudo-5'SS acts as a splicing silencer rather than a competing splice site.\nTo reconcile these observations, we propose the following: U1-70K knockdown globally impairs splicing (causing widespread exon skipping and intron retention), but the specific interaction between U1 snRNP and the Enox pseudo-5'SS promotes exon 4 inclusion. When U1-70K is depleted, this enhancing effect is lost; however, the relative impact on Enox exon 4 inclusion runs counter to the global trend, suggesting a gene-specific regulatory mechanism. In conclusion, we incorporated this new data in the revised Fig. S3f-g and added these findings in the revised manuscript on page 11.\nComparing splicing in Fig. 4a and 4c, there is a strong discrepancy in the control. In 4a there is 61% inclusion, in 4c in the control it is only 25%. This questions all splicing analyses.\nResponse: We would like to thank the reviewer for pointing out this discrepancy. In actuality, Fig. 4 includes three controls, with their respective inclusion ratios being 61%, 65%, and 25% in Fig. 4a, 4b, and 4c, respectively. When analyzing splicing of the endogenous DmEnox under the same S2 cell background, no significant differences were observed between the two experiments presented in Fig. 4a and 4b (61% vs. 65%).\nHowever, the detection target differs in Fig. 4c: here, we analyzed the splicing of DmEnox minigenes rather than that of endogenous DmEnox. These minigenes only contain a fragment of DmEnox (spanning exon 3 to exon 5) and are driven by the promoter contained within the vector (for details on the vector structure and intron sequences, please refer to Fig. 3b).\nIt is widely recognized that splicing efficiency is influenced by factors such as transcription rate and intron position. Furthermore, regardless of the baseline splicing efficiency of this intron in the two different controls, we consistently observed a comparable magnitude of increase in splicing efficiency following U1-70K knockdown.\nFig. 5c has also inconsistent controls. Splicing in the ‘-‘ controls differs from 55 to 36 % inclusion. This is too much variation.\nResponse: The reviewer’s concern here aligns with the one addressed above. The ‘-‘ controls in Fig. 5c are not truly identical, and this discrepancy arises from the distinct genetic background of the strains used in this study (see Supplement Table 4).\nFor example: The dTOR RNAi strain has a genetic background of y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMS00904}attP2; the Tsc1-RNAi strain background is y1 sc* v1 sev21; P{TRiP.HMC03672}attP40, and the RhebEx strain background is w*; P{UAS-Rheb.Pa}3. These strains were generated by different research groups. The observed variation in splicing efficiency among these controls reflects inherent genetic differences rather than experimental inconsistency, a phenomenon that is well-documented in Drosophila genetic research (Kurmangaliyev, et al., 2015, BMC Genomics; Guzman, et al., 2021, Genetics).\nTo avoid this confusion, we have added a note about the difference in genetic background to the revised manuscript (Page 12).\nIn 5e there is hardly a change in splicing in the Pten mutant but a strong change in protein expression. The change in the protein can’t then be mediated by the change in splicing.\nResponse: We thank the reviewer for pointing out this issue and agree that while the splicing change in the Pten mutant is less than that in other mutants, it exhibits a stronger alteration in protein expression. The exact mechanism underlying this phenomenon remains unclear. Several possibilities may exist in the Pten mutant background: 1) differences in RNA and protein stability and half-life; 2) Variations in post-transcriptional and post-translational regulatory mechanisms.\nWe have identified relevant clues from previous studies, which report significant changes in Pten mutants in the context of the human Insulin signaling pathway (Pal et al., 2012, PTEN mutations as a cause of constitutive insulin sensitivity and obesity. N Engl J Med). For PTEN specifically, protein expression is tightly regulated by two key mechanisms: 1) translational control mediated by PI3K/mTOR/4E-BP1 signaling (Mukherjee et al., Mol Cell 2021); 2) post-translational modifications, including ubiquitination, phosphorylation, and acetylation, that affect protein stability (Lee et al., Nat Commun 2015; González-García et al., Cancers 2022). Consistently, genome-wide studies demonstrate that mRNA levels are often poor predictors of protein abundance (Edfors et al., Mol Syst Biol 2016), as gene-specific RNA-to-protein conversion factors determine the relationship between transcript and protein levels.\nPlease show that the pseudo-splice site is really by the U1 snRNP, e.g. using CLIP. To make the claim that ‘UCP-5'SS is recognized by the U1 snRNP and their interaction impairs the intron definition…’ a spliceosome assembly gel should be shown comparing RNA with and without the pseudo splice site.\nResponse: We agree with this concern, which has also been raised by another reviewer.\nTo address the first question, we have performed an RNA immunoprecipitation (RIP) assay using three newly constructed plasmids and the U1-70K antibody in human Huh7 cells. Each plasmid transcribes a 250-nt RNA sequence containing either the WT UCP-5'SS, a mutated UCP-5'SS, or the deleted UCP-5'SS. Notably, only the RNA harboring the WT UCP-5'SS was co-immunoprecipitated by the U1-70K antibody. These new data have been incorporated into the revised Fig. 7f and the revised text on page 15.\nIn addition, we analyzed published CLIP-seq datasets and found that the U1A protein binds to Enox1 mRNA precisely at the UCP-5'SS site (Deng et al., 2020, Biochem Biophys Res Commun, A potential mechanism underlying U1 snRNP inhibition of the cleavage step of mRNA 3' processing). This finding is presented in a new supplementary figure (Fig. S7g) and the revised text on page 15. Taken together, these results provide direct support for the conclusion that the UCP-5'SS is recognized by U1 snRNP.\nRegarding the second question, we acknowledge the reviewer’s concern that the extent to which U1 snRNP recognition of the pseudo-5'SS impairs intron definition remains insufficiently elucidated. Therefore, we have revised the sentence to “…their interaction would impair the intron definition…”.\n‘Secondly, we found that the knockdown of dTOR resulted in a 38% decrease in the EE-inclusion, and the decrease was reduced to 21% when the…’ Please use PSI change.\nResponse: According to the reviewer’s suggestion, we have revised those numbers to PSI values on page 13.\n‘Consistently, the HsENOX1 protein was dramatically decreased in all the inhibitor-treated cells (Figs. 7d-e and Fig. S7b).’ This is not a ‘dramatic’ change.\nResponse: We revised it to “significantly decreased.”\n‘short ultraconserved RNA motif’ – Ultraconserved has been used before and is defines as conserved elements of at least 100 nts. I would call this short element ultraconserved.\nResponse: Thank you for this important terminological clarification. Addressing the author’s concern, we revised this to “ultraconserved short RNA element”.\nA direct connection of TOR and Insulin pathways with the translation of U1-70k is not shown. It is also not clear how the level of U1-70k regulates binding of U1 snRNP to the pseudo splice site.\nResponse: We thank the reviewer for these important questions, which highlight key aspects of our regulatory model.\nFirst, using a luciferase reporter containing the U1-70K 5'UTR sequence, we observed significantly increased luciferase activity following rapamycin treatment (see revised Figure S7f), confirming that the TOR pathway directly regulates U1-70K translation.\nSecond, as noted above, our two newly conducted experiments (the RIP assay using different RNA substrates and RNA-seq analysis of U1-70K knockdown S2 cells) indicate that reduced U1-70K levels impact the U1 snRNP binding to this pseudo-5'SS."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed the issues raised in my previous report and I am happy to recommend the paper for publication in Nature Communications as a very valuable addition to the literature on splice site recognition and regulation.", "reviewer_2": "The authors have answered my question very well.", "reviewer_3": "In the revised version of their manuscript ‘An ultraconserved pseudo 5' splice site fine-tunes development by regulating alternative splicing within TOR-related pathways’ Xu and colleagues have addressed most of my initial points. Some points remain:\n- ‘The total intron length per gene has expanded from ~200 nt to ~200K nt, while the length of exons (including UTRs) per gene has remained relatively constant at around 3K nt’ remains wrong. The paper/figure they cite specifically looks at the 10% longest genes. Also see labeling of the figure ‘long gene’.\n- As noted in the initial comment, there are 2 deletion lines (Fig. 3 L1 and L2). Please show the phenotype of both lines in Fig. 2c, d, e.\n- The gels in Fig 3c remain confusing. The only one that may allow a quantification is the second from top that shows all isoforms. This actually shows that IR and skipping of exon 10 are very rare events. Maybe add a note in the figure legend to acknowledge this.\n- The new Fig. 7f looks nice but, but how often was this done (n=?)? I would suggest additional qPCR as readout of at least 3 independent replicates."}, "author_response": "Response: We would like to thank the reviewer for all the helpful suggestions and insightful comments.\n- ‘The total intron length per gene has expanded from ~200 nt to ~200K nt, while the length of exons (including UTRs) per gene has remained relatively constant at around 3K nt’ remains wrong. The paper/figure they cite specifically looks at the 10% longest genes. Also see labeling of the figure ‘long gene’.\nResponse: We appreciate the reviewer’s suggestion and have revised the text as follows: “From budding yeast to humans, the number of introns in a genome has increased from hundreds to hundreds of thousands. The average length of introns per gene has also extended substantially. For example, in the 10% of longest genes of a genome, the average intron length per gene has increased from ~200 nt to ~200K nt over the course of evolution, whereas the average length of exons (including UTRs) per gene has remained relatively constant at around 3K nt 1.”\n- As noted in the initial comment, there are 2 deletion lines (Fig. 3 L1 and L2). Please show the phenotype of both lines in Fig. 2c, d, e.\nResponse: We thank the reviewer for this suggestion. Previously, we observed similar phenotypes between the two lines but did not perform a sufficient number of assays on L2 to enable statistical analysis. As requested, we have re-performed these assays with L2 and replaced new data into the revised Figs. 2c and 2d. These updated data demonstrate that both L1 and L2 exhibit significantly enlarged ovaries compared with WT controls, whereas testis size remains largely unchanged. Moreover, not much difference was detected between these two lines. Therefore, we consider it unnecessary to assess the fecundity of L2, as this would require labor-intensive and time-consuming crossing and counting experiments.\n- The gels in Fig 3c remain confusing. The only one that may allow a quantification is the second from top that shows all isoforms. This actually shows that IR and skipping of exon 10 are very rare events. Maybe add a note in the figure legend to acknowledge this.\nResponse: We appreciate the reviewer’s suggestion and have added the following note to the legend of Fig. 3c: “The frequency of intron 10 retention and exon 10 skipping in Huh7 cells is substantially lower than their counterparts in S2 cells.”\n- The new Fig. 7f looks nice but, but how often was this done (n=?)? I would suggest additional qPCR as readout of at least 3 independent replicates.\nResponse: According to the reviewer's suggestion, we have now performed this experiment with three independent biological replicates (n=3) and validated the results via qPCR quantification. As requested, the qPCR data generated from these replicates have been incorporated into the revised Fig.7f. These additional quantitative analyses further confirm the reproducibility and statistical significance of our findings."}]

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