hnRNPM cooperates with BCAS2 to modulate alternative splicing during oocyte development

Domain: neuroscience
Published: 2026-02-12
Editorial outcome: major_revision

Abstract
GrowingoocytesaccumulatematernalmRNAtosupportsubsequentmeioticmaturationandmaternal-to-zygotictransition.However,theregulatorymechanismsgoverningthefateofthesematernalmRNAsremainlargelyunknown.Here,weidentifiedheterogeneousnuclearribonucleoproteinM(hnRNPM)asacriticalregulatorofpre-mRNAalternativesplicingduringmouseoocytedevelopment.GeneticablationofhnRNPMleadstoseverecytoplasmicdefects,meioticarrest,andcompletefemaleinfertility.UsingSCAN-seq,weuncoverednoveltranscriptisoformsandsystematicallycharacterizedhnRNPM-regulatedalternativesplicingevents.Furthermore,LACE-seqrevealedhnRNPM-bindingsitesatsingle-nucleotideresolutioninoocytes,linkingitsRNA-bindingactivitytosplicingfidelity.Additionally,hnRNPMinteractswithBCAS2,aknownsplicingfactorcriticalforoocytedevelopment,andmodulatesitsbindingtopre-mRNAlocitopreciselycontrolthealternativesplicing.Overall,ourstudynotonlyuncoveranessentialroleofhnRNPMinmammalianoocytedevelopmentandfemalefertilitybutalsounveilsacriticalregulatorynetworkgoverningalternativesplicingduringoocytedevelopment.

Introduction
Oocytes, the largest mammalian cell type, accumulate substantial amounts of maternal transcripts during their growth phase to support subsequent meiotic maturation and early embryonic development1–3. The proper production, storage, and surveillance of mRNA are critical for oocyte and early embryonic development. We previously identified nuclear poly(A) domains as critical processing hubs that coordinate the maturation and storage of newly synthesized mRNAs in growing oocytes3. N-acetyltransferase 10 catalyzes the N4-acetylcytidine modification of mRNAs encoding key maternal factors4. RNA helicase mRNA transporter 4 functions as nuclear RNA surveillance machinery to ensure the clearance of aberrant transcripts in oocytes2. However, the post-transcriptional regulation of pre-mRNAs remains largely unknown.

Alternative pre-mRNA splicing represents a classical mechanism for enriching the diversity of the transcriptome and proteome5,6. Through this process, a single pre-mRNA can generate multiple mRNA isoforms, which subsequently translate into distinct protein variants7. mRNA undergoes significant alternative splicing dynamics during maternal-to-zygotic transition, a process referred to as zygotic splicing activation (ZSA)8. ZSA occurs concurrently with zygotic genome activation (ZGA) during early embryonic development in both humans and mice8. Precise expression of transcript isoforms is critical for successful preimplantation embryogenesis. Additionally, genetic studies in mice have identified several key splicing regulators, including RTCB, BCAS2, ESRP1, and PCBP1, that are essential for oogenesis9–12, further illustrating the significant role of alternative splicing in oocytes.

While pre-mRNA splicing is critical for oocyte and early embryonic development, capturing full-length transcripts and identifying splicing factor-binding sites remains technically challenging because of the limited quantity and scarcity of oocyte samples. Recent technological advances in RNA sequencing, particularly the emergence of third-generation sequencing platforms, have revolutionized our ability to detect and characterize splicing events with unprecedented accuracy13. The development of single-cell amplification and sequencing of full-length RNAs using an nanopore platform (SCAN-seq) based on third-generation sequencing platforms has overcome previous limitations by enabling comprehensive full-length transcriptome analysis from low-input samples and even on a single oocyte14. This method provides a sensitive and accurate approach for detecting full-length transcripts in oocytes. Additionally, linear amplification of cDNA ends and sequencing (LACE-seq), which combines linear cDNA end amplification with RNA-binding protein (RBP)-induced reverse transcription termination, allows precise mapping of RBP interaction sites at a single-nucleotide resolution in oocytes15.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of RBPs that form complexes with heterogeneous nuclear RNA to regulate pre-mRNA processing16. Although hnRNPs play crucial roles during spermatogenesis17–21, their functions in oogenesis remain largely unknown. In a systematic evaluation of hnRNP expression across multiple tissues, we observed that Heterogeneous nuclear ribonucleoprotein M (hnRNPM) is highly expressed in reproductive system22, suggesting a potentially critical role in germ cell development and reproductive physiology. Moreover, an independent study employing mRNA interactome capture (RIC) analysis on 50,000 fully-grown mouse oocytes identified hnRNPM as one of the core mRNA-binding proteins (RBPs) enriched in oocytes23. This finding indicates that hnRNPM may functionally engage in regulating the fate of maternal mRNAs in oocytes. hnRNPM contains three RNA recognition motif domains that bind to RNAs. Recent studies have demonstrated that hnRNPM preferentially binds to deep introns to repress cryptic splicing, thereby maintaining transcriptome integrity, which is important for regulating interferon-associated pathways and enhancing cancer surveillance24. Additionally, hnRNPM forms a complex with 5′-tRH-GlyGCC to regulate cancer cell proliferation25 and interacts with Fragile X Mental Retardation Protein and PARP4 to influence tumor progression26,27.

In this study, we demonstrated that hnRNPM coordinates with BCAS2 to regulate alternative pre-mRNA splicing during oogenesis and is essential for establishing competent oocytes capable of supporting oocyte maturation and early embryogenesis. We found that hnRNPM exhibited stage-specific nuclear localization throughout folliculogenesis, disappeared during meiotic resumption, and reappeared at the 2-cell stage. Genetic ablation of hnRNPM led to severe defects in cytoplasmic maturation, failure to enter metaphase I, and female infertility. Mechanistically, hnRNPM regulates alternative splicing of key factors involved in cytoplasmic lattice formation (Khdc3 and Nlrp14) and meiotic maturation (Cenph and Zar1l). We identified differential alternative splicing patterns in the novel Nlrp14 and Zar1l isoforms using SCAN-seq, overcoming the limitations of conventional sequencing technologies. Furthermore, we mapped the hnRNPM-binding sites and revealed their preferential association with transcripts governing organelle localization and meiotic regulation using LACE-seq analysis. These findings establish that hnRNPM is an indispensable regulator of oocyte development and provide a comprehensive analysis of hnRNPM-mediated splicing control during mammalian oogenesis.

Results
The dynamic nuclear expression of hnRNPM during oocyte development
Multi-alignment and phylogenetic analyses of the hnRNPM protein sequences showed that hnRNPM is highly conserved across multiple mammalian species, including human, rhesus monkey, mouse, Norway rat, pig, dog, domestic cattle, and bovine (Fig. 1a and Supplementary Fig. 1a, b). Strikingly, the sequence homology between mouse and human hnRNPM proteins was remarkably high (97.53%), suggesting that murine models may provide valuable insights for understanding and treating human diseases (Supplementary Fig. 1a). To elucidate the role of hnRNPM in female fertility, we systematically examined its spatiotemporal expression patterns during oogenesis. Immunofluorescence analysis revealed that hnRNPM was localized in oocyte nuclei. Its expression was weak in embryonic germ cells, increased after birth, and sustained throughout all subsequent follicular stages, including primordial, primary, secondary, and antral follicles (Fig. 1b and Supplementary Fig. 1c–e). To characterize the dynamics of hnRNPM expression following meiotic resumption, we conducted in vitro maturation (IVM) and in vitro fertilization (IVF). Immunofluorescence co-staining of hnRNPM with α-Tubulin (a marker of the spindle) and DAPI revealed its distinct spatiotemporal localization. hnRNPM was strongly expressed in the nuclei of germinal vesicle (GV) with non-surrounded nucleus (NSN) oocyte, whereas the signal was diminished in GV with surrounded nucleus (SN) oocyte and was completely absent in germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) oocytes (Fig. 1c). Following fertilization, hnRNPM remained undetectable in zygotes but reappeared in the nuclei of 2-cell embryos, persisting through the 4-cell, morula, and blastocyst stages (Fig. 1d).

Maternal Hnrnpm loss results in infertility
To illustrate the physiological role of hnRNPM during oocyte development, we conditionally deleted Hnrnpm in germ cells using Stra8-Cre transgenic mice (Stra8-Cre; Hnrnpmflox/Del, herein called Hnrnpm cKO), in which Cre specifically knockout the exon 7–12 of Hnrnpm in female germ cells from embryonic day 12.5 (Fig. 2a–c)28. Both the Hnrnpm mRNA and hnRNPM protein levels in cKO oocytes were significantly lower than those in the litter-matched controls (Hnrnpmflox/+; Stra8-Cre) mice (Fig. 2d, e and Supplementary Fig. 2a–c), indicating that hnRNPM was successfully inactivated in oocytes with high efficiency. The fertility test revealed that conditional deletion of Hnrnpm in germ cells led to complete female infertility (Fig. 2f).

To investigate the role of hnRNPM in oocyte development, we conducted comprehensive histological analyses of ovarian tissues from 3-month-old female mice. Comparative evaluation of control and Hnrnpm cKO ovarian tissues revealed no significant differences in ovarian size or maximum cross-sectional area (Fig. 2g, h). Quantitative assessment of follicle populations using hematoxylin-eosin staining demonstrated comparable numbers of follicles at all developmental stages, including the primordial, primary, secondary, and antral follicles (Fig. 2i).

To synchronize follicular growth and promote ovulation, control and Hnrnpm-deficient female mice were injected with pregnant mare serum gonadotropin (PMSG) at postnatal day 21 (P21), followed by human chorionic gonadotropin (hCG) 48 h later. After induction of superovulation, we collected cumulus-oocyte complexes (COCs) and performed IVF. While the number of oocytes obtained was comparable between control and Hnrnpm-deficient mice (Fig. 2j and Supplementary Fig. 2d), Hnrnpm-deficient oocytes failed to complete cleavage and embryonic development after IVF (Fig. 2k, l), indicating that Hnrnpm-deficient oocytes lacked developmental potential after fertilization.

hnRNPM regulates the formation of cytoplasmic lattice
Given the robust expression of hnRNPM during folliculogenesis and its complete absence from GVBD through the zygotic stage, we hypothesized that maternal hnRNPM deficiency causes early developmental impairments that subsequently compromise embryonic competence. To test this hypothesis, we collected GV-stage oocytes from 21-day-old female mice for statistical and morphological analysis. To synchronize follicular growth, control and Hnrnpm cKO female mice at P21 were injected with PMSG, and GV oocytes were collected from the ovaries at 14 h post-injection (Fig. 3a). Strikingly, although the number of GV oocytes collected from the ovaries was comparable between control and Hnrnpm cKO female mice (Fig. 3b), 88.3% of Hnrnpm-deficient oocytes exhibited abnormal dark cytoplasmic granules, in contrast to the homogeneous cytoplasm observed in the controls (Fig. 3c, d).

Cytoplasmic lattices serve as critical structural scaffolds that maintain proper organelle distribution by preventing the aberrant aggregation of cytoplasmic components29,30. To investigate the subcellular differences between control and hnRNPM knockout oocytes, we performed transmission electron microscopy analysis of GV-stage oocytes. Cytoplasmic lattices were abundant in control oocytes, whereas they were markedly reduced in Hnrnpm cKO oocytes (Fig. 3e), demonstrating that hnRNPM is essential for their formation.

Consistent with the observed loss of cytoplasmic lattices, transmission electron microscopy images revealed abnormal aggregation of lipid droplets and mitochondria in Hnrnpm cKO oocytes (Fig. 3e). To further characterize mitochondrial distribution patterns, we conducted MitoTracker staining. Control oocytes exhibited a homogeneous mitochondrial distribution throughout the cytoplasm, whereas Hnrnpm-deficient GV oocytes displayed pronounced mitochondrial clustering and uneven localization (Fig. 3f). Similarly, Nile red staining revealed significant lipid droplet aggregation in Hnrnpm cKO oocytes compared with controls (Fig. 3g). Some lipid droplets colocalized with dark cytoplasmic aggregates in Hnrnpm-deficient oocytes (Fig. 3g and Supplementary Fig. 2e), suggesting that these dark aggregates represented coalesced lipid droplets. Taken together, these findings demonstrate that Hnrnpm deficiency disrupts cytoplasmic lattices, leading to aberrant mitochondrial distribution and lipid droplet aggregation in GV-stage oocytes.

Hnrnpm deficiency impairs the meiotic maturation of oocytes
Acquisition of full developmental competence in oocytes depends on the coordinated progression of nuclear and cytoplasmic maturation31,32. To investigate the role of hnRNPM in oocyte maturation, we performed IVM assays using GV-stage oocytes. While the rates of GVBD were comparable between control and Hnrnpm-deficient oocytes (Fig. 4a, b), only 22.77% of Hnrnpm-deficient oocytes successfully progressed to the MII stage and extruded the first polar body compared with 77.20% of the controls (Fig. 4c). Consistent with IVM results, only ~30% of ovulated oocytes from Hnrnpm cKO mice reached the MII stage following hCG injection (Supplementary Fig. 2f, g), confirming severe maturation defects. Subsequently, we examined the nuclear behavior during oocyte meiosis. In control MI and MII oocytes, chromosomes are neatly aligned at the metaphase plate, accompanied by the establishment of a bipolar spindle, whereas Hnrnpm-deficient oocytes display misaligned chromosomes and exhibit apolar and multipolar spindles (Fig. 4d–g).

To determine whether hnRNPM regulates the assembly of a microtubule-organizing center (MTOC), we analyzed the expression of pericentrin, a key MTOC marker. Interestingly, pericentrin was localized around the spindle poles in control oocytes following 8 h of in vitro maturation, while most Hnrnpm-deficient oocytes failed to show detectable pericentrin expression (Fig. 4d and Supplementary Fig. 2h). This observation suggests that Hnrnpm deficiency may disrupt the timely expression of pericentrin after meiotic resumption. Remarkably, after in vitro maturation for 16 h, the expression of pericentrin can be observed in majority of Hnrnpm-deficient oocytes (Fig. 4f and Supplementary Fig. 2h). These results indicate that hnRNPM is required for the proper temporal regulation of pericentrin expression during oocyte maturation. We next asked whether aberrant spatiotemporal expression of pericentrin stemmed from defects in earlier growth phases. Fluorescence staining of GV oocytes revealed that well-defined puncta of pericentrin were present in controls but undetectable in KO oocytes (Supplementary Fig. 2h, i), indicating an early developmental defect. Collectively, these results demonstrate that hnRNPM is essential for meiotic maturation in oocytes.

hnRNPM regulates alternative splicing during oocyte development
To determine whether hnRNPM regulates alternative splicing in oocytes, we conducted SCAN-seq (single-cell amplification and sequencing of full-length RNAs using a nanopore platform) using control and Hnrnpm-deficient GV oocytes from postnatal day 21 mice (Fig. 5a and Supplementary Fig. 3a). Across all samples, we detected an average of 29,778 transcript isoforms per sample (Supplementary Fig. 3b). Interestingly, through full-length sequencing, novel transcripts in oocytes were detected (Supplementary Data 1). To study the function of these novel transcripts, we performed a Gene Ontology (GO) enrichment analysis using the international standard database (https://www.geneontology.org/) to investigate the biological processes involving these novel transcripts. Strikingly, the top enriched processes were closely related to female infertility—including female meiosis chromosome segregation, regulation of fertilization, and maternal-to-zygotic transition of gene expression (Supplementary Fig. 3c and Supplementary Data 1), suggesting that unique transcripts may exist in oocytes to regulate female fertility. To validate our sequencing results, four unannotated transcripts related to female fertility were amplified using RT-PCR and detected using Sanger sequencing (Fig. 5b–j). Meikin is a conserved regulator of kinetochores during meiosis I33. Nlrp14 regulates the subcellular localization of Dnmt1/Uhrf1 in mouse oocytes and is associated with cell lattice formation, oocyte maturation, and early embryonic development34–36. Vrk1 (Vaccinia-related kinase 1) mediates meiotic progression during oogenesis37. Zar1l, also known as Zar2, mediates maternal transcriptome and translational activation and regulates oocyte meiotic maturation38–41. The Sanger sequencing results for these four transcripts precisely matched the transcripts predicted by SCAN-seq (Fig. 5f–j). The remarkable accuracy in the identification of unannotated transcripts provided evidence that SCAN-seq was successful.

To characterize alternative splicing patterns, we systematically analyzed seven major types of splicing events: Skipping exon (SE), Mutually exclusive exons (MX), Alternative 5’ splice-site (A5), Alternative 3′ splice-site (A3), Retained intron (Rl), Alternative first exon (AF), and Alternative last exon (AL). Among these, the skipping exons and alternative first exons were the most prevalent splicing types (Supplementary Fig. 3d). Subsequently, we compared the splicing patterns between control and Hnrnpm-deficient GV oocytes and identified 1147 differentially spliced events, of which 992 were successfully annotated to known genes (Fig. 6a, Supplementary Fig. 4a, b and Supplementary Data 2). The gene ontology (GO) terms of these differentially spliced genes revealed multiple distinct gene clusters with a particular GO term enriched in the meiotic cell cycle (Fig. 6b). Other pathways included chromatin organization, protein transport, regulation of DNA-templated transcription, protein localization to microtubules, and spliceosomal complex assembly, implying that hnRNPM regulates multiple cellular processes in oocytes (Fig. 6b).

Among differentially spliced genes, those associated with cytoplasmic lattice formation (Khdc3 and Nlrp14), meiotic maturation (Cenph and Zar1l), and female fertility (Bcl2l1 and Prkaa1) have attracted attention. Khdc3 (also known as FILIA) is a component of the subcortical maternal complex (SCMC) that is essential for the formation of cytoplasmic lattices42. Nlrp14 has been implicated in cell lattice formation, oocyte maturation, and early embryonic development34–36. Cenph is a kinetochore component that regulates meiotic resumption in oocytes43. Zar1l mediates maternal transcriptome and translational activation and regulates oocyte meiotic maturation38. Prkaa1 (also known as AMPK) and Bcl2l1 are associated with female infertility44,45. Analysis of RNA sequencing data using sashimi plots revealed distinct alternative splicing patterns, including increased exon skipping in Cenph and Prkaa1, decreased exon skipping in Nlrp14 and Zar1l, elevated intron retention in Khdc3, and differential first-exon splicing in Bcl2l1, all of which were validated using RT-PCR (Fig. 6c–e and Supplementary Fig. 4c, d). These findings support the notion that hnRNPM is crucial for ensuring the fidelity of pre-mRNA splicing in genes that regulate cytoplasmic lattice formation and meiotic maturation. To assess the impact of alternative splicing defects on protein expression, we conducted Western blot analysis for two key downstream targets, CENPH and ZAR1L (Supplementary Fig. 4e). These data demonstrate that splicing defects caused by hnRNPM deficiency leads to a sharp decline in ZAR1L and a mild downregulation of CENPH protein, thereby bridging the observed molecular changes to downstream phenotypic consequences.

Profiling the RNA-binding landscape of hnRNPM in oocytes using Lace-seq
Given that hnRNPM is an RBP that regulates alternative splicing during oocyte development, we investigated the RNA-binding landscape of hnRNPM in oocytes. Lace-seq is a high-resolution method capable of identifying RBP-binding sites with high precision in dozens of cells15. In this technique, the binding site of the RBP is directly retrieved by linearly amplifying the termination signal of the reverse transcriptase precisely at the RBP-binding site (Fig. 7a). Compared with conventional methods, this technology achieves remarkable accuracy in identifying RBP-binding sites with single-base resolution at the single-cell level4,15. To profile the targets of hnRNPM at a single-nucleotide resolution in GV oocytes, we performed hnRNPM LACE-seq using GV oocytes. In parallel, we conducted hnRNPM LACE-seq using Hnrnpm cKO oocytes to eliminate false positive signals (Fig. 7a). Bioinformatics analyses of the LACE-seq results showed that the binding signals of hnRNPM were markedly decreased in Hnrnpm cKO oocytes, suggesting that our antibody exhibited high specificity (Fig. 7b). We identified 1691 hnRNPM-binding sites (corresponding to 1690 genes) in wild-type GV oocytes compared with Hnrnpm cKO oocytes (Supplementary Data 3). Genomic distribution analyses of the reads revealed that 69.67% of the putative binding sites were located in the exons of mRNAs, with 45.02% of reads mapped to the coding sequence (CDS), 9.57% to other exons, 11.75% to the 3′ untranslated region (3′ UTR), and 3.32% to the 5′ UTR, respectively, suggesting that hnRNPM preferentially binds to exons in oocytes (Fig. 7c). Previous eCLIP-seq analyses have demonstrated preferential binding of hnRNPM to the GU-rich hexamer46,47. To determine the consensus-binding sequence of hnRNPM, we performed motif analysis using the findMotifsGenome.pl tool from HOMER on the identified peak regions. Consistent with previous reports, hnRNPM was preferentially crosslinked with GU-rich sequences (Fig. 7d). GO terms indicated that hnRNPM-binding transcripts were enriched in establishment of organelle localization, heterochromatin formation, actin cytoskeleton organization, and regulation of synapse organization (Fig. 7e).

To further investigate the correlation between hnRNPM-targeted mRNA and alternative splicing, we compared the LACE-seq data with differential alternative splicing events identified by SCAN-seq. Among the 722 genes undergoing differential alternative splicing events, the mRNAs of 116 genes were identified as direct targets of hnRNPM based on LACE-seq data, indicating their potential role as direct targets of hnRNPM-alternative splicing regulatory complexes. Gene enrichment analysis revealed that the main functions of these overlapping genes were related to cell growth and mechanisms associated with pluripotency, microtubule nucleation, mitotic prometaphase, and sister chromatid cohesion (Fig. 7f). Given that hnRNPM regulates the alternative splicing of genes associated with cytoplasmic lattice formation (Khdc3 and Nlrp14), meiotic maturation (Cenph and Zar1l), and female fertility (Bcl2l1 and Prkaa1), we investigated whether hnRNPM could bind to the splicing sites of these transcripts. Strikingly, hnRNPM was specifically enriched at the alternative splicing sites of Khdc3, Nlrp14, Cenph, Bcl2l1, and Prkaa1 (but not Zar1l), indicating its direct regulation of these transcripts (Fig. 7g–i and Supplementary Fig. 5a, b). Furthermore, to obtain more direct evidence for hnRNPM’s role in regulating alternative splicing, we analyzed hnRNPM binding signals on differentially alternative spliced transcripts (AS) and unaffected alternative spliced transcripts (Un-AS). Strikingly, hnRNPM binding was significantly enriched on the exons of transcripts that underwent alternative splicing upon its depletion, compared to those that were unaffected (Supplementary Fig. 5c, d). This provides much more direct evidence that hnRNPM binding at these specific regions is functionally responsible for the splicing regulation.

hnRNPM cooperates with BCAS2 to regulate the alternative splicing
To unveil the protein-protein interaction network of hnRNPM in oocytes, we conducted immunoprecipitation-mass spectrometry using approximately 8000 GV oocytes (Fig. 8a and Supplementary Data 4). GO analysis revealed that hnRNPM-interacting proteins were significantly enriched in mRNA processing pathways (Fig. 8b), consistent with the known molecular functions of hnRNPM24. Among the interacting proteins enriched in the RNA splicing pathway, BCAS2 garnered attention because of its role as an RNA splicing factor that regulates cell lattice formation and oocyte maturation12 (Fig. 8c). Western blotting confirmed the robust interaction between hnRNPM and BCAS2, and this interaction does not rely on RNA (Fig. 8d and Supplementary Fig. 6a, b). Protein localization studies further revealed that BCAS2 displayed a nuclear distribution pattern similar to that of hnRNPM throughout follicular development (Fig. 8e). Previous studies reported that BCAS2 participates in pre-mRNA splicing by forming a complex with PRP19 and CDC5L12,48–52. We therefore asked whether hnRNPM associates with this complex. Strikingly, co-immunoprecipitation assays demonstrated strong interactions between hnRNPM and both PRP19 and CDC5L (Supplementary Fig. 6c, d), suggesting that hnRNPM and BCAS2 may function in a highly coordinated manner to regulate alternative splicing.

To investigate the potential cooperative functions of BCAS2 and hnRNPM, we compared our SCAN-seq data with published SCAN-seq data from control and Bcas2 cKO oocytes12. This analysis revealed that hnRNPM and BCAS2 shared 98 commonly affected alternative splicing genes (Fig. 8f). Of these 98 alternative splicing genes, 57 same alternative splicing events occurred in both Hnrnpm and Bcas2 cKO oocytes (Fig. 8f), demonstrating functional synergy between these two factors. To validate the co-regulation of splicing events by hnRNPM and BCAS2, we selected three representative targets for detailed analysis using sashimi plots and RT-PCR. These analyses confirmed consistent splicing alterations in both Bcas2 and Hnrnpm cKO oocytes, including increased exon skipping in Cenpf, decreased exon skipping in Prc1, and differential first-exon splicing in Hdlbp (Fig. 8f–k). Notably, Cenpf and Prc1 play significant roles in the meiotic maturation of oocytes. Prc1 is associated with spindle bipolarization in MI oocytes53, which is consistent with our observation of defective bipolarized spindle formation in Hnrnpm-deficient oocytes during the GVBD-to-MI transition. Cenpf mediates meiotic regulation by recruiting dynamin-related protein 1 (an atypical spindle assembly checkpoint protein) to the kinetochore during oocyte meiosis I54. To investigate whether a similar regulated role exists in human cells, we knocked down HNRNPM in 293T cells, and confirmed efficient protein depletion by western blotting (Supplementary Fig. 6e). Consistent with a conserved regulatory role, depletion of either hnRNPM or BCAS2 in human cells recapitulated the splicing changes of PRC1 and HDLBP, indicating that hnRNPM and BCAS2 also cooperate to control alternative splicing of human transcripts (Supplementary Fig. 6f).

Considering the ability of hnRNPM to co-regulate alternative splicing with BCAS2 in oocytes, we next asked whether transcripts with altered alternative splicing were bound by hnRNPM and BCAS2 proteins. RNA immunoprecipitation (RIP)-qPCR was performed using anti-hnRNPM and BCAS2 antibodies to immunoprecipitate RNAs from ovaries. Notably, Cenpf, Prc1, and Hdlbp transcripts were significantly enriched in both hnRNPM and BCAS2 immunoprecipitates (Fig. 8l, m). To elucidate how hnRNPM cooperates with BCAS2 during the alternative splicing process, we analyzed the expression and localization of BCAS2 protein upon hnRNPM depletion. The results showed that hnRNPM loss significantly decreased BCAS2 protein expression but did not affect its nuclear localization (Fig. 8n and Supplementary Fig. 6g). Interestingly, analysis of BCAS2-knockout oocytes revealed that BCAS2 depletion also led to a marked reduction in hnRNPM protein levels, without disrupting its nuclear localization (Supplementary Fig. 6h, i). These findings suggest that hnRNPM and BCAS2 likely form a complex and are mutually dependent for protein stability.

To examine whether the reduction of BCAS2 protein in hnRNPM‑deficient cells compromises its enrichment on pre‑mRNA, we conducted BCAS2 RNA immunoprecipitation assays in control and HNRNPM‑knockdown cells. The results showed a marked decrease in BCAS2 binding to CENPF, PRC1, and HDLBP transcripts upon hnRNPM depletion (Fig. 8o, p), indicating that hnRNPM deficiency impairs BCAS2 association with transcripts co‑regulated by both proteins. To further validate these findings, we analyzed six exon-skipping events (Arfip1, Bod1l, Cep43, Lrrc17, Mis18bp1, and Zcchc10) and seven alternative first-exon splicing events (Adh5, Eloc, Esyt1, Lonp2, Mapk6, Mrps33, and Snx15) co-regulated by hnRNPM and BCAS2. All 13 transcripts showed significant enrichment in both hnRNPM and BCAS2 immunoprecipitates (Supplementary Fig. 7a–d). Strikingly, hnRNPM knockdown led to a significant reduction in BCAS2 binding at 9 of these 13 co-regulated targets (69.2%) (Supplementary Fig. 7e–h). Together, these findings support a model in which hnRNPM cooperates with BCAS2 to regulate pre-mRNA alternative splicing in oocytes (Fig. 9).

Methods
Ethics statement
The mice used in this study were bred at the Animal Center of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The environment was rigorously controlled to adhere to specific pathogen-free standards, with the temperature maintained between 22 °C and 25 °C, humidity levels kept at 50% to 70%, and a regular 12-h day/night cycle of light. All experimental procedures involving animals were approved by the Animal Ethics Committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine (approval number: SRRSH2025-0029).

Mice
Hnrnpmflox/+ mice (strain no. T008736) were generated by GemPharmatech (Nanjing, China). Bcas2flox/+ mice were generously provided by Professor Lei Li at the State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences63. The Stra8-Cre mouse line was gifted by Professor Minghan Tong of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences (Shanghai, China)28. Vasa-Cre mice were obtained from The Jackson Laboratory. According to the structure of the Hnrnpm gene, exons 7–12 of Hnrnpm-206 (ENSMUST00000148178.7) transcript containing a 490-bp coding sequence were selected as the knockout region. Knocking out this region results in the impairment of protein function. To generate Hnrnpmflox/+ mice, loxP sites were inserted into introns 6 and 12 of Hnrnpm. Hnrnpmflox/flox mice were obtained by crossbreeding female Hnrnpmflox/+ and male Hnrnpmflox/+ mice. Hnrnpmflox/+; Stra8-Cre mice were generated by mating Stra8-Cre and Hnrnpmflox/+ mice. To produce Hnrnpmflox/Del; Stra8-Cre (designated Hnrnpm cKO) mice, male Hnrnpmflox/+; Stra8-Cre mice were mated with female Hnrnpmflox/flox or Hnrnpmflox/+ mice. To generate Bcas2flox/Del; Vasa-Cre (designated Bcas2 cKO) mice, we crossed male Bcas2flox/+; Vasa-Cre mice with female Bcas2flox/flox or Bcas2flox/+ mice. To genotype the mice, PCR amplification was performed on genomic DNA extracted from mouse tails. The primer sequences used for genotyping are listed in Supplementary Data 5. All mice used in this study were in C57BL/6J background.

Oocyte collection and IVM
Twenty-one-day-old female mice were intraperitoneally injected with 5 IU of PMSG (Ningbo Sansheng). After 44 to 48 h, the ovaries were punctured using a needle to release the oocytes. GV-stage mouse oocytes were manually retrieved using a mouth pipette. GV oocytes were harvested in M2 medium (Sigma, M7167) supplemented with 2.5 μM milrinone (MCE, HY-14252). Only full-grown GV oocytes were cultured in M16 medium (Sigma, M7292) and covered with mineral oil (Sigma, M5310). MI and MII oocytes were collected after 8 and 14 h of culture, respectively.

In vitro fertilization and culture
Three-week-old female mice were superovulated with 5 IU of hCG (Ningbo Sansheng), followed by 5 IU PMSG (Ningbo Sansheng) 44 h later. After 14 h, the female mice were euthanized, and COCs were collected from the ampulla of the fallopian tubes. Sperms were obtained from the epididymis of wild-type adult male mice and capacitated in TYH medium (Nanjing Aibei Biotechnology, M2035) for 50 min. Subsequently, the capacitated sperms and COCs were combined in HTF medium (Nanjing Aibei Biotechnology, M1135) for fertilization. After 6 h, fertilized oocytes were transferred to KSOM (Nanjing Aibei Biotechnology, M1435) for further culturing. Representative early embryos were photographed at 1, 2, 3, and 4 days post-IVF.

Cell culture, plasmid construction, and transfection
HEK293T cells (Procell, CL-0005) were maintained in Dulbecco’s Modified Eagle Medium supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum (NEST Biotechnology, 209111) at 37 °C in a 5% CO₂ humidified atmosphere. To passage cells, begin by removing the spent medium from the culture dish (Bioland, CCD06) and washing the cell layer gently with PBS. Dissociate the adherent cells by adding pre-warmed trypsin-EDTA and incubating at 37 °C. After cell detachment, inhibit trypsin activity by adding Fetal Bovine Serum (Jin Yuan Kang Biotechnology, FBS-300). Then, transfer the cell suspension to a 15 mL centrifuge tube (Bioland, ATS05-15) and pellet the cells via centrifugation at 300 × g for 3 min. Remove the supernatant and resuspend the cells in fresh complete medium. For cryopreservation, resuspend the cell pellet in Serum-free Cell Freezing Medium (Solarbio, C2950), distribute into cryovials (Bioland, AC05-20), and store at −80 °C.

To construct plasmids, the full-length coding sequences of mouse Cdc5l, Hnrnpm, and Prp19 were assembled into the pCMV-C-Flag, pEGFP-N1, or pCMV-HA-C vectors using the Clon Express Ultra One Step Cloning Kit (Vazyme, C115). Following this, each plasmid construct expressing a tagged protein was transfected into HEK293T cells via Polyethyleneimine. For RNA interference experiments, cells were transfected with human hnRNPM-targeting siRNA (Thermo Fisher Scientific, s9261) or human BCAS2-targeting siRNA (Thermo Fisher Scientific, 135537) using Lipofectamine RNAiMAX transfection reagent (Invitrogen, 13778150) according to the manufacturer’s protocol. Following 48 h of transfection, cells were harvested for subsequent analyses.

RT-PCR and qPCR
Total RNA was isolated from oocytes using the RNeasy Mini Kit (QIAGEN, 74104) following the manufacturer’s protocol. Genomic DNA was removed, and cDNA synthesis was performed using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, RR047A). For conventional PCR amplification, the 2X Accurate Taq Master Mix (Accurate Biotechnology, AG11009) was used. The samples were then subjected to agarose gel electrophoresis with a 100 bp DNA ladder (Amizona Scientific LLC, AMG2001-100) and imaged on a ChemiDoc Touch (Bio-Rad) and analyzed using Image Lab (Bio-Rad) software. qRT-PCR was conducted using TB Green® Premix Ex Taq™ II (Takara, RR820A) on a QuantStudio™ Real-Time PCR System (Thermo Fisher Scientific). All reactions were performed in technical triplicate with appropriate negative controls. The primer sequences are provided in Supplementary Data 5.

Immunofluorescence staining
Freshly collected oocytes were fixed in 2% paraformaldehyde for 30 min. After permeabilization with 0.25% Triton X-100 for 15 min, oocytes were blocked with 0.3% bovine serum albumin (BSA) for 30 min. The oocytes were then incubated with the corresponding primary antibodies overnight at 4 °C. Next, the oocytes were washed three times with washing buffer (0.1% Tween-20 and 0.04% Triton X-100 in PBS (MCE, HY-K1023)). The oocytes were subsequently transferred to the secondary antibody and incubated for 30–60 min. After washing three times with washing buffer containing 0.1% Hoechst 33342 (Invitrogen, H3570), the oocytes were mounted using the Slow Fade Diamond Antifade Mountant (Invitrogen, S36967).

For the immunolabeling of tissue slides, the samples were washed with PBS and blocked with 0.3% BSA (MCE, HY-D0842) for 30 min. They were then incubated with primary antibodies at 4 °C overnight. After washing with PBS three times, the samples were incubated with secondary antibodies for 30 to 60 min and then mounted with ProLong™ Diamond Antifade Mountant (Invitrogen, P36970). The image was captured by Zeiss laser-scanning confocal microscope and processed by Zen 2.3 (blue edition) software (Carl Zeiss). For the antibody information, please refer to Supplementary Data 6.

MitoTracker and Nile red staining
Oocytes were cultured in M2 medium containing MitoTracker Red CMXRos (Beyotime, C1049B) for 30 min or Nile red (MCE, 7385-67-3) for 15 min in a humidified chamber at 37 °C. Following three washes with fresh M2 medium, each lasting 5 min, the samples were imaged immediately using a Zeiss laser-scanning confocal microscope.

Transmission electron microscopy
GV oocytes from control and Hnrnpm cKO mice were collected and fixed overnight at 4 °C in 0.1 M cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde and 3% paraformaldehyde. After three 10-min washes with 0.1 M PB (pH 7.4), samples were post-fixed in 1% osmium tetroxide for 1.5 h and washed again with 0.1 M PB (3 × 10 min). Dehydration was performed using a graded ethanol series (50%, 70%, and 90%; 15 min each), followed by two 30-min immersions in 100% ethanol, a 30-min treatment with a 1:1 ethanol-acetone mixture, and two 30-min immersions in 100% acetone. The specimens were then infiltrated with mixtures of epon resin and acetone (1:1 for 2 h, 3:1 overnight), embedded, and polymerized at 65 °C for 24–48 h. Ultrathin sections (60 nm thick) were prepared and stained with uranyl acetate and lead citrate. Oocytes were sectioned into 70-nm ultrathin slices, stained with uranyl acetate and lead citrate, and imaged using a JEM-1400 transmission electron microscope (JEOL).

Western blotting
Samples were collected and lysed in RIPA Ultra Mix lysis buffer (X-blot, 100086) to extract proteins. Protein concentration was determined with the Bradford Protein Assay Kit (Absin, abs580304). The loading buffer containing β-mercaptoethanol was added to the lysates, and this mixture was heated at 95 °C for 10 min. The samples and protein standard were subsequently loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After separation, the proteins were transferred onto PVDF Transfer Membranes (Thermo Fisher, 88518) and blocked in Tris-buffered saline with Tween 20 (TBST) containing 5% non-fat milk for 1 h at room temperature (RT). Primary antibodies were incubated overnight at 4 °C. The membranes were washed with TBST and incubated with secondary antibodies for 1 h at RT. Protein bands were visualized using a Light Chemiluminescence Kit (Epizyme, SQ201) and imaged using a chemiluminescence detection device (Bio-Rad). The uncropped membranes of the blots are included in the Supplementary Information.

Immunoprecipitation coupled mass spectrometry
GV oocytes were snap-frozen in liquid nitrogen. A total of 8000 GV oocytes were pooled, lysed, and homogenized in 1 mL of IP buffer (NCM Biotech, P70100) containing a complete protease inhibitor (EDTA-free, Roche) and 25 µg/mL RNase A (Sigma) using a glass douncer, followed by incubation for 2 h at 4 °C. The lysates were cleared at 15,000 × g for 15 min, and the supernatant was incubated with 50 µL of pre-cross-linked antibody-magnetic beads (MCE, HY-K0202) overnight at 4 °C. The beads were washed five times in IP buffer and eluted for 10 min at 100 °C in 1× loading buffer. For immunoblotting analysis, protein complexes were eluted from the beads using 1× SDS loading buffer by heating at 98 °C for 5–10 min. For mass spectrometry, proteins were boiled in SDT buffer (4% SDS, 100 mM DTT, and 100 mM Tris-HCl) for 3 min with gentle agitation. The proteins eluted for mass spectrometry analysis were subjected to in-solution tryptic digestion following standard protocols. Briefly, proteins were reduced with DTT, alkylated with iodoacetamide, and digested overnight with sequencing-grade trypsin (Promega) at 37 °C. The resulting peptides were desalted using C18 StageTips and analyzed using liquid chromatography-tandem mass spectrometry on a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) equipped with a nano-electrospray ion source.

RNA immunoprecipitation
RIP assays were performed using the RNA Immunoprecipitation Kit (Bersin Bio, Bes5101), according to the manufacturer’s protocol, with minor modifications. Briefly, P21 mouse ovaries or HEK293T cells were lysed in RIP lysis buffer supplemented with 1× protease inhibitor cocktail (Roche, 04693159001) and 40 U/mL RNase inhibitor (Invitrogen, 10777019). The cleared lysates were incubated with 5 μg of IgG, anti-hnRNPM, or anti-BCAS2 antibodies at 4 °C for 16 h with rotation. After six stringent washes with ice-cold RIP wash buffer (5 min per wash at 4 °C), RNA-protein complexes were extracted using TRIzol™ LS Reagent (Invitrogen, 10296028) following the manufacturer’s protocol. The purity and integrity of RNA were verified using an Agilent 2100 bioanalyzer.

LACE-seq sequencing and data analyses
Ctrl and cKO Hnrnpm GV oocytes were collected into the bottom of 1.5 mL Eppendorf LoBind microcentrifuge tubes and irradiated twice with 400 mJ UV-C light on ice. The oocytes were lysed in 50 μL of wash buffer on ice for 10 min, followed by the addition of 1 μL of ribonuclease inhibitor (Ambion, AM2696) and 4 μL of RQ1 DNase (Promega, M6101), with incubation at 37 °C for 3 min. The tube was then snap-chilled on ice for 3 min, after which 10 µL of hnRNPM antibody-coupled beads were added to the lysate. The mixture was rotated at 4 °C for 1 h and subsequently placed on a magnetic stand for 1 min to separate the beads, with the supernatant being carefully discarded. Three washing steps were performed: washing with wash buffer (repeated thrice), followed by washing with high-salt wash buffer, and finally washing with PNK buffer. Immunoprecipitated RNAs were fragmented using MNase (Thermo Scientific, EN0181) diluted 300,000–600,000-fold in 1× MN reaction buffer. After thorough washing of the beads, 10 µL of diluted MNase was added, and the reaction was conducted at 37 °C for 3 min. Enzyme activity was quenched through sequential washes: twice with 1× PNK + EGTA buffer, twice with wash buffer, and twice with PNK buffer.

The beads were resuspended in 20 μL of FastAP reaction mixture (containing 2 μL 10× FastAP buffer, 1 μL FastAP alkaline phosphatase (Thermo Scientific, EF0651), and 17 μL nuclease-free water) and incubated at 37 °C for 10 min. Following washing, the beads were resuspended in 20 μL of ligation mixture (comprising 12.5 μL of water, 2 μL of 10× ligation buffer, 0.5 μL of 1 μM 3′ linker, 1 μL of T4 RNA ligase 2 truncated (NEB, M0242), and 4 μL of 50% PEG 8000), then incubated at 25 °C for 2.5 h with periodic vortexing. The 3′ linker was synthesized and high-performance liquid chromatography-purified by Integrated DNA Technologies. The beads were resuspended in 8.5 µL of DEPC-treated water containing 1 µL of biotinylated T7-RT primer. Following denaturation at 65 °C for 5 min and immediately cooling on ice for 2 min, the RT mixture was immediately transferred to a PCR tube and subjected to the following incubation protocols: 42 °C for 50 min, 70 °C for 15 min, followed by maintenance at 12 °C. The cDNA was then treated with 3 μL of Exonuclease I (NEB M0293) in 1× buffer (37 °C, 1 h) followed by heat inactivation (80 °C, 20 min). First-strand cDNAs were eluted from protein A/G beads and subsequently captured on streptavidin C1 beads for pre-PCR amplification. Following the addition of a 3′ cDNA linker, double-stranded DNA templates were generated for in vitro transcription (IVT). The IVT products were purified through sequential treatments: turbo deoxyribonuclease digestion to remove DNA templates, followed by purification using Agencourt RNA Clean beads, according to the manufacturer’s protocol. Linearly amplified RNA was reverse transcribed into cDNA and PCR-amplified using P7 and barcoded P5 index primers. Amplification products (130–300 bp) were size-selected using electrophoresis on a 2% agarose gel and purified using a MinElute Gel Extraction Kit (Qiagen, 28604) following the manufacturer’s protocol. LACE-seq libraries were subjected to single-end sequencing on an Illumina HiSeq 2500 platform by Novogene.

Raw sequence reads were first processed using Fastp (v0.23.4) for quality control66, after which poor-quality reads and adapter sequences were removed. Quality filtered reads were then mapped to the mm39 (Gencode, vM33) reference genome using STAR (v2.7.11b)67 with the parameters “--alignEndsType Local --alignIntronMin 20 --alignIntronMax 20000 --outFilterMismatchNmax 2 --outFilterMultimapNmax 10 --alignMatesGapMax 700 --alignSJoverhangMin 10 --outFilterIntronMotifs RemoveNoncanonicalUnannotated.” Samtools (v1.19.2)67 was used to calculate the read coverage and depth. Duplicate PCR reads and reads in the blacklist regions68 were filtered using BedTools. The filtered reads were exported as a BED file for peak calling using Piranha (v1.2.1)69. Statistical significance was calculated based on a Zero-Truncated Negative Binomial distribution (parameters: “-s -p 0.001 -b 20”). Specifically, a one-sided (right-tailed) test was employed to identify regions with a significant enrichment of reads. To account for multiple comparisons across genomic bins, p values were adjusted using the Benjamini-Hochberg method, and a False Discovery Rate (FDR) threshold of <0.001 was applied. Subsequently, the peak regions were extensively annotated using the reference annotation dataset Gencode_vM33 with ChIPSeeker (v1.42.0)70. The genomic distribution frequency of annotated peaks across gene body features (including UTRs, 1st exons, and introns) was statistically analyzed using the “TxDb.Mmusculus.UCSC.mm39.refGene” database. Finally, findMotifsGenome.pl from HOMER (v4.11)71 was used with default parameters to identify known motifs within the peak regions. Additionally, the DeepTools (v3.5.4)72 bamCoverage function was used to generate the LACE-seq signal track files in the BigWig format. Subsequently, heatmaps of peak signals across all TSS were generated using the computeMatrix function and plotHeatmap from DeepTools, along with aligned peak signal heatmaps.

SCAN-seq single-cell amplification
GV oocytes from control and Hnrnpm cKO mice were collected using a mouth pipette and placed in a lysis buffer containing 0.1% Triton X-100 (Sangon, A110694-0100), dNTP mixture (Invitrogen, 00575484), barcode oligo (dT) primers, and an RNase inhibitor (Takara, 2313A). Immediately, the tubes underwent vigorous vortexing for 60 s and then incubated at 72 °C for 3 min to promote cell lysis and release of linearized RNA. Then, each sample was mixed with 2.83 μL of reverse transcription mixture, comprising SuperScript IV reverse transcriptase (Invitrogen, 18090050), RNase inhibitor (Takara, Cat. 2313A), SuperScript IV first-strand buffer, 1 M betaine (Sigma-Aldrich, B0300-1VL), 100 mM DTT (Invitrogen, 18090050), 50 mM MgCl₂ (Sangon, A610328-0500), and template-switching oligo primers. The reverse transcription reaction was executed under the conditions detailed below: 25 °C for 5 min, 42 °C for 60 min, 50 °C for 30 min, and 70 °C for 10 min. Subsequently, RT samples were mixed with 12.5 μL of PCR mixture containing ISPCR primers, the first-strand reaction product, 2× KAPA HiFi Hot-Start Ready Mix, and nuclease-free water. Amplification was performed under the following conditions: 98 °C for 20 s, followed by four cycles of 98 °C for 20 s, 65 °C for 30 s, and 72 °C for 5 min, and then 16 cycles of 98 °C for 20 s, 67 °C for 15 s, and 72 °C for 5 min, with a final extension at 72 °C for 5 min. Following PCR amplification, cDNA products from different cell barcodes were pooled and subjected to dual-round purification with 0.6× volume of AMPure XP beads. Finally, 1 μg of cDNA was used for subsequent library construction.

SCAN-seq library preparation and sequencing
The nanopore sequencing library was prepared using the Ligation Sequencing Kit 1D (Oxford Nanopore Technologies, ONT, SQK-LSK114), following the manufacturer’s instructions. Briefly, the cDNA fragments were subjected to end repair and dA-tailing using the Ultra II End Prep module. Subsequently, the dA-tailed cDNA fragments were ligated to the 1D adapter using the Quick Ligation Module. The resulting libraries were precisely quantified, quality-checked, and loaded onto a PromethION flow cell for sequencing on an Oxford Nanopore PromethION platform.

Bioinformatics analyses of SCAN-seq data
MinKNOW (v3.6.3) and Guppy (v3.1.5) were used to convert electrical signals from the Oxford Nanopore PromethION platform into FASTQ sequence data. Quality control was performed using Nanofilt (v2.8.0, https://github.com/wdecoster/nanofilt) and NanoPlot (v1.42.0, https://github.com/wdecoster) to filter out low-quality reads, defined as those with a Q-score < 7 or length shorter than 100 bp. The remaining reads were identified, oriented, and trimmed using Pychopper (v2.7.10, https://github.com/nanoporetech/pychopper). Full-length reads were then extracted based on cell barcodes and PCR anchor sequences (AAGCAGTGGTATCAACGCAGAGTAC), followed by alignment to the mouse genome (Ensembl, GRCm39) using minimap2 (v2.25)73 with the parameters “-ax splice -uf --junc-bed.” The resulting BAM file was used for downstream analysis.

Differentially expressed genes in the full-length transcriptome were analyzed using the DESeq2 (v1.46.0)74 package with default parameters, screening thresholds set at padj < 0.05 and |log2(FoldChange)| > 1. GffCompare (v0.12.6) was used to detect unannotated novel transcripts across individual samples. The SUPPA2 (v2.4, https://github.com/comprna/SUPPA) was used to identify alternative splicing events in each sample. Differential alternative splicing events across samples were visualized using sashimi plots generated using ggsashimi (v1.1.5; https://github.com/guigolab/ggsashimi).

Statistics and reproducibility
GraphPad Prism 9 and R Studio were used for statistical evaluations. Differences between two groups were assessed using a two-sided unpaired Student’s t-test. Data are presented as mean ± SEM. ns not significant. *p < 0.05, **p < 0.01, ***p < 0.001. Detailed descriptions of the statistical tests used for each experiment are available in the respective figure legends. All experiments, including histology, electron microscopy, immunofluorescence, western blotting, immunoprecipitation, PCR, and qPCR, were executed with a minimum of three biological replicates with similar results. The complete, unprocessed images for all Western blots are provided in the Sourcedata file and Supplementary Information.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript provides compelling and insightful data to reveal the crucial role of hnRNPM in pre-mRNA alternative splicing during oocyte development. Using a germ cell-specific knockout mouse model, the authors confirmed in vivo that HnRNPM deficiency results in complete infertility in female mice. Furthermore, the results of SCAN-seq and LACE-seq demonstrated that hnRNPM regulates pre-mRNA alternative splicing in oocytes by directly binding to transcripts. Additionaly, IP-MS and RIP assays revealed that hnRNPM regulates alternative splicing in oocytes by promoting the binding of BCAS2 to pre-mRNA. Overall, the conclusion is robust,and the manuscript is well-written and clear in logic. I have the following comments.\n1) IVM experiments confirmed that hnRNPM deficiency affects oocyte maturation. However, in the IVF assay, the authors only described that Hnrnpm-deficient oocytes fail to complete cleave. It is unclear whether the maturation and fertilization rate of ovulated oocytes are affected. Whether the maturation phenotype is consistent between in vivo and in vitro? If not, please give an explanation.\n2) The author introduces zygotic splicing activation (ZSA) in the background. What is the reason for the failure of zygotic cleavage in hnRNPM -deficient mice, is it due to the AS in oocytes or ZSA in zygotes? Whether knockdown of hnRNPM in zygotes using siRNA or Trim-Away affect embryonic development?\n3) A previous study showed that hnRNPH1 recruits PTBP2 and SRSF3 to modulate alternative splicing in germ cells (Nat Commun. 2022 Jun 23;13(1): 3588.doi: 10.1038/s41467-022-31364-7), what is the relationship between hnRNPM and hnRNPH1? Are they co-localized in the oocytes?\n4) Line 202-203, the author found most Hnrnpm -deficient oocytes displayed abnormal pericentrin localization. Since pericentrin is a key MTOC marker, I suggest to evaluate whether the MTOCs were destroyed in hnRNPM-deficient oocytes before GVBD using pericentrin Immunofluorescence.\n5) Whether hnRNPM CKO affect the protein level or the localization of BCAS2? In addition, BCAS2-deficient mice show more severe phenotype than hnRNPM-deficient mice, please discussion the difference.", "reviewer_2": "Zhou et al. investigate the role of heterogeneous nuclear ribonucleoprotein M (hnRNPM) in oocyte development, presenting its splicing regulatory role of maternal transcripts essential for meiosis. The authors demonstrate that the conditional knockout of hnRNPM in female germ cells leads to significant cytoplasmic defects, impaired meiotic maturation, and complete female infertility. Through single-cell amplification and sequencing of full-length RNAs using the Oxford nanopore platform (SCAN-seq), the study reveals that hnRNPM modulates alternative splicing of genes crucial for the meiotic cell cycle. Furthermore, the authors identified BCAS2 as an interacting partner of hnRNPM and propose a model where hnRNPM recruits BCAS2 to regulate the splicing of specific transcripts. The research is well-designed, and the findings are interesting, but several points could be strengthened to provide more solid evidence for the authors’ conclusion.\nMajor:\n1. Although the manuscript discusses the regulatory potential of hnRNP proteins, the introduction should explain why hnRNPM was chosen for study in the context of oocyte development.\n2. In line 211, the authors report 89 novel transcripts linked to female infertility, yet Supplementary Fig. 3b shows ~6,000–7,000 novel isoforms per sample. Is female infertility specifically enriched, and were other pathways enriched in hnRNPM-deficient GV oocytes?\n3. In Fig. 5b, it would be helpful to show gel images with both control and cKO oocytes.\n4. In Fig. 6a, indicating the direction of splicing changes (e.g., increased vs. decreased inclusion) would be helpful.\n5. In Fig. 6d and 6e, while the study convincingly demonstrates that hnRNPM depletion leads to alternative splicing of various transcripts, such as Khdc3, Nlrp14, Cenph, and Zar1l, it is not clear from the data presented whether these splicing changes result in significant alterations to the levels of corresponding intact proteins. Providing evidence, perhaps through Western blotting for some of these key affected proteins in hnRNPM-depleted oocytes, would more directly link the observed splicing defects to the downstream cellular phenotypes.\n6. The analysis in Fig. 7f shows an overlap between hnRNPM-bound genes and differentially spliced genes. To provide more direct evidence for a regulatory role, the analysis could be extended to compare hnRNPM binding signals specifically on exons and introns that are alternatively spliced versus those unaffected by hnRNPM depletion.\n7. A recent study cited by the authors (Zheng et al., 2024) demonstrated that hnRNPM protects against interferon response by repressing LINE-associated cryptic splicing. It would be a valuable addition to investigate whether the hnRNPM binding sites identified in oocytes are also enriched for LINE1 elements, and whether the key splice events affected by hnRNPM depletion are associated with these retrotransposons.\n8. In Fig. 8d, the authors propose that hnRNPM recruits Bcas2 to regulate splicing. To clarify the nature of this interaction, a co-IP experiment including an RNase treatment step is necessary. This would determine whether the interaction is a direct protein-protein association or is mediated by RNA.\n9. In Fig. 8f, the study proposes a “strong functional synergy” between hnRNPM and Bcas2 based on the overlap of 98 commonly affected alternative splicing genes and 57 identical splicing events in the respective knockout oocytes. Are these overlaps statistically significant? While suggestive, further evidence is needed to solidify this claim, or toning down is necessary.\nMinor:\n1. In Fig. 1c and 1d, the study demonstrates that hnRNPM is absent from the GVBD stage through the zygote stage. It would be interesting to speculate on the functional implications of this dynamic expression. Is the dynamic expression during oocyte maturation important for ZSA? What might be the consequences if hnRNPM is consistently expressed during these stages?\n2. In Fig. 2k and 2l, there appears to be a discrepancy between the dramatic development arrest shown in Fig. 2k and the moderate, albeit significant, changes in developmental rates presented in Fig. 2l. The visual evidence in Fig. 2k suggests a near-complete failure of Hnrnpm-deficient oocytes to develop beyond the 2-cell stage after IVF. An explanation for this difference would strengthen the interpretation of the impaired developmental phenotype.\n3. In Fig. 8g, the basis of claiming “increased exon skipping in Cenpf” could be more quantitatively supported.\n4. Based on the study, we can hypothesize but cannot definitively conclude that human hnRNPM and BCAS2 form the same interaction network to control splicing in human oocytes. While the RIP-qPCR analyses in Fig. 8n-p are suggestive, the splicing regulatory consequences in human transcripts tested here should be examined in a human cell model as well.\n5. A minor but important point for clarity is the consistent use of nomenclature for genes, transcripts, and proteins. The study frequently uses human-centric or nomenclature for proteins (e.g., BCAS2) in a mouse model or mouse-centric nomenclature for transcripts (e.g. Cenpf, Prc1, and Hdlbp in Fig. 8) in a human model, while correctly italicizing mouse gene names (e.g., Hnrnpm, Bcas2). Adhering to the correct organism-specific nomenclature throughout the manuscript would improve precision.\n6. To better understand the specificity of the hnRNPM-Bcas2 interaction, it would be informative to examine whether it also occurs at later developmental stages where both proteins are re-expressed, such as the 2-cell stage or a fully developed cell line. This would clarify if the interaction is unique to oogenesis or part of a more general splicing regulatory mechanism. While additional experiments are not necessary for this manuscript, the authors could consider discussing this point.\n7. The color key is missing in Fig. 2f.\n8. Should the column labels in Suppl Table 1b be KO-1 and KO-2?\n9. A typo in line 408 “Notably, tThe meiotic“\n10. In the affiliation, “8Sir Run Shaw Hospital” is likely to be “Sir Run Run Shaw Hospital.”", "reviewer_3": "The author studied the function of hnRNPM during oocyte development and thoroughly analyzed its molecular mechanism of action. The results demonstrate that hnRNPM is a key regulatory factor for the alternative splicing of pre-mRNA during oocyte development. hnRNPM recruits BCAS2 to control the splicing of crucial pre-mRNA related to meiosis. These findings have significant academic value for understanding the regulation mechanisms of alternative splicing during oocyte development, and provide important references for understanding the pathogenesis of related diseases. The findings are innovative but still need to be further improved.\nMajor concerns:\n1.Stra8 Cre activation occurs at embryonic day 12.5. I am curious about the expression pattern of hnRNPM in oocytes during different developmental stages of the embryo and postnatally. If its expression level is high and it can be effectively knocked out after birth, does this indicate that it has no function in the establishment of primordial follicle pool and activation of primordial follicles? Additionally, previous studies have shown that BCAS2 plays an important role in the first meiotic division of oocytes. Does hnRNPM function independently of BCAS2 in early ovarian oocytes?\n2.In oocytes and granulosa cells, BCAS2 exerts important regulatory functions by forming complexes with corresponding interacting proteins to influence the alternative splicing of precursor mRNAs. In this study, does the recruitment of BCAS2 by hnRNPM require the formation of a complex with PRP19 and CDC5L to perform splicing functions? If possible, provide relevant evidence, or discuss this in the revised version.\n3.After the absence of hnRNPM in oocytes, how do the gene and protein levels of BCAS2, as well as its protein localization? The related results may be helpful. BCAS2 might undergo degradation or positional changes, which can also be reflected in the working model diagram.\n4.In vitro OR in vivo experiments demonstrate whether Bcas2 knockdown or knockout affects the expression level and localization of hnRNPs, and alternative splicing of precursor mRNA regulated by hnRNPs?\n5.Is BCAS2 cKO stra8 cre or vasa cKO? The authors should provide the breeding and acquisition process of BCAS2 cKO mice in the Materials and Methods section.\nMinor concerns:\n1.The abstract does not end with a period.\n2.Line 116, revise 'cattle' for repetition.\n3.Line 126, Ensure that 'tubulin' starts with an uppercase letter throughout the MS, including Figures and Legends.\n4.Ensure that Stra8-Cre and Stra8-GFPcre are consistently used throughout the MS, e.g., Line 458, 465, Figure 2a, b, c and figure legends.\n5.Gene writing should be uniform throughout the MS，e.g., Line 168, 312.\n6.The controls in Figure 3c are WT? What’s the difference between WT and CTRL?\n7.Line 227, missed spaces should be added after ‘site’.\n8.Line 280-281, Revise repeated word usage according to the highlighted certain information in the Figure.\n9.Ensure consistency between 'Western blot' and 'Western blotting' throughout the MS.\n10.Modification of citation format in the main text，e.g., Line 52, 58, 70, 96, 312, 349-350, 373, 421.\n11.References should be modified according to NC format, such as using both the full name and abbreviation of journal titles.；\n12.Figure 1a, ‘Bovin’ should be corrected.\n13.The annotation information on the image is either left-aligned or centered, maintaining consistency; as shown in Figure 8e (which can be aligned similarly to the figure editing in Figure 1) it is left-aligned, see also Figures 1c, etc.\n14.In the figures, should there be a space before the parentheses in the horizontal and vertical axes? Please ensure consistency across all figures by the author. For example, Figure 4b has a space, but other figures do not.\n15.Figure S1，Modify the title of the figure legend to cover all key information in the figure; change the letters annotated in Figure S1 to black; ‘Bovin’ should be corrected.\n16.The spacing between the upper and lower figures in Figure 5f should be adjusted, causing some letters to be covered by the lower figure; in Figure 5h, the Chinese text should be deleted or replaced.\n17.In Figure legends, ‘Error bars, mean ± SEM’ should be revised to ‘Error bars indicate SEM’, OR ‘Data are presented as mean ± SEM’.\n18.Figure 3e, can the blue arrowhead be identified as mitochondrial aggregation? What are the morphological characteristics of mitochondrial aggregation? Modify the position of the yellow arrow (below).\n19.In Figure 2e, DAPI signal should be added.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Dear reviewers,\nThank you for careful review of our manuscript entitled “hnRNPM recruits BCAS2 to modulate\nalternative splicing during oocyte development” (NCOMMS-25-42705A). We greatly\nappreciate your valuable comments and suggestions, which have significantly helped us\nrevise and improve our paper. Your feedback has also provided important insights for guiding\nour further research.\nWe have carefully addressed all your comments and made every effort to revise the\nmanuscript accordingly. The main corrections in the revised manuscript are highlighted in\nyellow, and our point-by-point responses to the reviewers' comments are presented in blue\ntext.\nOnce again, on behalf of my co-authors, I sincerely thank you for your kind assistance in\nimproving our manuscript. We deeply appreciate your efforts and apologize for taking up so\nmuch of your time. We look forward to your further suggestions.\nBest regards,\nFei Sun\nHeng-Yu Fan\nThe point-by-point response was listed below.\nReviewer #1 (Remarks to the Author)\nThis manuscript provides compelling and insightful data to reveal the crucial role of hnRNPM\nin pre-mRNA alternative splicing during oocyte development. Using a germ cell-specific\nknockout mouse model, the authors confirmed in vivo that HnRNPM deficiency results in\ncomplete infertility in female mice. Furthermore, the results of SCAN-seq and LACE-seq\ndemonstrated that hnRNPM regulates pre-mRNA alternative splicing in oocytes by directly\nbinding to transcripts. Additionaly, IP-MS and RIP assays revealed that hnRNPM regulates\nalternative splicing in oocytes by promoting the binding of BCAS2 to pre-mRNA. Overall, the\nconclusion is robust, and the manuscript is well-written and clear in logic. I have the following\ncomments.\nResponse: We sincerely thank Reviewer #1 for the positive and insightful comments on our\nmanuscript, as well as for the constructive suggestions that have significantly helped us\nimprove our work.\n1) IVM experiments confirmed that hnRNPM deficiency affects oocyte maturation. However,\nin the IVF assay, the authors only described that Hnrnpm-deficient oocytes fail to complete\ncleave. It is unclear whether the maturation and fertilization rate of ovulated oocytes are\naffected. Whether the maturation phenotype is consistent between in vivo and in vitro? If not,\nplease give an explanation.\nResponse: We sincerely thank the reviewer for this insightful suggestion. hnRNPM\ndeficiency severely compromises both oocyte maturation and fertilization competence.\nSpecifically, only ~30% of ovulated oocytes from Hnrnpm cKO mice reached the MII stage\nfollowing hCG injection, confirming severe maturation defects (Supplementary Fig. 2f-g), a\nfinding consistent with our in vitro results. In addition, Hnrnpm-deficient oocytes fail to form\npronucleus (Fig. 2k-l), suggesting a fertilization failure. These results have been incorporated\ninto the revised manuscript to provide a more comprehensive understanding of hnRNPM's\ncritical role in oocyte development.\n2) The author introduces zygotic splicing activation (ZSA) in the background. What is the\nreason for the failure of zygotic cleavage in hnRNPM -deficient mice, is it due to the AS in\noocytes or ZSA in zygotes? Whether knockdown of hnRNPM in zygotes using siRNA or Trim-\nAway affect embryonic development?\nResponse: We thank the reviewer for this critical question. Hnrnpm deficiency severely\nimpairs oocyte maturation, leading to completely failure of pronucleus formation (Fig. 2k-l).\nSince this defect occurs prior to the timing of ZSA, the failure of zygotic cleavage in Hnrnpm-\ndeficient mice is primarily caused by defective AS in oocytes, rather than a failure of ZSA.\nTo directly evaluate the role of hnRNPM during ZSA, we performed siRNA-mediated\nknockdown in zygotes. This acute depletion did not block the first cleavage but resulted in a\n2-cell arrest (unpublished), a phenotype consistent with ZSA failure, suggesting that hnRNPM\nis required for ZSA and development beyond the 2-cell stage.\n3) A previous study showed that hnRNPH1 recruits PTBP2 and SRSF3 to modulate\nalternative splicing in germ cells (Nat Commun. 2022 Jun 23;13(1): 3588.doi:\n10.1038/s41467-022-31364-7), what is the relationship between hnRNPM and hnRNPH1?\nAre they co-localized in the oocytes?\nResponse: We thank the reviewer for raising an interesting question regarding the potential\nfunctional relationship between hnRNPM and hnRNPH1. Both hnRNPM and hnRNPH1\nbelong to the heterogeneous nuclear ribonucleoprotein (hnRNP) family and function as key\nRNA-binding proteins to regulate pre-mRNA splicing. Our co-immunoprecipitation\nexperiments demonstrate a physical interaction between hnRNPM and hnRNPH1 (Fig. R1a).\nFurthermore, immunofluorescence co-localization analysis reveals significant nuclear co-\nlocalization of hnRNPM and hnRNPH1 in oocytes (Fig. R1b), providing cytological evidence\nsupporting their potential functional collaboration in splicing regulation.\nFig. R1 hnRNPM interacts and colocalizes with hnRNPH1. a Co-immunoprecipitation assays analyze\nthe interaction between hnRNPM and hnRNPH1. b Immunofluorescence staining of GV oocytes showing\nhnRNPH1 (purple) and hnRNPM (green).\n4) Line 202-203, the author found most Hnrnpm-deficient oocytes displayed abnormal\npericentrin localization. Since pericentrin is a key MTOC marker, I suggest to evaluate\nwhether the MTOCs were destroyed in hnRNPM-deficient oocytes before GVBD using\npericentrin Immunofluorescence.\nResponse: We thank the reviewer for this excellent suggestion. To directly assess whether\nMTOCs are destroyed in Hnrnpm-deficient oocytes before GVBD, we perform pericentrin\nimmunofluorescence using GV-stage oocytes. Strikingly, single large pericentrin were\npresent in controls but undetectable in most of Hnrnpm-deficient oocytes (Supplementary\nFig. 2h-i), suggesting a pre-existing MTOC defect prior to meiotic resumption.\nIn addition, we performed quantitative analysis of oocytes with pericentrin signals at 8\nand 16 h after culture. Interestingly, MTOCs were detected in approximately 37.9% of cKO\ncells at 8 hours post-release, with the rate increasing to about 80.5% by 16 hours\n(Supplementary Fig. 2h). These findings indicate that hnRNPM deficiency leads to a\ndelayed appearance of pericentrin signals in oocytes. Thank you once again for your\nconstructive feedback.\n5) Whether hnRNPM CKO affect the protein level or the localization of BCAS2? In addition,\nBCAS2-deficient mice show more severe phenotype than hnRNPM-deficient mice, please\ndiscussion the difference.\nResponse: We thank the reviewer for this insightful comment. To determine whether\nhnRNPM depletion affects BCAS2, we first examined the protein level and subcellular\nlocalization of BCAS2 in control and Hnrnpm-deficient oocytes. Our results clearly show that\nthe protein level of BCAS2 was significantly reduced in Hnrnpm CKO oocytes compared to\ncontrols (Fig. 8n). However, the subcellular localization of the remaining BCAS2 protein did\nnot appear to be altered. it still exhibited a nuclear localization pattern similar to that in control\noocytes (Supplementary Fig. 6g).\nThe complete deficiency of BCAS2 likely abolishes its entire function, leading to a\ncatastrophic failure in spliceosome assembly and mRNA processing, thus manifesting a more\nsevere phenotype. In contrast, the loss of hnRNPM results in a partial reduction, but not a\ncomplete ablation, of BCAS2 protein. We propose that the residual BCAS2 in Hnrnpm-\ndeficient oocytes retains a degree of functionality, thereby mitigating the phenotypic severity\ncompared to a full BCAS2 knockout. We have now included a detailed discussion on this\npoint in the revised manuscript (Lines 362-365 & 474-479).\nReviewer #2 (Remarks to the Author)\nZhou et al. investigate the role of heterogeneous nuclear ribonucleoprotein M (hnRNPM) in\noocyte development, presenting its splicing regulatory role of maternal transcripts essential\nfor meiosis. The authors demonstrate that the conditional knockout of hnRNPM in female\ngerm cells leads to significant cytoplasmic defects, impaired meiotic maturation, and\ncomplete female infertility. Through single-cell amplification and sequencing of full-length\nRNAs using the Oxford nanopore platform (SCAN-seq), the study reveals that hnRNPM\nmodulates alternative splicing of genes crucial for the meiotic cell cycle. Furthermore, the\nauthors identified BCAS2 as an interacting partner of hnRNPM and propose a model where\nhnRNPM recruits BCAS2 to regulate the splicing of specific transcripts. The research is well-\ndesigned, and the findings are interesting, but several points could be strengthened to\nprovide more solid evidence for the authors’ conclusion.\nResponse: We sincerely appreciate the reviewer’s thoughtful summary and positive\nfeedback on our work. We agree that providing more robust evidence is essential and have\ntherefore conducted extensive new experiments in accordance with the comments below.\nIn addition, We apologize for the lack of clarity regarding the novel transcripts mentioned\nin Major Comments #2, #3, and Minor Comment #8. In Figure 5, we presented novel\ntranscript data only for WT1 and WT2, and not for KO1 and KO2, as the aim of this figure\nwas to highlight previously overlooked transcripts present in normal oocytes, emphasizing a\nfinding with broader biological relevance. In KO samples, a proportion of the novel transcripts\nrepresent abnormal isoforms resulting from dysregulated splicing, which is why they were not\nincluded them in the analysis presented in Figure 5. In contrast, the differentially expressed\ntranscripts in cKO groups—including novel transcripts—is comprehensively presented and\ninterpreted in Figure 6. Collectively, Fig. 5b-e and Supplementary Table 1b present data\nexclusively from WT samples (detailed in Major Comment #3 and Minor Comment #8), and\nthe identification of 89 novel transcripts linked to female infertility is also based on analysis\nof WT samples (detailed in Major Comment #2).\nWe deeply regret any confusion our original presentation may have caused and have\nthoroughly revised the relevant figures and text to improve clarity. We hope the current\nversion satisfactorily addresses your concerns.\nMajor:\n1. Although the manuscript discusses the regulatory potential of hnRNP proteins, the\nintroduction should explain why hnRNPM was chosen for study in the context of oocyte\ndevelopment.\nResponse: We sincerely thank the reviewer for raising this important point. Our selection of\nhnRNPM for this study was based on the following compelling reasons. First, our research\ngroup has long been dedicated to investigating the functions of hnRNP family proteins in the\nreproductive system1–6. In a previous systematic evaluation of hnRNP expression across\nmultiple tissues, we observed that the expression of HNRNPM in the testis and ovary was\nsignificantly higher compared to other tissues (Fig. R2)7, suggesting a potentially critical role\nin germ cell development and reproductive physiology.\nFig. R2 Western blot analyses of the protein expression levels of 12 hnRNP family proteins in\nmultiple mouse tissues7.\nFurthermore, an independent study employing mRNA interactome capture (RIC)\nanalysis on 50,000 fully-grown mouse oocytes identified hnRNPM as one of the core mRNA-\nbinding proteins (RBPs) enriched in oocytes8. This finding indicates that hnRNPM may\nfunctionally engage in regulating the fate of maternal mRNAs in oocytes. Therefore, based\non both its tissue-enriched expression pattern and functional evidence from oocyte-specific\nRBP profiling, we focused on hnRNPM to elucidate its potential functions in oocyte\ndevelopment and reproductive health. This description has been added to introduction (Lines\n84-90).\n2. In line 211, the authors report 89 novel transcripts linked to female infertility, yet\nSupplementary Fig. 3b shows ~6,000–7,000 novel isoforms per sample. Is female infertility\nspecifically enriched, and were other pathways enriched in hnRNPM-deficient GV oocytes?\nResponse: We thank the reviewer for raising this important point regarding the specificity\nand breadth of pathway enrichment among the novel transcripts. Our initial identification of\n89 novel transcripts linked to female infertility was based on a systematic review of published\nliterature. However, this approach did not allow us to confirm whether these transcripts were\nspecifically enriched in female infertility or to analyze other potential pathways.\nTo address this, we performed a Gene Ontology (GO) enrichment analysis using the\ninternational standard database (https://www.geneontology.org/) to investigate the biological\nprocesses involving these novel transcripts. Strikingly, the top enriched processes were\nclosely related to female infertility—including female meiosis chromosome segregation,\nregulation of fertilization and maternal-to-zygotic transition of gene expression\n(Supplementary Fig. 3c).\nInterestingly, alongside the female-specific terms, one of the most strongly enriched\nterms was \"male meiosis chromosome segregation\" (Supplementary Fig. 3c), suggesting\nthat hnRNPM’s role in meiotic regulation may be conserved across sexes. Beyond\ngametogenesis, the analysis also revealed significant enrichment in other key biological\nprocesses, including regulation of stress granule assembly, tRNA surveillance and mRNA 3'-\nend processing by stem-loop binding and cleavage (Supplementary Fig. 3c), indicating that\nhnRNPM deficiency may influence a broader range of RNA metabolic functions.\nIn summary, GO enrichment analysis revealed that the novel transcripts are specifically\nassociated with female infertility and are also enriched in pathways related to male meiosis\nand RNA processing.\n3. In Fig. 5b, it would be helpful to show gel images with both control and cKO oocytes.\nResponse: We thank the reviewer for this suggestion. We apologize for the lack of clarity\nregarding the novel transcripts mentioned. We only presented representative data of WT\noocytes, as the primary aim of Fig. 5b-e was to validate the effectiveness of our technical\napproach for oocyte samples. Therefore, we only presented representative data of WT\noocytes.\nIn Fig. 6, we focused on comparing differences between Control and cKO groups,\nincluding analysis of novel transcripts. Among the four genes shown in Fig. 5b-e,\nalternative splicing differences of Nlrp14 and Zar1l were observed. Accordingly, we have\nincluded gel images from both control and cKO oocytes for these two genes in Fig. 6d-e to\ndirectly demonstrate these differential splicing events.\nWe deeply regret any confusion our original presentation may have caused and have\nthoroughly revised the text to improve clarity. We hope the current version satisfactorily\naddresses your concerns.\n4. In Fig. 6a, indicating the direction of splicing changes (e.g., increased vs. decreased\ninclusion) would be helpful.\nResponse: This is an excellent suggestion. We fully agree that indicating the direction of\nsplicing changes will improve the clarity of Fig. 6a. Accordingly, the number of splicing events\nwith increased inclusion or decreased inclusion is now explicitly indicated in Supplementary\nFig. 4b.\n5. In Fig. 6d and 6e, while the study convincingly demonstrates that hnRNPM depletion leads\nto alternative splicing of various transcripts, such as Khdc3, Nlrp14, Cenph, and Zar1l, it is\nnot clear from the data presented whether these splicing changes result in significant\nalterations to the levels of corresponding intact proteins. Providing evidence, perhaps through\nWestern blotting for some of these key affected proteins in hnRNPM-depleted oocytes, would\nmore directly link the observed splicing defects to the downstream cellular phenotypes.\nResponse: We thank the reviewer for raising this important point regarding the impact of\nsplicing alterations. As suggested, we performed Western blot analysis to evaluate protein-\nlevel changes for two key affected candidates, CENPH and ZAR1L (KHDC3 and NLRP14\nwere not tested due to the lack of commercially available antibodies). The results confirm a\nslight downregulation of CENPH and a dramatic reduction in ZAR1L in hnRNPM-depleted\noocytes (Supplementary Fig. 4e). These data provide direct evidence that the splicing\ndefects caused by hnRNPM ablation lead to reduced expression of proteins, thereby bridging\nthe observed molecular changes to potential phenotypic consequences.\n6. The analysis in Fig. 7f shows an overlap between hnRNPM-bound genes and differentially\nspliced genes. To provide more direct evidence for a regulatory role, the analysis could be\nextended to compare hnRNPM binding signals specifically on exons and introns that are\nalternatively spliced versus those unaffected by hnRNPM depletion.\nResponse: We thank the reviewer for this excellent suggestion. Accordingly, we analyzed\nhnRNPM binding signals on exons and introns that are alternatively spliced versus those\nunaffected by hnRNPM depletion. Strikingly, hnRNPM binding was significantly enriched on\nthe exons of transcripts that underwent alternative splicing upon its depletion, compared to\nthose that were unaffected (Supplementary Fig. 5e-f). This provides much more direct\nevidence that hnRNPM binding at these specific locations is functionally responsible for the\nsplicing regulation.\n7. A recent study cited by the authors (Zheng et al., 2024) demonstrated that hnRNPM\nprotects against interferon response by repressing LINE-associated cryptic splicing. It would\nbe a valuable addition to investigate whether the hnRNPM binding sites identified in oocytes\nare also enriched for LINE1 elements, and whether the key splice events affected by\nhnRNPM depletion are associated with these retrotransposons.\nResponse: We thank the reviewer for this insightful suggestion. Accordingly, we performed\nthe recommended analysis and found that hnRNPM exhibits no significant binding preference\nfor LINE1 elements (Fig. R3). We propose two possible explanations for this result. First,\nhnRNPM may not operate in oocytes through the same mechanism as reported by Zheng et\nal. Alternatively, the discrepancy may arise from methodological differences in sequencing.\nIn the study by Zheng et al., nuclear RNA was used for sequencing, thereby enriching pre-\nmRNAs and increasing sequencing depth for nascent transcripts. In our study, however, the\nlimited quantity of oocyte samples necessitated the use of total RNA, which is dominated by\nmature cytoplasmic mRNAs.\nFig. R3 Comparison of LINE1 intensity on long introns between hnRNPM-binding genes and\nbackground genes.\n8. In Fig. 8d, the authors propose that hnRNPM recruits Bcas2 to regulate splicing. To clarify\nthe nature of this interaction, a co-IP experiment including an RNase treatment step is\nnecessary. This would determine whether the interaction is a direct protein-protein\nassociation or is mediated by RNA.\nResponse: We thank the reviewer for raising this critical point. We fully agree that\ndistinguishing between a direct protein-protein interaction and an RNA-facilitated one is\nessential. In direct response to this comment, we performed the suggested co-IP experiment\nwith RNase treatment. As shown in Fig. 8d, the interaction between hnRNPM and BCAS2\nremained intact following RNase treatment, indicating that their association is a direct protein-\nprotein interaction and not dependent on RNA.\n9. In Fig. 8f, the study proposes a “strong functional synergy” between hnRNPM and Bcas2\nbased on the overlap of 98 commonly affected alternative splicing genes and 57 identical\nsplicing events in the respective knockout oocytes. Are these overlaps statistically significant?\nWhile suggestive, further evidence is needed to solidify this claim, or toning down is\nnecessary.\nResponse: We thank the reviewer for raising this important point regarding statistical\nsignificance. In direct response, we performed hypergeometric tests to rigorously evaluate\nthe overlap. The results revealed that both overlaps are overwhelmingly significant, with p-\nvalues of 2 × 10 ²⁷ for the 98 commonly affected genes and 4 × 10 ²⁷ for the 57 identical\nsplicing events, indicating they are far beyond random chance. We have now included these\nstatistical results in the revised manuscript (Fig. 8f legend).\nMinor:\n1. In Fig. 1c and 1d, the study demonstrates that hnRNPM is absent from the GVBD stage\nthrough the zygote stage. It would be interesting to speculate on the functional implications\nof this dynamic expression. Is the dynamic expression during oocyte maturation important for\nZSA? What might be the consequences if hnRNPM is consistently expressed during these\nstages?\nResponse: We thank the reviewer for this interesting suggestion. Accordingly, we have\nincorporated the following paragraph to speculate on the functional implications of hnRNPM's\ndynamic expression pattern (Lines 393-401): \"The specific absence of hnRNPM from the\nGVBD to zygote stages implies that its expression may be precisely regulated to ensure\nproper ZSA. We propose that this transient downregulation serves at least two critical\nfunctions. First, The absence of hnRNPM, a known splicing regulator, may protect the pre-\ndeposited maternal mRNA pool from spurious or aberrant splicing. Second, its removal might\nbe essential to 'reset' the splicing landscape, allowing the nascent zygotic transcriptome to\nbe established under a new regulatory framework. Conversely, persistent hnRNPM\nexpression potentially cause mis-splicing of both maternal transcripts and early zygotic genes,\nultimately lead to failure of ZSA and subsequent embryonic developmental arrest.\"\n2. In Fig. 2k and 2l, there appears to be a discrepancy between the dramatic development\narrest shown in Fig. 2k and the moderate, albeit significant, changes in developmental rates\npresented in Fig. 2l. The visual evidence in Fig. 2k suggests a near-complete failure of\nHnrnpm-deficient oocytes to develop beyond the 2-cell stage after IVF. An explanation for this\ndifference would strengthen the interpretation of the impaired developmental phenotype.\nResponse: We thank the reviewer for noting the apparent discrepancy between Fig. 2k and\n2l. In response, we wish to clarify that the developmental rates of the cKO group are indeed\n0% at all stages, which is fully consistent with the phenotype shown in Fig. 2k. We\nacknowledge that in the original Fig. 2l, the cKO data trajectory was completely overlapped\nwith the X-axis, potentially hindering visual interpretation. To address this issue, we have\nadjusted the axis scaling in the revised panel to clearly display the 0% trend above the\nbaseline. We apologize for any confusion caused by the initial presentation and thank the\nreviewer for this opportunity to improve our figure.\n3. In Fig. 8g, the basis of claiming “increased exon skipping in Cenpf” could be more\nquantitatively supported.\nResponse: We thank the reviewer for this important suggestion. In response, we have\nreplaced the representative images in Fig. 8g to support the increased exon skipping in Cenpf.\n4. Based on the study, we can hypothesize but cannot definitively conclude that human\nhnRNPM and BCAS2 form the same interaction network to control splicing in human oocytes.\nWhile the RIP-qPCR analyses in Fig. 8n-p are suggestive, the splicing regulatory\nconsequences in human transcripts tested here should be examined in a human cell model\nas well.\nResponse: We thank the reviewer for this excellent suggestion. We agree that validating the\nsplicing regulatory function of hnRNPM and BCAS2 on the identified human transcripts in a\nhuman cell model is crucial for strengthening our conclusions. Our new results clearly\ndemonstrate that depletion of either hnRNPM or BCAS2 in human cell recapitulates the\nsplicing changes of PRC1 and HDLBP (Supplementary Fig. 6f). We also apologize that,\ndespite extensive efforts, we were unable to successfully amplify CENPF in human cells\nunder our experimental conditions. Nevertheless, the consistent and reproducible splicing\nalterations in PRC1 and HDLBP upon HNRNPM or BCAS2 knockdown strongly support the\nconclusion that the hnRNPM/BCAS2 network functions to regulate alternative splicing in\nhuman cells.\n5. A minor but important point for clarity is the consistent use of nomenclature for genes,\ntranscripts, and proteins. The study frequently uses human-centric or nomenclature for\nproteins (e.g., BCAS2) in a mouse model or mouse-centric nomenclature for transcripts (e.g.\nCenpf, Prc1, and Hdlbp in Fig. 8) in a human model, while correctly italicizing mouse gene\nnames (e.g., Hnrnpm, Bcas2). Adhering to the correct organism-specific nomenclature throughout the manuscript would improve\nprecision.\nResponse: We thank the reviewer for highlighting this issue. We have carefully reviewed the\nentire manuscript and revised the nomenclature to adhere to organism-specific conventions.\nHuman genes are now presented in uppercase italics (e.g., BCAS2), mouse genes in\ncapitalized italics (e.g., Bcas2), and all protein names are in uppercase non- italics (e.g.,\nBCAS2). These corrections have been made consistently throughout the manuscript.\n6. To better understand the specificity of the hnRNPM-Bcas2 interaction, it would be\ninformative to examine whether it also occurs at later developmental stages where both\nproteins are re-expressed, such as the 2-cell stage or a fully developed cell line. This would\nclarify if the interaction is unique to oogenesis or part of a more general splicing regulatory\nmechanism. While additional experiments are not necessary for this manuscript, the authors\ncould consider discussing this point.\nResponse: We thank the reviewer for this insightful suggestion. To directly address whether\nthe hnRNPM-BCAS2 interaction is specific to oogenesis or represents a more general\nmechanism, we performed additional experiments in HEK 293T cells. hnRNPM and BCAS2\nalso interact in this somatic cell line (Fig. R4). This new finding indicates that the hnRNPM-\nBCAS2 interaction is indeed a conserved feature of a broader splicing regulatory mechanism.\nFig. R4 Co-immunoprecipitation assays confirm the interaction of hnRNPM with BCAS2 in HEK\n293T cells.\n7. The color key is missing in Fig. 2f.\nResponse: Thank you for noticing this. We have now included the missing color key in Fig.\n2f in the revised figure panel.\n8. Should the column labels in Suppl Table 1b be KO-1 and KO-2?\nResponse: Thank you for your comment. We sincerely apologize for the lack of clarity in our\nprevious description, which may have caused confusion. The purpose of analyzing novel\ntranscripts in this study is to highlight previously overlooked transcripts present in normal\noocytes, emphasizing a broader and more generalizable research finding. Since some novel\ntranscripts detected in the KO samples may represent aberrant products resulting from gene\ndeletions, we present data only for WT1 and WT2 in Suppl Table 1b to ensure a focus on\nauthentic transcripts. We deeply regret any confusion our original presentation may have\ncaused and have thoroughly revised the text to improve clarity. We hope the current version\nsatisfactorily addresses your concerns.\n9. A typo in line 408 “Notably, tThe meiotic“\nResponse: This has been corrected to “Notably, the meiotic…” in the revised manuscript.\n10. In the affiliation, “8Sir Run Shaw Hospital” is likely to be “Sir Run Run Shaw Hospital.”\nResponse: We thank the reviewer for spotting this error. The affiliation has been corrected\nto “Sir Run Run Shaw Hospital.”\nReviewer #3 (Remarks to the Author)\nThe author studied the function of hnRNPM during oocyte development and thoroughly\nanalyzed its molecular mechanism of action. The results demonstrate that hnRNPM is a key\nregulatory factor for the alternative splicing of pre-mRNA during oocyte development.\nhnRNPM recruits BCAS2 to control the splicing of crucial pre-mRNA related to meiosis. These\nfindings have significant academic value for understanding the regulation mechanisms of\nalternative splicing during oocyte development, and provide important references for\nunderstanding the pathogenesis of related diseases. The findings are innovative but still need\nto be further improved.\nResponse: We thank the reviewer for the positive evaluation of our work and for the valuable\nsuggestions. We have performed additional experiments and analyses to address the\nconcerns as detailed below.\nMajor concerns:\n1. Stra8 Cre activation occurs at embryonic day 12.5. I am curious about the expression\npattern of hnRNPM in oocytes during different developmental stages of the embryo and\npostnatally. If its expression level is high and it can be effectively knocked out after birth, does\nthis indicate that it has no function in the establishment of primordial follicle pool and\nactivation of primordial follicles? Additionally, previous studies have shown that BCAS2 plays\nan important role in the first meiotic division of oocytes. Does hnRNPM function independently of BCAS2 in early ovarian oocytes?\nResponse: This is a great suggestion. We examined the expression of hnRNPM in oocytes\nacross multiple developmental stages: from embryonic days (E13.5, E15.5, E17.5) to\npostnatal stages (P0, P3, P7) using immunofluorescence. In the embryonic stages (E13.5,\nE15.5, E17), the expression of hnRNPM in germ cells was weak and lower compared to\nSertoli cells (Supplementary Fig. 1d). After birth (P0), hnRNPM expression in germ cells\nincreased significantly (Supplementary Fig. 1e). In primordial and primary follicles at P3 and\nP7, the hnRNPM signal was notably stronger and exceeded the expression levels seen in\nSertoli cells (Supplementary Fig. 1e).\nTo verify the knockout efficiency, we assessed hnRNPM expression in P0 oocytes from\nboth control and cKO mice and confirmed that hnRNPM was completely absent in the germ\ncells of cKO mice (Supplementary Fig. 2a). Importantly, despite the perinatal expression of\nhnRNPM, its knockout had no effect on the number of primordial or primary follicles (Fig. 2i).\nThis suggests that hnRNPM is dispensable for the establishment and activation of the\nprimordial follicle pool.\nIn addition, we sincerely appreciate your insightful comment regarding the potential\ninterplay between hnRNPM and BCAS2, especially considering that previous studies have\nhighlighted BCAS2's crucial role in the first meiotic division of oocytes. In contrast to BCAS2,\nconditional knockout of hnRNPM using Stra8-Cre did not lead to significant defects in the first\nmeiotic division in either male or female germ cells9. This suggests that hnRNPM functions\nindependently of BCAS2 in the regulation of meiosis, particularly during the first meiotic\ndivision. We have added these results and clarified this point in the Discussion (Lines 468–\n474).\n2.In oocytes and granulosa cells, BCAS2 exerts important regulatory functions by forming\ncomplexes with corresponding interacting proteins to influence the alternative splicing of\nprecursor mRNAs. In this study, does the recruitment of BCAS2 by hnRNPM require the\nformation of a complex with PRP19 and CDC5L to perform splicing functions? If possible,\nprovide relevant evidence, or discuss this in the revised version.\nResponse: We thank the reviewer for this insightful comment. Our new data reveal that\nhnRNPM could interact with PRP19 and CDC5L (Supplementary Fig. 6c-d), suggesting its\nassociation with the entire PRP19/CDC5L/BCAS2 complex to coordinate splicing. These\nresults provide a deeper mechanistic understanding for the role of hnRNPM in modulating\nthe splicing machinery. We have included these new data and discussed their implications.\n3.After the absence of hnRNPM in oocytes, how do the gene and protein levels of BCAS2,\nas well as its protein localization? The related results may be helpful. BCAS2 might undergo\ndegradation or positional changes, which can also be reflected in the working model diagram.\nResponse: We thank the reviewer for this insightful suggestion. Our investigation showed\nthat loss of hnRNPM led to reduced BCAS2 protein expression without affecting its\nsubcellular localization (Fig. 8n and Supplementary Fig. 6g), suggesting that BCAS2 may\nundergo degradation. The working model has been updated accordingly to indicate that\nhnRNPM is necessary for maintaining normal BCAS2 protein levels (Fig. 9).\n4.In vitro OR in vivo experiments demonstrate whether Bcas2 knockdown or knockout affects\nthe expression level and localization of hnRNPs, and alternative splicing of precursor mRNA\nregulated by hnRNPs?\nResponse: We thank the reviewer for these insightful questions. Our data show that Bcas2\nknockout reduces hnRNPM protein levels without affecting its subcellular localization\n(Supplementary Fig. 6h-i), Together with the finding that loss of hnRNPM decreases BCAS2\nprotein expression (Major comment 3), suggesting that hnRNPM and BCAS2 likely form a\ncomplex and are mutually dependent for protein stability. Furthermore, loss of Bcas2 in\noocyte recapitulates the aberrant splicing of hnRNPM targets transcripts (Fig. 8g-k),\nindicating that BCAS2 and hnRNPM collaborate closely in regulating alternative splicing.\nThese data have been included and discussed in the revised manuscript.\n5.Is BCAS2 cKO stra8 cre or vasa cKO? The authors should provide the breeding and\nacquisition process of BCAS2 cKO mice in the Materials and Methods section.\nResponse: We apologize for this omission. We have now added the detailed information on\nthe breeding strategy to obtain Bcas2flox/Del; Vasa-Cre (designated Bcas2 cKO) mice in the\nMaterials and Methods section (Lines 520-521).\nMinor concerns:\n1.The abstract does not end with a period.\nResponse: Thank you for pointing this out. We have added a period at the end of the abstract\nto ensure correct punctuation.\n2.Line 116, revise 'cattle' for repetition.\nResponse: Corrected.\n3.Line 126, Ensure that 'tubulin' starts with an uppercase letter throughout the MS, including\nFigures and Legends.\nResponse: Thank you for bringing this up. We have corrected to \"Tubulin\" throughout the\nmanuscript, figures, and legends.\n4.Ensure that Stra8-Cre and Stra8-GFPcre are consistently used throughout the MS, e.g.,\nLine 458, 465, Figure 2a, b, c and figure legends.\nResponse: Thank you for pointing this out. We have carefully revised the manuscript to\nensure that \"Stra8-Cre\" are used consistently throughout the manuscript, figures, and\nlegends.\n5.Gene writing should be uniform throughout the MS，e.g., Line 168, 312.\nResponse: We sincerely thank the reviewer for this careful observation. We have now\nstandardized the gene and protein nomenclature throughout the text by adhering to the\nfollowing format: italicized, first-letter-capitalized for mouse genes (e.g., Hnrnpm, Bcas2);\nitalicized, all-capitals for human genes (e.g., HNRNPM, BCAS2); and non-italicized for\nproteins (e.g., hnRNPM, BCAS2).\n6.The controls in Figure 3c are WT? What’s the difference between WT and CTRL?\nResponse: We apologize for the inconsistency in the genotype annotation and have\nstandardized it to \" CTRL \". \" WT \" in Figure 3c refers to the control oocytes from Hnrnpmflox/+\nmice, which are phenotypically wild-type.\n7.Line 227, missed spaces should be added after ‘site’.\nResponse: Thank you for pointing this out. We have added the necessary spaces after the\nword “site” as suggested.\n8.Line 280-281, Revise repeated word usage according to the highlighted certain information\nin the Figure.\nResponse: Corrected.\n9.Ensure consistency between 'Western blot' and 'Western blotting' throughout the MS.\nResponse: Thank you for pointing this out. We have standardized to \"Western blot\"\nthroughout the manuscript.\n10.Modification of citation format in the main text, e.g., Line 52, 58, 70, 96, 312, 349-350, 373,\n421.\nResponse: We thank the reviewer for careful reading and have adjusted the citation format\nin the main text to ensure consistency with the required style.\n11.References should be modified according to NC format, such as using both the full name\nand abbreviation of journal titles.\nResponse: Thank you for pointing this out. We have thoroughly reformatted the entire\nreference list according to the Nature Communications guidelines\n12.Figure 1a, ‘Bovin’ should be corrected.\nResponse: We thank the reviewer for careful reading. We have corrected the typo “Bovin” to\n“Bovine” in Figure 1a and any other relevant sections.\n13.The annotation information on the image is either left-aligned or centered, maintaining\nconsistency; as shown in Figure 8e (which can be aligned similarly to the figure editing in\nFigure 1) it is left-aligned, see also Figures 1c, etc.\nResponse: Thank you for your valuable advice. We have adjusted Figure 8e to match the\nalignment style in Figure 1 (left-aligned), as well as other figures that may require alignment\nadjustments.\n14.In the figures, should there be a space before the parentheses in the horizontal and\nvertical axes? Please ensure consistency across all figures by the author. For example,\nFigure 4b has a space, but other figures do not.\nResponse: Thank you for your comments. We have checked all axis labels and ensured\nconsistency.\n15.Figure S1,Modify the title of the figure legend to cover all key information in the figure;\nchange the letters annotated in Figure S1 to black; ‘Bovin’ should be corrected.\nResponse: We thank the reviewer for careful reading. We have modified the figure legend\ntitle to be more comprehensive. Additionally, we have changed the annotated letters to\nblack in Figure S1 and correct the typo \"Bovin\" to “Bovine” as suggested.\n16.The spacing between the upper and lower figures in Figure 5f should be adjusted, causing\nsome letters to be covered by the lower figure; in Figure 5h, the Chinese text should be\ndeleted or replaced.\nResponse: Thank you for your important suggestion. We have adjusted the spacing between\nthe upper and lower figures in Figure 5f to prevent overlap. We have also removed the\nChinese text in Figure 5h as reviewer’s suggestion.\n17.In Figure legends, ‘Error bars, mean ± SEM’ should be revised to ‘Error bars indicate SEM’,\nOR ‘Data are presented as mean ± SEM’.\nResponse: Thank you for your valuable feedback. We have checked and corrected all Figure\nlegends.\n18.Figure 3e, can the blue arrowhead be identified as mitochondrial aggregation? What are\nthe morphological characteristics of mitochondrial aggregation? Modify the position of the\nyellow arrow (below).\nResponse: Thank you for this interesting and important question. The structure indicated by\nthe blue arrowhead can indeed be identified as mitochondrial aggregation. Mitochondrial\naggregation is currently not clearly defined in the literature. Based on our research\nexperience10, it is characterized by the clustering of multiple mitochondria—typically more\nthan ten—tightly grouped together without being separated by cytoplasmic lattice structures.\nIn addition, as suggested, we have adjusted the position of the yellow arrow to improve\nlabeling accuracy. We sincerely appreciate your careful observation and hope this response\naddresses your concerns.\n19.In Figure 2e, DAPI signal should be added.\nResponse: Thank you for bringing this up. We have added the DAPI (white) signal to Figure\n2e as suggested.\nReviewer #4 (Remarks to the Author)\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: On behalf of my co-authors and myself, I would like to extend our sincere\ngratitude for your thorough assessment and helpful suggestions for our manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The positive responses satisfied all my concerns, I suggest the publication of this study.", "reviewer_2": "The authors have thoroughly revised the manuscript and successfully addressed all of our comments. We have no further concerns.", "reviewer_3": "My concerns have been addressed in the revised version.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter