Alternative Splicing Research Articles

HnRNPC Functions with HuR to Regulate Alternative Splicing in an m6A-Dependent Manner and is Essential for Meiosis.

N6-methyladenosine (m6A) and its reader proteins are involved in pre-mRNA processing and play a variety of roles in numerous biological processes. However, much remains to be understood about the regulation of m6A and the function of its specific readers during meiotic processes. Here, this study shows that the potential m6A reader protein hnRNPC is essential for both male and female meiosis in mice. Germ cell-specific knockout of Hnrnpc causes meiotic arrest at pachynema in male mice. Specifically, hnRNPC-deficient males show abnormal meiosis initiation and defective meiotic progression, ultimately leading to meiotic arrest at the pachytene stage. Interestingly, hnRNPC-null females show similar meiotic defects to males. Mechanistically, this study discovers that in male germ cells, hnRNPC works with HuR to directly bind and modulate alternative splicing of meiotic-related genes (e.g., Sycp1, Brca1, and Smc5) in an m6A-dependent manner during spermatogenesis. Collectively, these findings reveal hnRNPC as a critical factor for meiosis and contribute to a mechanistic understanding of the hnRNPC-HuR interaction in alternative splicing of mRNAs during germ cell development.

Genome-Wide Identification of Alternative Splicing in Botrytis cinerea During Infection Stage of Solanum lycopersicum

Genome-Wide Identification of Alternative Splicing in Botrytis cinerea During Infection Stage of Solanum lycopersicum

TAp63γ is the primary isoform of TP63 for tumor suppression but not development

TP63 is expressed as TAp63 and ΔNp63 from the P1 and P2 promoters, respectively. While TAp63 and ΔNp63 are expressed as three TAp63α/β/γ and ΔNp63α/β/γ due to alternative splicing, only p63α (TA and ΔN) and p63γ (TA and ΔN) proteins are found to be detectable and likely to be responsible for p63-dependent activity. Previous studies implied and/or demonstrated that TAp63α, which contains an N-terminal activation domain conserved in p53, functions as a tumor suppressor by regulating an array of genes for growth suppression. By contrast, ΔNp63α, which also contains an N-terminal activation domain but is different from that in TAp63, regulates a unique set of genes and functions as a master regulator for development of epidermis and other stratified epithelial tissues. However, the biological function of p63γ is largely unexplored. To explore this, we generated a mouse model in that exon 10’, a coding exon specific for p63γ, was deleted by CRISPR-cas9. We showed that mice deficient in p63γ are viable and futile, which is different from mice deficient in total TP63 or p63α. Like TAp63-deficient mice, p63γ-deficient mice have a short lifespan and are prone to spontanenous tumors. Additionally, loss of p63γ shortens the lifespan of tumor-free mice potentially via increased cellular senescence. Moreover, mice deficient in p63γ are prone to chronic inflammation in multiple organs and liver steatosis potentially via altered lipid metabolism. Single-cell RNA-seq revealed that loss of p63γ increases the expression of SCD1, a rate-limiting enzyme for synthesis of monounsaturated fatty acids, leading to altered lipid homeostasis. Together, our data indicate that TP63γ is the primary isoform of TP63 for tumor suppression but not development by maintaining normal inflammatory response and lipid homeostasis.

Landscapes of alternative splicing genes/events in the gills of Mozambique tilapia (Oreochromis mossambicus) and their roles in high-salinity adaptation

Landscapes of alternative splicing genes/events in the gills of Mozambique tilapia (Oreochromis mossambicus) and their roles in high-salinity adaptation

Full-length mRNA sequencing resolves novel variation in 5' UTR length for genes expressed during human CD4 T-cell activation.

Isoform sequencing (Iso-Seq) uses long-read technology to produce highly accurate full-length reads of mRNA transcripts. Visualization of individual mRNA molecules can reveal new details of transcript variation within understudied portions of mRNA, such as the 5' untranslated region (UTR). Differential 5' UTRs may contain motifs, upstream open reading frames (uORFs), and secondary structures that can serve to regulate translation or further indicate changes in promoter usage, where transcriptional control may impact protein expression levels. To begin to explore isoform variation during T-cell activation, we generated the first Iso-Seq reference transcriptome of activated human CD4 T cells. Within this dataset, we discovered many novel splice- and end-variant transcripts. Remarkably, one in every eight genes expressed in our dataset was found to have a notable proportion of transcripts with 5' UTR lengthened by over 100bp compared to the longest corresponding UTR within the Gencode dataset. Among these end-variant transcripts, two novel isoforms were identified for CXCR5, a chemokine receptor associated with T follicular helper cell (Tfh) function and differentiation. When investigated in a model cell system, these lengthened UTR conferred reduced transcript stability and, for one of these isoforms, short uORFs introduced by the added length altered protein expression kinetics. This study highlights instances in which current reference databases are incomplete relative to the information obtained by long-read sequencing of intact mRNA. Iso-Seq is thus a promising approach to better understanding the plasticity of promoter usage, alternative splicing, and UTR sequences that influence RNA stability and translation efficiency.

Update of Aging Hallmarks in Idiopathic Pulmonary Fibrosis

Idiopathic Pulmonary Fibrosis (IPF) is an epithelial-driven interstitial lung disease of unknown etiology characterized by the excessive proliferation of fibroblast populations that synthesize large amounts of extracellular matrix. In this devastating disorder, all aging hallmarks appear prematurely or are altered. This review highlights key findings about IPF characteristics recently recognized as hallmarks of aging, including mechanical alterations, inflammaging, dysbiosis, alternative splicing, and disabled macroautophagy. It also revisits the classic hallmarks of aging, which encompass stem cell exhaustion, cellular senescence, and altered intercellular communication. Enhancing our understanding of the fundamental processes that underlie the altered hallmarks of aging in IPF may facilitate the development of innovative experimental strategies to improve therapeutic outcomes.

Targeting the splicing factor SNRPB inhibits endometrial cancer progression by retaining the POLD1 intron.

Dysregulated alternative splicing has been closely linked to the initiation and progression of tumors. Nevertheless, the precise molecular mechanisms through which splicing factors regulate endometrial cancer progression are still not fully understood. This study demonstrated elevated expression of the splicing factor SNRPB in endometrial cancer samples. Furthermore, our findings indicate that high SNRPB expression is correlated with poor prognosis in patients with endometrial cancer. Functionally, SNRPB inhibition hindered the proliferative and metastatic capacities of endometrial cancer cells. Mechanistically, we revealed that SNRPB knockdown decreased POLD1 expression and that POLD1 intron 22 was retained after SNRPB silencing in endometrial cancer cells, as determined via RNA sequencing data analysis. The retained intron 22 of POLD1 created a premature termination codon, leading to the absence of amino acids 941-1,107 and the loss of the site of interaction with PCNA, which is essential for POLD1 enzyme activity. In addition, POLD1 depletion decreased the increase in the malignancy of endometrial cancer cells overexpressing SNRPB. Furthermore, miR-654-5p was found to bind directly to the 3' untranslated region of SNRPB, resulting in SNRPB expression inhibition in endometrial cancer. Antisense oligonucleotide-mediated SNRPB inhibition led to a decrease in the growth capacity of a cell-derived xenograft model and a patient with endometrial cancer-derived xenograft model. Overall, SNRPB promotes the efficient splicing of POLD1 by regulating intron retention, ultimately contributing to high POLD1 expression in endometrial cancer. The oncogenic SNRPB-POLD1 axis is an interesting therapeutic target for endometrial cancer, and antisense oligonucleotide-mediated silencing of SNRPB may constitute a promising therapeutic approach for treating patients with endometrial cancer.

Understanding HLA-DQ in renal transplantation: a mini-review

Human leukocyte antigen (HLA) mismatching, particularly with HLA-DQ, significantly impacts the development of donor-specific antibodies (DSA) and transplant outcomes. HLA-DQ antibodies are highly immunogenic and detrimental, necessitating advanced high-resolution HLA typing to improve mismatch assessment and clinical risk evaluation. Traditional serological or low-resolution typing often misclassifies mismatches, leading to inaccuracies in assessing immunogenicity and predicting outcomes. Emerging molecular mismatch algorithms refine immunogenicity assessments by analyzing amino acid differences and structural interactions. These tools show promise for personalizing transplant protocols but have limitations, such as variability in predicting individual patient outcomes. Immunogenicity of mismatches also depends on evolutionary divergence and specific amino acid differences, with studies revealing that certain evolutionary lineages and polymorphisms influence T-cell alloreactivity and DSA development. Complexities in HLA-DQ protein expression, including combinatorial diversity of heterodimers and inter-isotypic heterodimers, further complicate risk evaluation. Expression levels, influenced by tissue specificity and inflammatory stimuli, and alternative splicing of HLA-DQ transcripts add additional layers of variability. Future clinical applications, enabled by high-resolution HLA typing, may include refined graft selection, improved DSA monitoring, and individualized therapy. However, understanding the precise mechanisms of HLA-DQ immunogenicity remains a priority for advancing transplantation science and enhancing patient outcomes.

An alternative splicing caused by a natural variation in BnaC02.VTE4 gene affects vitamin E and glucosinolate content in rapeseed (Brassica napus L.).

Vitamin E (VE) is essential for plants and animals. Rapeseed oil is rich in α-tocopherol (α-T), which is the most bioactive form of VE in human body. This study demonstrated that VE in rapeseed seeds was mainly controlled by embryo genotype through incomplete diallel hybridization. By genome-wide association study, the QTL-qVE.C02 associated with VE and α-T contents was detected in a Brassica napus association population, and the phenotypic contribution rate was up to 18.71%. BnaC02.VTE4, encoding gama-tocopherol methyltransferase, was proved as the target gene of qVE.C02 by genetic complementation. Two BnaC02.VTE4 haplotypes were identified in the population. Compared with BnaC02.VTE4HapH, a point mutation from A to G at the 3' splicing site of the second intron were found in BnaC02.VTE4HapL, resulting in alternative splicing and early termination of translation. HapL1052(G-A), the site-directed mutagenesis fragment of BnaC02.VTE4HapL, was introduced into Arabidopsis vte4 mutant and 8S088 (a BnaC02.VTE4HapL accession), and the contents of VE and α-T in atvte4-4 and 8S088 seeds were increased by 90.10% to 307.29%. These demonstrated the point mutation as the causal for the difference in VE biosynthesis in rapeseed. Further, this variation also led to the significant difference in glucosinolate content between BnaC02.VTE4HapH and BnaC02.VTE4HapL accessions. Multi-omics analysis suggested that the expression of some genes and the accumulation of several metabolites related to the glucosinolate biosynthesis pathway were significantly increased in BnaC02.VTE4HapL group. Moreover, by functional marker identification, the BnaC02.VTE4HapH was found to be selected during domestication. Our findings offered promising opportunities for enhancing rapeseed quality traits.

Global dysregulation of circular RNAs in frontal cortex and whole blood from DM1 and DM2.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular disorders associated with expansions of microsatellites, respectively, in DMPK and CNBP. Their pathogenesis is linked to the global aberrant alternative splicing (AAS) of many genes and marks mostly muscular and neuronal tissues, while blood is the least affected. Recent data in DM1 skeletal muscles indicated that abnormalities in RNA metabolism also include global upregulation of circular RNAs (circRNAs). CircRNAs are a heterogeneous group considered splicing errors and by-products of canonical splicing. To elucidate whether circRNA dysregulation is an inherent feature of the myotonic environment, we perform their analysis in the frontal cortex and whole blood of DM1 and DM2 patients. We find a global elevation of circRNAs in both tissues, and its magnitude is neither correlated with the differences in their parental gene expression nor is associated with AAS published earlier. Aberrantly spliced cassette exons of linear transcripts affected in DM1 and DM2 are not among the circularized exons, which unique genomic features prerequisite back-splicing. However, the blueprint of the AAS of linear RNAs is found in a variety of circRNA isoforms. The heterogeneity of circRNAs also originates from the utilization of exonic and intronic cryptic donors/acceptors in back splice junctions, and intron-containing circRNAs are more characteristic of the blood. Overall, this study reveals circRNA dysregulation in various tissues from DM1 and DM2; however, their levels do not correlate with the AAS in linear RNAs, suggesting a potential independent regulatory mechanism underlying circRNA upregulation in myotonic dystrophy.

Alternative Splicing Options for Ultra-High-Performance Concrete (UHPC) H-Piles

Pile splicing is generally considered in construction because of transportation limits, length requirements, construction means and methods, and strength capacity. A major challenge in the use of precast prestressed UHPC piles is the lack of efficient and effective splicing solutions. To address the problem, this study evaluated different pile splicing methods for UHPC H-piles and their constructability. The analysis and design for strength capacity and detailing presented here are based on relevant established guidelines and design codes for UHPC. This study assessed two pile splicing methods: epoxy-bonded dowels and near-surface mounted bars (NSMBs). The analysis demonstrated that the epoxy-bonded dowel method provides a moment capacity that is 127% of the pile moment capacity in the strong direction and 139% of the pile moment capacity in the weak direction. In comparison, the NSMB method achieved 121% in the strong direction and 106% in the weak direction. Both methods developed the established strength capacity requirements. The constructability of both pile splicing options was evaluated to provide practical guidelines for their preparation in preplanned and unplanned situations. The results reported are for 18-inch UHPC H-piles; however, the construction and analytical approach applies to other pile sizes as well. The pile splicing options developed are recommended for further experimental investigations.

Multi-omics profiling in spinal muscular atrophy (SMA): investigating lipid and metabolic alterations through longitudinal CSF analysis of Nusinersen-treated patients.

Spinal muscular atrophy (SMA) is a rare neuromuscular disease caused by biallelic mutations in the SMN1 gene, leading to progressive muscle weakness due to degeneration of the anterior horn cells. Since 2017, SMA patients can be treated with the anti-sense oligonucleotide Nusinersen, which promotes alternative splicing of the SMN2 gene, by regular intrathecal injections. In this prospective study, we applied metabolomic, lipidomic, and proteomic analysis to examine sequential CSF samples from 13 SMA patients and controls. This multi-omic approach identified over 800 proteins and 400 small molecules including lipids. Multivariate analysis of multi-omic data successfully discriminated between the CSF derived from SMA patients and control subjects. Lipidomic analysis revealed increased levels of cholesteryl esters and lyso-phospholipids, along with reduced levels of cholesterol and phospholipids in the CSF of SMA patients as compared to healthy controls. These data, combined with results from functional assays, led us to conclude that SMA patients exhibit altered levels and function of high-density-lipoprotein (HDL)-like particles in the CSF. Notably, Nusinersen therapy was observed to reverse disease-specific profile changes toward a physiological state, potentially explicable by restoring HDL function.

Assessment of PRMT6-dependent alternative splicing in pluripotent and differentiating NT2/D1 cells.

Protein arginine methyltransferase 6 (PRMT6) is a well-characterized epigenetic regulator that methylates histone H3 at arginine 2 (H3R2me2a) in both promoter and enhancer regions, thereby modulating transcriptional initiation. We report here that PRMT6 also regulates gene expression at the post-transcriptional level in the neural pluripotent state and during neuronal differentiation of NT2/D1 cells. PRMT6 knockout causes widespread alternative splicing changes in NT2/D1 cells, most frequently cassette exon alterations. Most of the PRMT6-dependent splicing targets are not transcriptionally affected by the enzyme and regulated in an H3R2me2a-independent manner. However, for a small subset of splicing events, the PRMT6-mediated deposition of H3R2me2a overlaps with the splice site, suggesting a potential dual function in both transcriptional and co-/post-transcriptional regulation. The splicing targets of PRMT6 include ribosomal proteins, splicing factors, and chromatin-modifying enzymes such as PRMT4, DNMT3B, and ASH2L, some of which are associated with differentiation decisions. Taken together, our results in NT2/D1 cells show that PRMT6 exerts predominantly H3R2me2a-independent functions in RNA splicing, which may contribute to pluripotency and neuronal identity.

PRMT5 Maintains Homeostasis of the Intestinal Epithelium by Modulating Cell Proliferation and Survival.

Intestinal homeostasis is sustained by self-renewal of intestinal stem cells, which continuously divide and produce proliferative transit-amplifying (TA) and progenitor cells. Protein arginine methyltransferases 5 (PRMT5) plays a crucial role in regulating homeostasis of various mammalian tissues. However, its function in intestinal homeostasis remains elusive. In this study, conditional knockout of Prmt5 in the mouse intestinal epithelium leads to a reduction in stem cell population, suppression of cell proliferation, and increased cell apoptosis within the intestinal crypts, accompanied with shortened gut length, decreased mouse body weight, and eventual animal mortality. Additionally, Prmt5 deletion or its enzymatic inhibition in intestinal organoids in vitro also shows resembling cellular phenotypes. Methylome profiling identifies 90 potential Prmt5 substrates, which are involved in RNA-related biological processes and cell division. Consistently, Prmt5 depletion in intestinal organoids leads to aberrant alternative splicing in a subset of genes related to the mitotic cell cycle. Furthermore, Prmt5 loss triggers p53-mediated apoptosis in the intestinal epithelium. Collectively, the findings uncover an indispensable role of PRMT5 in promoting cell proliferation and survival, as well as maintaining stem cells in the gut epithelium.

Transcriptomic Analysis Reveals Patterns of Expression of Stage-Specific Genes in Early Apis cerana Embryos

Background/Objectives: Apis cerana development is described as comprising four stages: embryo, larva, pupa, and adult. There are significant differences between workers and drones in terms of physiological functions and social roles, and the formation of the organ primordia occurs during the embryonic stage. Therefore, the objective of this study is to investigate the differential expression of and alternative splicing of genes in worker and drone embryos and to explain their unique developmental patterns. Methods: Long-read sequencing (PacBio Iso-Seq) and short-read sequencing (Illumina RNA-Seq) were used to investigate worker and drone embryo gene expression differences in A. cerana across five developmental points (12, 24, 36, 48, and 60 h). Results: The study identified 59,254 common isoforms, with 5744 and 5106 isoforms specific to worker and drone embryos, respectively. Additionally, a new transcript of the csd gene was identified. The number of differentially expressed genes (3391) and differential splicing events (470 genes) peaked at the 24-h embryonic stage. Differential splicing events of csd, dsx, and Y-y were observed in the worker and drone embryos. Conclusions: The gene expression results indicated that the 24-h embryonic point is a critical period for the expression of genes related to developmental and behavioral differences between workers and drones. The findings provide a theoretical basis for future research on the developmental differences between workers and drones.

Hypoxia-induced CTCF mediates alternative splicing via coupling chromatin looping and RNA Pol II pause to promote EMT in breast cancer.

Hypoxia influences the epithelial-mesenchymal transition (EMT) through the remodeling of the chromatin structure, epigenetics, and alternative splicing. Hypoxia drives CCCTC-binding factor (CTCF) induction through hypoxia-inducible factor 1-alpha (HIF1α), which promotes EMT, although the underlying mechanisms remain unclear. We find that hypoxia significantly increases CTCF occupancy at various EMT-related genes. We present a CTCF-mediated intricate mechanism promoting EMT wherein CTCF binding at the collagen type V alpha 1 chain (COL5A1) promoter is crucial for COL5A1 upregulation under hypoxia. Additionally, hypoxia drives exon64A inclusion in a mutually exclusive alternative splicing event of COL5A1exon64 (exon64A/64B). Notably, CTCF mediates COL5A1 promoter-alternatively spliced exon upstream looping that regulates DNA demethylation at distal exon64A. This further regulates the CTCF-mediated RNA polymerase II pause at COL5A1exon64A, leading to its inclusion in promoting the EMT under hypoxia. Genome-wide study indicates the association of gained CTCF occupancy with the alternative splicing of many cancer-related genes, similar to the proposed model. Specifically, disrupting the HIF1α-CTCF-COL5A1exon64A axis through the dCas9-DNMT3A system alleviates the EMT in hypoxic cancer cells and may represent a novel therapeutic target in breast cancer.

Clinical and Prognostic Implications of an Alternative Splicing-related Risk Model Based on TP53 Status in Breast Cancer.

Breast Cancer (BRCA) is one of the most common cancers worldwide. Abnormal Alternative Splicing (AS) is frequently observed in cancers. Understanding the intricate relationship between gene mutations and abnormal AS is vital for developing novel diagnostic and therapeutic strategies to effectively target cancer. This study aimed to focus on the analysis of transcriptomic splicing events in patients with Breast Cancer (BRCA), particularly those with mutations in the TP53 gene. Understanding the role of AS may be helpful in revealing potential predictive indicators for survival and treatment strategies. The splicing data were downloaded from the Cancer Genome Atlas (TCGA) breast cancer project, incorporating 972 patients in the study, classified according to TP53 mutation status. A comprehensive splicing profile of these breast tumors was outlined, and an interaction network of Alternative Splicing (AS) events and splicing factors was constructed. This allowed for the identification of specific AS events associated with TP53-mutant breast cancer. A prognostic risk model based on AS events was established, using univariate and multivariate Cox regression analyses. To understand the molecular heterogeneity, consensus clustering analyses of prognostic AS events were performed. We also investigated the association of AS patterns with the immune microenvironment and drug sensitivity. A total of 4519 significant Alternative Splicing (AS) events were distributed among 2729 genes that were altered in TP53 mutant tumors. Based on the analysis of these events, a prognostic risk model was created involving ten AS events from ten genes (such as NKTR, CD46, VCAN, etc.). The survival analysis showed that patients with high-risk scores had significantly poorer overall survival (p<0.001, HR=2.46, 95% CI 1.90-3.18) than those with low-risk scores. Furthermore, the study identified four molecular subtypes related to AS events (C1, C2, C3, and C4), which showed significant differences in immune cell infiltration, with C1 and C4 clusters having a higher degree of immune cell infiltration than C2 and C3. The chemosensitivity analysis revealed that these different AS clusters have different sensitivities to several anticancer drugs, such as docetaxel, paclitaxel, and doxorubicin, with C1 and C4 clusters being more sensitive than the other clusters. We have demonstrated differential transcriptomic splicing events between TP53 mutant and wild-type cases of breast cancer, establishing an effective prognostic risk model based on AS events. These findings provide new insights that may aid in understanding the biological behavior of breast cancer and potentially in optimizing treatment strategies for breast cancer.

MiR-448-3p/miR-1264-3p Participates in Intermittent Hypoxic Response in Hippocampus by Regulating Fam76b/hnRNPA2B1.

Intermittent hypoxia (IH), as a key pathogenic factor of obstructive sleep apnea syndrome (OSAS), can cause many diseases, such as increased inflammation and oxidative stress, diabetes, cardiovascular disease, and Alzheimer's disease (AD). The response of cells to hypoxia involves multiple levels of regulatory mechanisms, including transcriptional regulation of gene expression, regulation of mRNA stability, post-transcriptional regulation, and post-translational modification regulation. The regulation of miRNA and alternative splicing (AS) in neuronal response to intermittent hypoxia deserve further study. By establishing a mouse model of intermittent hypoxia, we conducted functional studies on key miRNAs and splicing factor using methods such as miRNA sequencing, bioinformatics, and molecular biology. In the mouse hippocampus, intermittent hypoxia altered the expression of many miRNAs, with miR-448-3p and miR-1264-3p changing over the course of more than three time periods. Interestingly, the expression of Fam76b, the common target gene of these two miRNAs, also changed under intermittent hypoxia. Further studies showed that Fam76b may regulate the ratio of Nbr1 and Dph3 transcripts in response to hypoxia by affecting the localization of hnRNPA2B1 protein within cells. Research into intermittent hypoxia-induced disorders, including Alzheimer's disease and other neurodegenerative diseases, might benefit from a better understanding of the regulatory mechanisms of miRNA and alternative splicing in hypoxic response at the animal and cell levels. This study demonstrates that intermittent hypoxia alters the expression of miR-448-3p and miR-1264-3p, as well as the localization of the splicing factor hnRNPA2B1 in the cell nucleus. These findings enhance our understanding of the molecular mechanisms of neuronal responses to hypoxia and hold potential implications for treating hypoxia-related diseases like Alzheimer's disease.

ZYG-12/Hook's dual role as a dynein adaptor for early endosomes and nuclei is regulated by alternative splicing of its cargo binding domain.

The microtubule motor cytoplasmic dynein-1 transports and positions various organelles, but the molecular basis of this functional diversity is not fully understood. Cargo adaptors of the Hook protein family recruit dynein to early endosomes (EE) in fungi and human cells by forming the FTS-Hook-FHIP (FHF) complex. By contrast, the Caenorhabditis elegans Hook homologue ZYG-12 recruits dynein to the nuclear envelope (NE) in the meiotic gonad and mitotic early embryo by forming a Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Here, we demonstrate that ZYG-12 recruits dynein to EE in epithelia. We identify and functionally characterize the homologues of FTS (UBC-19) and FHIP (FHIP-1) that constitute the C. elegans FHF complex, validate the predicted FHIP-1-RAB-5 binding interface in vivo, and show that ZYG-12 forms FHF via a conserved segment that precedes, and is distinct from, its C-terminal NE targeting domain. Finally, we show that C-terminal ZYG-12 splice isoforms differ in their ability to target to the NE and EE. We conclude that the C. elegans Hook adaptor evolved to recruit dynein to two distinct organelles, and that cargo specificity of ZYG-12 is regulated by alternative splicing.

IGF2BP1 stabilizes Akt2 mRNA to promote glucose metabolism and maintain spermatogonial proliferation.

Insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1), as a newly identified N6-methyladenosine (m6A) methylation reader, plays a key role in mRNA stability, alternative splicing, and translation. However, the function of IGF2BP1 and its target genes in the development of male germ cells remains unclear. In the present study, Western blotting, immunofluorescence, and other cellular methods were used to confirm whether IGF2BP1 is expressed throughout the male germline with high abundance in spermatogonia of mice, and whether its downregulation inhibits spermatogonia proliferation in vitro. Through RNA sequencing, dual luciferase reporter assays, RIP-qPCR, and mRNA stability experiments, it was identified that Akt2 is one of the target genes of IGF2BP1. By stabilizing Akt2 mRNA in an m6A-dependent manner, IGF2BP1 maintains spermatogonial proliferation. Furthermore, co-immunoprecipitation and mass spectrometry analyses revealed that PABPC1, DDX5, and FXR1 interact with IGF2BP1, and that the interaction between IGF2BP1 and PABPC1 promotes AKT2 expression. Finally, following the knockdown of both IGF2BP1 and AKT2, glucose and ATP levels in C18-4 and GC-1 spermatogonia cells were found to be reduced, indicating that IGF2BP1 regulates glucose metabolism homeostasis by stabilizing Akt2 expression, thereby influencing spermatogonia proliferation. Additionally, analysis of the cryptorchidism database from NCBI revealed that the expression of IGF2BP1 and AKT2 is significantly downregulated in cryptorchid patients with oligospermia or azoospermia. In conclusion, our study elucidates the role and underlying mechanism of IGF2BP1 in spermatogonia, providing a candidate target for male infertility.