TransGCN: a semi-supervised graph convolution network-based framework to infer protein translocations in spatio-temporal proteomics.

Protein subcellular localization (PSL) is very important in order to understand its functions, and its movement between subcellular niches within cells plays fundamental roles in biological process regulation. Mass spectrometry-based spatio-temporal proteomics technologies can help provide new insights of protein translocation, but bring the challenge in identifying reliable protein translocation events due to the noise interference and insufficient data mining. We propose a semi-supervised graph convolution network (GCN)-based framework termed TransGCN that infers protein translocation events from spatio-temporal proteomics. Based on expanded multiple distance features and joint graph representations of proteins, TransGCN utilizes the semi-supervised GCN to enable effective knowledge transfer from proteins with known PSLs for predicting protein localization and translocation. Our results demonstrate that TransGCN outperforms current state-of-the-art methods in identifying protein translocations, especially in coping with batch effects. It also exhibited excellent predictive accuracy in PSL prediction. TransGCN is freely available on GitHub at https://github.com/XuejiangGuo/TransGCN.

Cellular nucleic acid binding protein facilitates cardiac repair after myocardial infarction by activating ß-catenin signaling.

The regenerative capacity of the adult mammalian heart is limited, while the neonatal heart is an organ with regenerative and proliferative ability. Activating adult cardiomyocytes (CMs) to re-enter the cell cycle is an effective therapeutic method for ischemic heart disease such as myocardial infarction (MI) and heart failure. Here, we aimed to reveal the role and potential mechanisms of cellular nucleic acid binding protein (CNBP) in cardiac regeneration and repair after heart injury. CNBP is highly expressed within 7 days post-birth while decreases significantly with the loss of regenerative ability. In vitro, overexpression of CNBP promoted CM proliferation and survival, whereas knockdown of CNBP inhibited these processes. In vivo, knockdown of CNBP in CMs robustly hindered myocardial regeneration after apical resection in neonatal mice. In adult MI mice, CM-specific CNBP overexpression in the infarct border zone ameliorated myocardial injury in acute stage and facilitated CM proliferation and functional recovery in the long term. Quantitative proteomic analysis with TMT labeling showed that CNBP overexpression promoted the DNA replication, cell cycle progression, and cell division. Mechanically, CNBP overexpression increased the expression of ß-catenin and its downstream target genes CCND1 and c-myc; Furthermore, Luciferase reporter and Chromatin immunoprecipitation (ChIP) assays showed that CNBP could directly bind to the ß-catenin promoter and promote its transcription. CNBP also upregulated the expression of G1/S-related cell cycle genes CCNE1, CDK2, and CDK4. Collectively, our study reveals the positive role of CNBP in promoting cardiac repair after injury, providing a new therapeutic option for the treatment of MI.

Maternal KLF17 controls zygotic genome activation by acting as a messenger for RNA Pol II recruitment in mouse embryos.

Initiation of timely and sufficient zygotic genome activation (ZGA) is crucial for the beginning of life, yet our knowledge of transcription factors (TFs) contributing to ZGA remains limited. Here, we screened the proteome of early mouse embryos after cycloheximide (CHX) treatment and identified maternally derived KLF17 as a potential TF for ZGA genes. Using a conditional knockout (cKO) mouse model, we further investigated the role of maternal KLF17 and found that it promotes embryonic development and full fertility. Mechanistically, KLF17 preferentially binds to promoters and recruits RNA polymerase II (RNA Pol II) in early 2-cell embryos, facilitating the expression of major ZGA genes. Maternal Klf17 knockout resulted in a downregulation of 9% of ZGA genes and aberrant RNA Pol II pre-configuration, which could be partially rescued by introducing exogenous KLF17. Overall, our study provides a strategy for screening essential ZGA factors and identifies KLF17 as a crucial TF in this process.

STYXL1 regulates CCT complex assembly and flagellar tubulin folding in sperm formation.

Tubulin-based microtubule is a core component of flagella axoneme and essential for sperm motility and male fertility. Structural components of the axoneme have been well explored. However, how tubulin folding is regulated in sperm flagella formation is still largely unknown. Here, we report a germ cell-specific co-factor of CCT complex, STYXL1. Deletion of Styxl1 results in male infertility and microtubule defects of sperm flagella. Proteomic analysis of Styxl1-/- sperm reveals abnormal downregulation of flagella-related proteins including tubulins. The N-terminal rhodanese-like domain of STYXL1 is important for its interactions with CCT complex subunits, CCT1, CCT6 and CCT7. Styxl1 deletion leads to defects in CCT complex assembly and tubulin polymerization. Collectively, our findings reveal the vital roles of germ cell-specific STYXL1 in CCT-facilitated tubulin folding and sperm flagella development.

Germ cell-specific deletion of Pex3 reveals essential roles of PEX3-dependent peroxisomes in spermiogenesis.

Peroxisomes are organelles enclosed by a single membrane and are present in various species. The abruption of peroxisomes is correlated with peroxisome biogenesis disorders and single peroxisomal enzyme deficiencies that induce diverse diseases in different organs. However, little is known about the protein compositions and corresponding roles of heterogeneous peroxisomes in various organs. Through transcriptomic and proteomic analyses, we observed heterogenous peroxisomal components among different organs, as well as between testicular somatic cells and different developmental stages of germ cells. As Pex3 is expressed in both germ cells and Sertoli cells, we generated Pex3 germ cell- and Sertoli cell-specific knockout mice. While Pex3 deletion in Sertoli cells did not affect spermatogenesis, the deletion in germ cells resulted in male sterility, manifested as the destruction of intercellular bridges between spermatids and the formation of multinucleated giant cells. Proteomic analysis of the Pex3-deleted spermatids revealed defective expressions of peroxisomal proteins and spermiogenesis-related proteins. These findings provide new insights that PEX3-dependent peroxisomes are essential for germ cells undergoing spermiogenesis, but not for Sertoli cells.

scapGNN: A graph neural network-based framework for active pathway and gene module inference from single-cell multi-omics data.

Although advances in single-cell technologies have enabled the characterization of multiple omics profiles in individual cells, extracting functional and mechanistic insights from such information remains a major challenge. Here, we present scapGNN, a graph neural network (GNN)-based framework that creatively transforms sparse single-cell profile data into the stable gene-cell association network for inferring single-cell pathway activity scores and identifying cell phenotype-associated gene modules from single-cell multi-omics data. Systematic benchmarking demonstrated that scapGNN was more accurate, robust, and scalable than state-of-the-art methods in various downstream single-cell analyses such as cell denoising, batch effect removal, cell clustering, cell trajectory inference, and pathway or gene module identification. scapGNN was developed as a systematic R package that can be flexibly extended and enhanced for existing analysis processes. It provides a new analytical platform for studying single cells at the pathway and network levels.

Epididymal segment-specific miRNA and mRNA regulatory network at the single cell level.

Spermatozoa released from the testis cannot fertilize an egg before becoming mature and motile in the epididymis. Based on three bulk and one single-cell RNA-seq (scRNA-seq) data series, we compared mRNA or miRNA expression between epididymal segment-specific samples and the other samples. Hereby, we identified 570 differentially expressed mRNAs (DE-mRNAs) and 23 differentially expressed miRNAs (DE-miRNAs) in the caput, 175 DE-mRNAs and 15 DE-miRNAs in the corpus, 946 DE-mRNAs and 12 DE-miRNAs in the cauda. In accordance with respective DE-miRNAs, we predicted upstream transcription factors (TFs) and downstream target genes. Subsequently, we intersected target genes of respective DE-miRNAs with corresponding DE-mRNAs, thereby obtaining 127 upregulated genes in the caput and 92 upregulated genes in cauda. Enriched upregulated pathways included cell motility-related pathways for the caput, smooth muscle-related pathways for the corpus, and immune-associated pathways for the cauda. Protein-protein interaction (PPI) network was constructed to extract key module for the caput and cauda, followed by identifying hub genes through cytohubba. Epididymis tissues from six mice were applied to validate hub genes expression using qRT-PCR, and 7 of the 10 genes displayed identical expression trends in mice caput/cauda. These hub genes were found to be predominantly distributed in spermatozoa using scRNA-seq data. In addition, target genes of DE-miRNAs were intersected with genes in the PPI network for each segment. Subsequently, the miRNA and mRNA regulatory networks for the caput and cauda were constructed. Conclusively, we uncover segment-specific miRNA-mRNA regulatory network, upstream TFs, and downstream pathways of the human epididymis, warranting further investigation into epididymal segment-specific functions.

Correction of a homoplasmic mitochondrial tRNA mutation in patient-derived iPSCs via a mitochondrial base editor.

Pathogenic mutations in mitochondrial DNA cause severe and often lethal multi-system symptoms in primary mitochondrial defects. However, effective therapies for these defects are still lacking. Strategies such as employing mitochondrially targeted restriction enzymes or programmable nucleases to shift the ratio of heteroplasmic mutations and allotopic expression of mitochondrial protein-coding genes have limitations in treating mitochondrial homoplasmic mutations, especially in non-coding genes. Here, we conduct a proof of concept study applying a screened DdCBE pair to correct the homoplasmic m.A4300G mutation in induced pluripotent stem cells derived from a patient with hypertrophic cardiomyopathy. We achieve efficient G4300A correction with limited off-target editing, and successfully restore mitochondrial function in corrected induced pluripotent stem cell clones. Our study demonstrates the feasibility of using DdCBE to treat primary mitochondrial defects caused by homoplasmic pathogenic mitochondrial DNA mutations.

Zinc oxide nanoparticles-induced testis damage at single-cell resolution: Depletion of spermatogonia reservoir and disorder of Sertoli cell homeostasis.

The widespread application of zinc oxide nanoparticles (ZnO NPs) in our daily life has initiated an enhanced awareness of their biosafety concern. An incredible boom of evidence of organismal disorder has accumulated for ZnO NPs, yet there has been no relevant study at the single-cell level. Here, we profiled > 28,000 single-cell transcriptomes and assayed > 25,000 genes in testicular tissues from two healthy Sprague Dawley (SD) rats and two SD rats orally exposed to ZnO NPs. We identified 10 cell types in the rat testis. ZnO NPs had more deleterious effects on spermatogonia, Sertoli cells, and macrophages than on the other cell types. Cell-cell communication analysis indicated a sharp decrease of interaction intensity for all cell types except macrophages in the ZnO NPs group than in the control group. Interestingly, two distinct maturation states of spermatogonia were detected during pseudotime analysis, and ZnO NPs induced reservoir exhaustion of undifferentiated spermatogonia. Mechanically, ZnO NPs triggered fatty acid accumulation in GC-1 cells through protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling and peroxisome proliferator-activated receptor alpha (PPARα)/acyl-CoA oxidase 1 (Acox1) axis, contributing to cell apoptosis. In terms of Sertoli cells, downregulated genes were highly enriched for tight junction. In vitro and in vivo experiments verified that ZnO NPs disrupted blood-testis barrier formation and growth factors synthesis, which subsequently inhibited the proliferation and induced the apoptosis of spermatogonia. As for the macrophages, ZnO NPs activated oxidative stress of Raw264.7 cells through nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway and promoted cell apoptosis through extracellular signal-regulated kinase (ERK) 1/2 pathway. Collectively, our work reveals the cell type-specific and cellularly heterogenetic mechanism of ZnO NPs-induced testis damage and paves the path for identifying putative biomarkers and therapeutics against this disorder.

Oocytes orchestrate protein prenylation for mitochondrial function through selective inactivation of cholesterol biosynthesis in murine species.

Emerging research and clinical evidence suggest that the metabolic activity of oocytes may play a pivotal role in reproductive anomalies. However, the intrinsic mechanisms governing oocyte development regulated by metabolic enzymes remain largely unknown. Our investigation demonstrates that geranylgeranyl diphosphate synthase1 (Ggps1), the crucial enzyme in the mevalonate pathway responsible for synthesizing isoprenoid metabolite geranylgeranyl pyrophosphate from farnesyl pyrophosphate, is essential for oocyte maturation in mice. Our findings reveal that the deletion of Ggps1 that prevents protein prenylation in fully grown oocytes leads to subfertility and offspring metabolic defects without affecting follicle development. Oocytes that lack Ggps1 exhibit disrupted mitochondrial homeostasis and the mitochondrial defects arising from oocytes are inherited by the fetal offspring. Mechanistically, the excessive farnesylation of mitochondrial ribosome protein, Dap3, and decreased levels of small G proteins mediate the mitochondrial dysfunction induced by Ggps1 deficiency. Additionally, a significant reduction in Ggps1 levels in oocytes is accompanied by offspring defects when females are exposed to a high-cholesterol diet. Collectively, this study establishes that mevalonate pathway-protein prenylation is vital for mitochondrial function in oocyte maturation and provides evidence that the disrupted protein prenylation resulting from an imbalance between farnesyl pyrophosphate and geranylgeranyl pyrophosphate is the major mechanism underlying impairment of oocyte quality induced by high cholesterol.

Multi-omics reveal the metabolic patterns in mouse cumulus cells during oocyte maturation.

Bi-directional communication between cumulus cells and the surrounded oocytes is important for the development and functions of both compartments. However, the metabolic framework in cumulus cells has not been systematically described. In the present study, cumulus cells from cumulus-oocyte complexes (COCs) at three key time points were isolated (arrested GV stage, post-hCG 0h; meiotic resumption GVBD stage, post-hCG 3h; and metaphase II stage, post-hCG 12h), and the temporal metabolomic and proteomic profiling were performed. Integrated multi-omics analysis reveals the global metabolic patterns in cumulus cells during mouse oocyte maturation. In particular, we found the active hyaluronic acid metabolism, steroid hormone synthesis, and prostaglandin E2 (PGE2) production in cumulus cells. Meanwhile, accompanying the oocyte maturation, a progressive increase in nucleotide and amino acid metabolism was detected in the surrounding cumulus cells. In sum, the data serve as a valuable resource for probing metabolism during terminal differentiation of ovarian granulosa cells, and provide the potential biomarkers for improving and predicting oocyte quality.

DeepSP: A Deep Learning Framework for Spatial Proteomics.

The study of protein subcellular localization (PSL) is a fundamental step toward understanding the mechanism of protein function. The recent development of mass spectrometry (MS)-based spatial proteomics to quantify the distribution of proteins across subcellular fractions provides us a high-throughput approach to predict unknown PSLs based on known PSLs. However, the accuracy of PSL annotations in spatial proteomics is limited by the performance of existing PSL predictors based on traditional machine learning algorithms. In this study, we present a novel deep learning framework named DeepSP for PSL prediction of an MS-based spatial proteomics data set. DeepSP constructs the new feature map of a difference matrix by capturing detailed changes between different subcellular fractions of protein occupancy profiles and uses the convolutional block attention module to improve the prediction performance of PSL. DeepSP achieved significant improvement in accuracy and robustness for PSL prediction in independent test sets and unknown PSL prediction compared to current state-of-the-art machine learning predictors. As an efficient and robust framework for PSL prediction, DeepSP is expected to facilitate spatial proteomics studies and contributes to the elucidation of protein functions and the regulation of biological processes.

RNA-binding protein complex AMG-1/SLRP-1 mediates germline development and spermatogenesis by maintaining mitochondrial homeostasis in Caenorhabditis elegans.

The mechanisms of RNA-binding proteins (RBPs)-mediated post-transcriptional regulation of pre-existing mRNAs, which is essential for spermatogenesis, remain poorly understood. In this study, we identify that a germline-specific mitochondrial RBP AMG-1(abnormal mitochondria in germline 1), a homolog of mammalian leucine-rich PPR motif-containing protein (LRPPRC), is required for spermatogenesis in Caenorhabditis elegans. The amg-1 mutation hinders germline development without affecting somatic development and leads to the aberrant mitochondrial morphology and structure associated with mitochondrial dysfunctions specifically in the germline. We demonstrate that AMG-1 is most frequently bound to mtDNA-encoded 12S and 16S ribosomal RNA, the essential components of mitochondrial ribosomes, and that 12S rRNA expression mediated by AMG-1 is crucial for germline mitochondrial protein homeostasis. Furthermore, steroid receptor RNA activator (SRA) stem loop interacting RNA binding protein (SLRP-1), a homolog of mammalian SRA stem loop interacting RNA binding protein (SLIRP) in C. elegans, interacts with AMG-1 genetically to regulate germline development and reproductive success in C. elegans. Overall, these findings reveal the novel function of mtRBP, specifically in regulating germline development.

STK33 Phosphorylates Fibrous Sheath Protein AKAP3/4 to Regulate Sperm Flagella Assembly in Spermiogenesis.

Spermatogenesis defects are important for male infertility; however, the etiology and pathogenesis are still unknown. Herein, we identified two loss-of-function mutations of STK33 in seven individuals with non-obstructive azoospermia. Further functional studies of these frameshift and nonsense mutations revealed that Stk33-/KI male mice were sterile, and Stk33-/KI sperm were abnormal with defects in the mitochondrial sheath, fibrous sheath, outer dense fiber, and axoneme. Stk33KI/KI male mice were subfertile and had oligoasthenozoospermia. Differential phosphoproteomic analysis and in vitro kinase assay identified novel phosphorylation substrates of STK33, fibrous sheath components A-kinase anchoring protein 3 and A-kinase anchoring protein 4, whose expression levels decreased in testis after deletion of Stk33. STK33 regulated the phosphorylation of A-kinase anchoring protein 3/4, affected the assembly of fibrous sheath in the sperm, and played an essential role in spermiogenesis and male infertility.

PATL2 regulates mRNA homeostasis in oocytes by interacting with EIF4E and CPEB1.

The accumulation and storage of maternal mRNA is crucial for oocyte maturation and embryonic development. PATL2 is an oocyte-specific RNA-binding protein, and previous studies have confirmed that PATL2 mutation in humans and knockout mice cause oocyte maturation arrest or embryonic development arrest, respectively. However, the physiological function of PATL2 in the process of oocyte maturation and embryonic development is largely unknown. Here, we report that PATL2 is highly expressed in growing oocytes and couples with EIF4E and CPEB1 to regulate maternal mRNA expression in immature oocytes. The germinal vesicle oocytes from Patl2-/- mice exhibit decreasing maternal mRNA expression and reduced levels of protein synthesis. We further confirmed that PATL2 phosphorylation occurs in the oocyte maturation process and identified the S279 phosphorylation site using phosphoproteomics. We found that the S279D mutation decreased the protein level of PATL2 and led to subfertility in Palt2S279D knock-in mice. Our work reveals the previously unrecognized role of PATL2 in regulating the maternal transcriptome and shows that phosphorylation of PATL2 leads to the regulation of PATL2 protein levels via ubiquitin-mediated proteasomal degradation in oocytes.

Regulation of Miwi-mediated mRNA stabilization by Ck137956/Tssa is essential for male fertility.

BACKGROUND: Sperm is formed through spermiogenesis, a highly complex process involving chromatin condensation that results in cessation of transcription. mRNAs required for spermiogenesis are transcribed at earlier stages and translated in a delayed fashion during spermatid formation. However, it remains unknown that how these repressed mRNAs are stabilized. RESULTS: Here we report a Miwi-interacting testis-specific and spermiogenic arrest protein, Ck137956, which we rename Tssa. Deletion of Tssa led to male sterility and absence of sperm formation. The spermiogenesis arrested at the round spermatid stage and numerous spermiogenic mRNAs were down-regulated in Tssa-/- mice. Deletion of Tssa disrupted the localization of Miwi to chromatoid body, a specialized assembly of cytoplasmic messenger ribonucleoproteins (mRNPs) foci present in germ cells. We found that Tssa interacted with Miwi in repressed mRNPs and stabilized Miwi-interacting spermiogenesis-essential mRNAs. CONCLUSIONS: Our findings indicate that Tssa is indispensable in male fertility and has critical roles in post-transcriptional regulations by interacting with Miwi during spermiogenesis.

A novel protein encoded by circRsrc1 regulates mitochondrial ribosome assembly and translation during spermatogenesis.

BACKGROUND: Circular RNAs (circRNAs) are a large class of mammalian RNAs. Several protein products translated by circRNAs have been reported to be involved in the development of various tissues and systems; however, their physiological functions in male reproduction have yet not been explored. RESULTS: Here, we report an endogenous circRNA (circRsrc1) that encodes a novel 161-amino-acid protein which we named Rsrc1-161aa through circRNA sequencing coupled with mass spectrometry analysis on mouse testicular tissues. Deletion of Rsrc1-161aa in mice impaired male fertility with a significant decrease in sperm count and motility due to dysfunctions of mitochondrial energy metabolism. A series of in vitro rescue experiments revealed that circRsrc1 regulates mitochondrial functions via its encoded protein Rsrc1-161aa. Mechanistically, Rsrc1-161aa directly interacts with mitochondrial protein C1qbp and enhances its binding activity to mitochondrial mRNAs, thereby regulating the assembly of mitochondrial ribosomes and affecting the translation of oxidative phosphorylation (OXPHOS) proteins and mitochondrial energy metabolism. CONCLUSIONS: Our studies reveal that Rsrc1-161aa protein encoded by circRsrc1 regulates mitochondrial ribosome assembly and translation during spermatogenesis, thereby affecting male fertility.

A tissue specific-infection mouse model of SARS-CoV-2.

Animal models play crucial roles in the rapid development of vaccines/drugs for the prevention and therapy of COVID-19, but current models have some deficits when studying the pathogenesis of SARS-CoV-2 on some special tissues or organs. Here, we generated a human ACE2 and SARS-CoV-2 NF/F knockin mouse line that constitutively expresses human ACE2 and specifically expresses SARS-CoV-2 N gene induced by Cre-recombinase. By crossing with Cre transgenic lines allowing for lung-specific and constitutive expression, we generated lung-specific (Sftpc-hACE2-NF/F) and constitutive SARS-CoV-2 N (EIIa-hACE2-NF/F) expressing mice. Upon intranasal infection with a SARS-CoV-2 GFP/ΔN strain which can only replicate in SARS-CoV-2 N expressed cells, we demonstrated that both the Sftpc-hACE2-NF/F and EIIa-hACE2-NF/F mice support viral replication. Consistent with our design, viral replication was limited to the lung tissues in Sftpc-hACE2-NF/F mice, while the EIIa-hACE2-NF/F mice developed infections in multiple tissues. Furthermore, our model supports different SARS-CoV-2 variants infection, and it can be successfully used to evaluate the effects of therapeutic monoclonal antibodies (Ab1F11) and antiviral drugs (Molnupiravir). Finally, to test the effect of SARS-CoV-2 infection on male reproduction, we generated Sertoli cell-specific SARS-CoV-2 N expressed mice by crossing with AMH-Cre transgenic line. We found that SARS-CoV-2 GFP/ΔN strain could infect Sertoli cells, led to spermatogenic defects due to the destruction of blood-testis barrier. Overall, combining with different tissue-specific Cre transgenic lines, the human ACE2 and SARS-CoV-2 NF/F line enables us to evaluate antivirals in vivo and study the pathogenesis of SARS-CoV-2 on some special tissues or organs.

A conditional knockout rat resource of mitochondrial protein-coding genes via a DdCBE-induced premature stop codon.

Hundreds of pathogenic variants of mitochondrial DNA (mtDNA) have been reported to cause mitochondrial diseases, which still lack effective treatments. It is a huge challenge to install these mutations one by one. We repurposed the DddA-derived cytosine base editor to incorporate a premature stop codon in the mtProtein-coding genes to ablate mitochondrial proteins encoded in the mtDNA (mtProteins) instead of installing pathogenic variants and generated a library of both cell and rat resources with mtProtein depletion. In vitro, we depleted 12 of 13 mtProtein-coding genes with high efficiency and specificity, resulting in decreased mtProtein levels and impaired oxidative phosphorylation. Moreover, we generated six conditional knockout rat strains to ablate mtProteins using Cre/loxP system. Mitochondrially encoded ATP synthase membrane subunit 8 and NADH:ubiquinone oxidoreductase core subunit 1 were specifically depleted in heart cells or neurons, resulting in heart failure or abnormal brain development. Our work provides cell and rat resources for studying the function of mtProtein-coding genes and therapeutic strategies.

Absence of CEP78 causes photoreceptor and sperm flagella impairments in mice and a human individual.

Cone-rod dystrophy (CRD) is a genetically inherited retinal disease that can be associated with male infertility, while the specific genetic mechanisms are not well known. Here, we report CEP78 as a causative gene of a particular syndrome including CRD and male infertility with multiple morphological abnormalities of sperm flagella (MMAF) both in human and mouse. Cep78 knockout mice exhibited impaired function and morphology of photoreceptors, typified by reduced ERG amplitudes, disrupted translocation of cone arrestin, attenuated and disorganized photoreceptor outer segments (OS) disks and widen OS bases, as well as interrupted connecting cilia elongation and abnormal structures. Cep78 deletion also caused male infertility and MMAF, with disordered '9+2' structure and triplet microtubules in sperm flagella. Intraflagellar transport (IFT) proteins IFT20 and TTC21A are identified as interacting proteins of CEP78. Furthermore, CEP78 regulated the interaction, stability, and centriolar localization of its interacting protein. Insufficiency of CEP78 or its interacting protein causes abnormal centriole elongation and cilia shortening. Absence of CEP78 protein in human caused similar phenotypes in vision and MMAF as Cep78-/- mice. Collectively, our study supports the important roles of CEP78 defects in centriole and ciliary dysfunctions and molecular pathogenesis of such multi-system syndrome.