Translational regulation plays a central role during post-meiotic development of male germ cells. Previous studies suggested that P-element induced wimpy testis like 1 (PIWIL1), a PIWI-interacting RNA (piRNA) binding protein that is critical for sperm development, participates in the maintenance and translational regulation of post-meiotic mRNAs in haploid spermatids. However, how PIWIL1 regulates protein translation remains largely unclear. Using biochemical assays, we show here that PIWIL1 utilizes different domains to interact with post-meiotic mRNAs and Poly-A binding protein cytoplasmic 1 (PABPC1), a general protein translation regulator. PIWIL1 binds 3′-untranslated regions (3′-UTRs) of several spermiogenic mRNAs via its N-terminal domain, whereas its interactions with PABPC1 are mediated through its N- and C-terminal domains in an RNA-dependent manner. Using a heterologous cell system, we analyzed its effects on protein translation via luciferase reporter assay and sucrose gradient sedimentation. It was found that PIWIL1 augments protein translation with PABPC1 in the presence of 3′-UTRs of post-meiotic mRNAs. While both the N-terminal RNA recognition motif (RRM) domain and the central linker region of PABPC1 stimulate translation, only the PIWI Argonaute and Zwille (PAZ) domain of PIWIL1 positively affects translation of reporter mRNAs. Interestingly, the PAZ domain was found absent from polysomal fractions, in contrast to the N- and C-terminal domains of PIWIL1. Taken together, the results suggest that PIWIL1 interacts with various partners using different domains and participates in translational regulation partly through 3′-UTRs. It will be of interest to further explore how PIWIL1 elicit its versatile functions, including translational regulation of post-meiotic mRNAs through intrinsic structural changes and extrinsic signals during mouse spermiogenesis under more physiological settings.
Background and Purpose An increasing number of autoimmune encephalitis (AE)-associated autoantibodies have been successfully characterized. However, many cases of AE remain unexplained on account of unknown antibodies. The aim of the present study was to identify a novel antibody against collapsin response mediator protein 2 (CRMP2) in suspected AE patients. Methods A patient’s serum and cerebrospinal fluid samples tested negative for known AE antibodies; however, strong immunolabel signals were observed in the neuronal cytoplasm of the cortex, hippocampus, and Purkinje cells on rat brain sections. Immunoprecipitation from the rat brain protein lysate, followed by mass spectrometry analysis, was used to identify the targeting antigen. Western blotting and cell-based assay with antigen-overexpressing HEK293T cells were used for antibody specificity, epitope, IgG subtype determination, and retrospective study. Results An antibody against CRMP2, a synaptic protein involved in axon guidance, was identified. The immunostains of the patient’s samples on rat brain sections were eliminated by pre-absorption with HEK293T cells overexpressing CRMP2. The samples specifically immunoreacted with CRMP2, but not with CRMP1, CRMP3, CRMP4, and CRMP5. The C-terminus of CRMP2 with 536 amino acids contained the epitope for antibody binding. The subtype analysis showed that the anti-CRMP2 antibody was IgG4. Furthermore, a screening of 46 patients with neurological disoders and neuro-cytoplasm immunostainings on rat brain sections resulted in the identification of anti-CRMP2 antibodies in a case of encephalomyelitis. The two patients responded well to immunotherapies. Conclusions This study discovered that a novel anti-CRMP2 antibody was associated with suspected AE and thus should be included in the testing list for AE.
The development and maintenance of the correct morphology of sperm is important for their functions. Cellular morphogenesis of sperm occurs during the post-meiotic developmental stage; however, little is known about what coordinates this process. In the present study, we investigated the role of A-kinase anchoring protein 3 (AKAP3) during mouse spermiogenesis, using both mouse genetics and proteomics. It was found that AKAP3 is essential for the formation of the specific subcellular structure of the sperm flagellum, motility of sperm and male fertility. Additionally, lack of AKAP3 caused global changes of the sperm proteome and mislocalization of sperm proteins, including accumulation of RNA metabolism and translation factors and displacement of PKA subunits in mature sperm, which may underlie misregulated PKA activity and immotility in sperm. Interestingly, sperm lacking a complete fibrous sheath from both Akap3 and Akap4 null mice accumulated F-actin filaments and morphological defects during post-testicular maturation in the epididymis. These results suggest that the subcellular structures of sperm could be formed via independent pathways, and elucidate the roles of AKAP3 during the coordinated synthesis and organization of the sperm proteome and sperm morphology.
Abstract The protein kinase A (PKA) signaling pathway, which mediates protein phosphorylation, is important for sperm motility and male fertility. This process relies on A-kinase anchoring proteins that organize PKA and its signalosomes within specific subcellular compartments. Previously, it was found that the absence of A-kinase anchoring protein 3 (AKAP3) leads to multiple morphological abnormalities in mouse sperm. But how AKAP3 regulates sperm motility is yet to be elucidated. AKAP3 has two amphipathic domains, here named dual and RI, in its N-terminus. These domains are responsible for binding regulatory subunits I alpha (RIα) and II alpha (RIIα) of PKA and for RIα only, respectively. Here, we generated mutant mice lacking the dual and RI domains of AKAP3. It was found that the deletion of these domains caused male mouse infertile, accompanied by mild defects in the fibrous sheath of sperm tails. Additionally, the levels of serine/threonine phosphorylation of PKA substrates and tyrosine phosphorylation decreased in the mutant sperm, which exhibited a defect in hyperactivation under capacitation conditions. The protein levels of PKA subunits remained unchanged. But, interestingly, the regulatory subunit RIα was mis-localized from principal piece to midpiece of sperm tail, whereas this was not observed for RIIα. Further protein–protein interaction assays revealed a preference for AKAP3 to bind RIα over RIIα. Collectively, our findings suggest that AKAP3 is important for sperm hyperactivity by regulating type-I PKA signaling pathway mediated protein phosphorylation via its dual and RI domains.
Background: Cerebral malaria (CM) is the most severe complication caused by Plasmodium falciparum infection. The pathophysiological changes caused by parasite virulent factors and human immune response to parasites contribute to CM. To date, very few parasite virulent proteins have been found to participate in CM. Identifying new parasite proteins involved in CM may provide new directions for possible intervention.Methods: Comparative genomic analysis was performed and parasite-infected erythrocyte specific protein 2 (PIESP2) was identified to be a CM-related candidate protein. The immunogenic property of PIESP2 was investigated. Its expression and localization in infected red blood cells (IRBCs) during intraerythrocytic stage were characterized. Adhesion assay and RNA-seq were adopted to evaluate the role of PIESP2 in the development of CM.Findings: PIESP2 is an immunogenic protein. Its expression begins at the early trophozoite stage and progressively increases with parasite development. PIESP2 mainly resides inside IRBCs but few of them are present on IRBC surface at the late trophozoite stage. Blockage of PIESP2 by antiserum apparently inhibited the adhesion of IRBCs to brain microvascular endothelial cells (BMECs). Moreover, PIESP2 could directly bind to BMECs, subsequently leading to an increase in the expression of genes involved in inflammatory response in BMECs and a decrease in the expression of genes related to anchoring junction.Interpretation: The P. falciparum specific protein PIESP2 may contribute to CM by mediating the sequestration of IRBCs to BMECs, inducing inflammatory response and impairing the integrity of the blood–brain barrier.Funding Statement: This work was supported by a grant from the National Natural Science Foundation of China (Grant No. 31600615).Declaration of Interests: The authors declare that they have no conflict of interest.Ethics Approval Statement: Ethical approval was given by the First Affiliated Hospital Ethics Committee of Fourth Military Medical University with reference number KY20163289-1. All experimental procedures were performed in accordance with relevant institutional and national guidelines and approved by the Institutional Animal Care and Use Committee of Fourth Military Medical University.
Mammalian spermatogenesis is regulated by coordinated gene expression in a spatiotemporal manner. The spatiotemporal regulation of major sperm proteins plays important roles during normal development of the male gamete, of which the underlying molecular mechanisms are poorly understood. A-kinase anchoring protein 3 (AKAP3) is one of the major components of the fibrous sheath of the sperm tail that is formed during spermiogenesis. In the present study, we analyzed the expression of sperm-specific Akap3 and the potential regulatory factors of its protein synthesis during mouse spermiogenesis. Results showed that the transcription of Akap3 precedes its protein synthesis by about 2 wk. Nascent AKAP3 was found to form protein complex with PKA and RNA binding proteins (RBPs), including PIWIL1, PABPC1, and NONO, as revealed by coimmunoprecipitation and protein mass spectrometry. RNA electrophoretic gel mobility shift assay showed that these RBPs bind sperm-specific mRNAs, of which proteins are synthesized during the elongating stage of spermiogenesis. Biochemical and cell biological experiments demonstrated that PIWIL1, PABPC1, and NONO interact with each other and colocalize in spermatids' RNA granule, the chromatoid body. In addition, NONO was found in extracytoplasmic granules in round spermatids, whereas PIWIL1 and PABPC1 were diffusely localized in cytoplasm of elongating spermatids, indicating their participation at different steps of mRNA metabolism during spermatogenesis. Interestingly, type I PKA subunits colocalize with PIWIL1 and PABPC1 in the cytoplasm of elongating spermatids and cosediment with the RBPs in polysomal fractions on sucrose gradients. Further biochemical analyses revealed that activation of PKA positively regulates AKAP3 protein synthesis without changing its mRNA level in elongating spermatids. Taken together, these results indicate that PKA signaling directly participates in the regulation of protein translation in postmeiotic male germ cells, underscoring molecular mechanisms that regulate protein synthesis during mouse spermiogenesis.
Abstract Objectives Mammalian spermatogenesis is a biological process of male gamete formation. Gonocytes are the only precursors of spermatogonial stem cells (SSCs) which develop into mature spermatozoa. DDX5 is one of DEAD‐box RNA helicases and expresses in male germ cells, suggesting that Ddx5 plays important functions during spermatogenesis. Here, we explore the functions of Ddx5 in regulating the specification of gonocytes. Materials and Methods Germ cell‐specific Ddx5 knockout ( Ddx5 ‐/‐ ) mice were generated. The morphology of testes and epididymides and fertility in both wild‐type and Ddx5 ‐/‐ mice were analysed. Single‐cell RNA sequencing (scRNA‐seq) was used to profile the transcriptome in testes from wild‐type and Ddx5 ‐/‐ mice at postnatal day (P) 2. Dysregulated genes were validated by single‐cell qRT‐PCR and immunofluorescent staining. Results In male mice, Ddx5 was expressed in germ cells at different stages of development. Germ cell‐specific Ddx5 knockout adult male mice were sterile due to completely devoid of germ cells. Male germ cells gradually disappeared in Ddx5 ‐/‐ mice from E18.5 to P6. Single‐cell transcriptome analysis showed that genes involved in cell cycle and glial cell line‐derived neurotrophic factor (GDNF) pathway were significantly decreased in Ddx5 ‐deficient gonocytes. Notably, Ddx5 ablation impeded the proliferation of gonocytes. Conclusions Our study reveals the critical roles of Ddx5 in fate determination of gonocytes, offering a novel insight into the pathogenesis of male sterility.
Deafness-dystonia-optic neuronopathy (DDON) syndrome, a condition that predominantly affects males, is caused by mutations in translocase of mitochondrial inner membrane 8A (TIMM8A)/deafness dystonia protein 1 (DDP1) gene and characterized by progressive deafness coupled with other neurological abnormalities. In a previous study, we demonstrated the phenotype of male mice carrying the hemizygous mutation of Timm8a1-I23fs49X. In a follow-up to that study, this study aimed to observe the behavioral changes in the female mutant (MUT) mice with homologous mutation of Timm8a1 and to elucidate the underlying mechanism for the behavioral changes.Histological analysis, transmission electron microscopy (EM), Western blotting, hearing measurement by auditory brainstem response (ABR), and behavioral observation were compared between the MUT mice and wild-type (WT) littermates.The weight of the female MUT mice was less than that of the WT mice. Among MUT mice, both male and female mice showed hearing impairment, anxiety-like behavior by the elevated plus maze test, and cognitive deficit by the Morris water maze test. Furthermore, the female MUT mice exhibited coordination problems in the balance beam test. Although the general neuronal loss was not found in the hippocampus of the MUT genotype, EM assessment indicated that the mitochondrial size showing as aspect ratio and form factor in the hippocampus of the MUT strain was significantly reduced compared to that in the WT genotype. More importantly, this phenomenon was correlated with the upregulation of translation of mitochondrial fission process protein 1(Mtfp1)/mitochondrial 18 kDa protein (Mtp18), a key fission factor that is a positive regulator of mitochondrial fission and mitochondrial size. Interestingly, significant reductions in the size of the uterus and ovaries were noted in the female MUT mice, which contributed to significantly lower fertility in the MUT mice.Together, a homologous mutation in the Timm8a1 gene caused the hearing impairment and psychiatric behavioral changes in the MUT mice; the latter phenotype might be related to a reduction in mitochondrial size regulated by MTP18.
ABSTRACT Objective Autoimmune encephalitis (AE) is a large category disorder urging antibody characterization. The aim was to identify a novel AE related autoantibody targeting an intracellular synaptic protein. Methods Suspected AE patients had negative conventional antibodies screening but strong immunolabel signals on rat brain sections with the serum and cerebrospinal fluid (CSF) samples were considered burdening unkown antibody. Immunoprecipitation from the rat brain protein lysate followed by mass spectrometry analysis was used to identify the targeting antigen. Western blotting and/or cell-based assay (CBA) with antigen-overexpressing HEK293T cells were used for antibody specificity, epitope and IgG subtype determination. Patients with similar immunostaining pattern on rat brain sections were retrospectively screened for the antibody. Results The antibody against collapsin response mediator protein 2 (CRMP2), a synaptic protein involved in axon guidance, was identified in a patient with suspected AE. The patient samples reactivated with HEK293T cell overexpressed CRMP2, rather than CRMP1, 3, 4, and 5. The patient samples mainly stained neuronal cytoplasm of the cortex, hippocampus and cerebellum Purkinje cells. This reactivity was eliminated by pre-immunoabsorption with CRMP2-overexpressing HEK293T cells. CRMP2 truncation experiments indicated that 536 amino acids at C-terminus was necessary for the epitope. Subtype analysis showed that anti-CRMP2 antibody was IgG4. Moreover, screening from 19 suspected AE patients led to identification of anti-CRMP2 antibody in another patient with a diagnosis of encephalomyelitis. The two patients responded to immunotherapy. Conclusions This study discovered a novel anti-CRMP2 antibody associated with AE. Testing of the antibody might be promising for AE diagnosis and treatment.
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