Arginine–serine (RS) domain(s) in splicing factors are critical for protein–protein interaction in pre‐mRNA splicing. Phosphorylation of RS domain is important for splicing control and nucleocytoplasmic transport in the cell. RNA‐binding motif 20 (RBM20) is a splicing factor primarily expressed in the heart. A previous study using phospho‐antibody against RS domain showed that RS domain can be phosphorylated. However, its actual phosphorylation sites and function have not been characterized. Using middle‐down mass spectrometry, we identified 16 phosphorylation sites, two of which (S638 and S640 in rats, or S637 and S639 in mice) were located in the RSRSP stretch in the RS domain. Mutations on S638 and S640 regulated splicing, promoted nucleocytoplasmic transport and protein‐RNA condensates. Phosphomimetic mutations on S638 and S640 indicated that phosphorylation was not the major cause for RBM20 nucleocytoplasmic transport and condensation in vitro. We generated a S637A knock‐in (KI) mouse model (Rbm20S637A) and observed the reduced RBM20 phosphorylation. The KI mice exhibited aberrant gene splicing, protein condensates, and a dilated cardiomyopathy (DCM)‐like phenotype. Transcriptomic profiling demonstrated that KI mice had altered expression and splicing of genes involving cardiac dysfunction, protein localization, and condensation. Our in vitro data showed that phosphorylation was not a direct cause for nucleocytoplasmic transport and protein condensation. Subsequently, the in vivo results reveal that RBM20 mutations led to cardiac pathogenesis. However, the role of phosphorylation in vivo needs further investigation.
Chlorpyrifos (CPF) is the most frequently applied insecticide. Aside from effects on the neuronal cholinergic system, previous studies suggested a potential relationship between CPF exposure and male infertility; however, the molecular mechanism remains elusive. The aim of this study was to investigate the toxic effect of CPF on testicular cells and the potential mechanism via in vitro and in vivo experiments. The cytotoxic effects of CPF on mouse-derived spermatogonial cell lines (GC-1), Sertoli cell lines (TM4) and Leydig cell lines (TM3) were assessed by a CCK-8 assay, flow cytometry, a TUNEL assay, quantitative RT-PCR, and Western blotting. Exposure to CPF (10−50 μM) for 12 or 24 h resulted in significant death in all three testicular cell lines. The number of TUNEL-positive apoptotic cells were dose-dependent and increased with raised CPF concentrations. Further investigation indicated that CPF induced cell-cycle arrest and then promoted cell apoptosis. Additionally, CPF increased reactive-oxygen-species (ROS) production and lipid peroxidation (MDA) and reduced mitochondrial-membrane potential. The mechanism of cell apoptosis induced by CPF involved an increase in phosphorylated-AMP-activated-protein-kinase (p-AMPK) levels in the tested cells. In vivo, the expression of steroidhormone-biosynthesis-related genes in testis, spleen, and lung in F0 and F1 mice were downregulated when there was intraperitoneal injection or dietary supplementation of CPF. This study provides a potential molecular mechanism of CPFinduced toxicity in testicular cells and a theoretical basis for future treatment of male infertility.
POU (Pit-Oct-Unc) class 1 homeobox 1 (POU1F1, or Pit-1) is a transcription factor that directly regulates pituitary hormone-related genes, as well as affects the reproduction and growth in mammals. Thus, POU1F1 gene was investigated as a candidate gene for litter size and growth performance in goats. In the current study, using direct DNA sequencing, c.682G > T, c.723T > G and c.837T > C loci were genotyped in Shaanbei white cashmere (SBWC) goats (n = 609), but c.876 + 110T > C was monomorphic. Besides, the c.682G > T locus was first identified by HinfI (Haemophilus influenzae Rf) restriction endonuclease. Association analysis results showed that the c.682G > T, c.837T > C loci and diplotypes were significantly associated with goat litter size (p < 0.05). The positive genotypes were GT and TT for the two SNPs, respectively, and the optimal diplotype was H3H7 (GTTT-TTTT). On the other hand, the c.682G > T, c.723T > G and c.837T > C strongly affected growth traits and body measurement indexes in SBWC goats (p < 0.05). The positive genotypes or allele of these SNPs were GT, G and TT, respectively. Additionally, the goats with H3H7 diplotype also had a greater growth status than others (p < 0.05). Here, individuals with same genotype had both a better litter size and growth traits, showing a positive correlation between these economic traits. Meanwhile, the positive genotypes of four SNPs were combined to obtain the optimal diplotype, which was also H3H7. These SNPs, especially the diplotype, could be used for the genomic selection of excellent individuals with a greater litter size and better growth status in goat breeding.
Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop a clinically aggressive dilated cardiomyopathy (DCM). RBM20 is a splicing factor with two canonical domains, an RNA recognition motif (RRM) and an arginine-serine rich (RS) domain. RRM loss-of-function disrupts the splicing of RBM20 target transcripts and leads to systolic dysfunction without overt DCM, while mutations in the RS domain precipitate DCM. We show that mice lacking the RS domain (Rbm20deltaRS) manifest DCM with mis-splicing of RBM20 target transcripts. We found that RBM20 is mis-localized in Rbm20ΔRS mice but not in mice lacking the RRM, which are also deficient in RBM20 splicing. We determine that the RS domain, not other domains including the RRM, is critical for RBM20 nuclear import and define the core nuclear localization signal (NLS) within this domain. Mutation analysis of phosphorylation sites within the RS domain indicate that phosphorylation is dispensable for RBM20 nuclear import. Collectively, our findings establish disruption of the NLS in RBM20 as a causative mechanism in DCM through nucleocytoplasmic transport.
Abstract. Cell division cycle 25A (CDC25A), a member of the CDC25 family of phosphatases, is
required for progression from G1 to the S phase of the cell cycle. CDC25A provides an essential function during early embryonic development in mice,
suggesting that it plays an important role in growth and development. In
this study, we used mathematical expectation (ME) methods to identify a
20-bp insertion/deletion (indel) polymorphism of CDC25A gene in Shaanbei White
Cashmere (SBWC) goats. We also investigated the association between this
20-bp indel and growth-related traits in SBWC goats. Association results
showed that the indel was related to growth traits (height at hip cross,
cannon circumference, and cannon circumference index) in SBWC goats. The
height at hip cross of individuals with insertion/insertion (II) genotype
was higher than those with insertion/deletion (ID) genotype (P=0.02); on
the contrary, the cannon circumference and cannon circumference index of
individuals with ID genotype were superior when compared with those with II
genotype (P=0.017 and P=0.009). These findings suggest that the 20-bp
indel in the CDC25A gene significantly affects growth-related traits, and could be
utilized as a candidate marker for marker-assisted selection (MAS) in
the cashmere goat industry.
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