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Dilated cardiomyopathy (DCM) is a heritable and genetically heterogenous disease often idiopathic and a leading cause of heart failure with high morbidity and mortality. DCM caused by RNA binding motif protein 20 (RBM20) mutations is diverse and needs a more complete mechanistic understanding. RBM20 mutation S637G (S639G in mice) is linked to severe DCM and early death in human patients. In this study, we generated a RBM20 S639G mutation knock-in (KI) mouse model to validate the function of S639G mutation and examine the underlying mechanisms. KI mice exhibited severe DCM and premature death with a ~ 50% mortality in two months old homozygous (HM) mice. KI mice had enlarged atria and increased ANP and BNP biomarkers. The S639G mutation promoted RBM20 trafficking and ribonucleoprotein (RNP) granules in the sarcoplasm. RNA Seq data revealed differentially expressed and spliced genes were associated with arrhythmia, cardiomyopathy, and sudden death. KI mice also showed a reduction of diastolic stiffness and impaired contractility at both the left ventricular (LV) chamber and cardiomyocyte levels. Our results indicate that the RBM20 S639G mutation leads to RNP granules causing severe heart failure and early death and this finding strengthens the novel concept that RBM20 cardiomyopathy is a RNP granule disease.

Backfat trait is an important economic trait and highly heritable, but difficult to evaluate. Thus, it is of great significance to explore optimal backfat thickness of pigs by using marker-assisted selection (MAS) to speed up its breeding process and improve economic efficiency. This study aimed to investigate the relationship between genetic variations (e.g., SSRs) and backfat of Qinghai Bamei pigs using MALDI-TOF Mass Spectrometry (MALDI-TOF-MS). Herein, five alternative SSR loci (namely V1, V2, V3, V4 and V5) were selected for subsequent detection. The results suggested that 3 (141-, 143- and 145-), 3 (128-, 130- and 132-), 2 (160- and 162-), 2 (136- and 139-) and 3 (170-, 184- and 192-) alleles of V1, V2, V3, V4 and V5 were found, respectively. Subsequent analysis showed that there was linkage equilibrium among five SSRs and Hap19 (13.1%) (141-/132-/160-/139-/192-) had the highest haplotype frequency. Among these five SSR loci, V1, V2 and V3 loci were significantly associated to the backfat of Qinghai Bamei sows. These findings enriched the study of SSRs in Qinghai Bamei pigs, and (AC)

Label-free quantitative proteomics identifies molecular correlates of dilated cardiomyopathy in rats lacking the muscle-specific splicing factor Rbm20.

(1) Background: RNA binding motif 20 (RBM20) regulates mRNA splicing specifically in muscle tissues. Missense mutations in the arginine/serine (RS) domain of RBM20 lead to abnormal gene splicing and have been linked to severe dilated cardiomyopathy (DCM) in human patients and animal models. Interestingly, many of the reported DCM-linked missense mutations in RBM20 are in a highly conserved RSRSP stretch within the RS domain. Recently, it was found that the two Ser residues within this stretch are constitutively phosphorylated, yet the identity of the kinase(s) responsible for phosphorylating these residues, as well as the function of RSRSP phosphorylation, remains unknown. (2) Methods: The ability of three known SR protein kinases (SRPK1, CLK1, and AKT2) to phosphorylate the RBM20 RSRSP stretch and regulate target gene splicing was evaluated by using both in vitro and in vivo approaches. (3) Results: We found that all three kinases phosphorylated S638 and S640 in the RSRSP stretch and regulated RBM20 target gene splicing. While SRPK1 and CLK1 were both capable of directly phosphorylating the RS domain in RBM20, whether AKT2-mediated control of the RS domain phosphorylation is direct or indirect could not be determined. (4) Conclusions: Our results indicate that SR protein kinases regulate the splicing of a cardiomyopathy-relevant gene by modulating phosphorylation of the RSRSP stretch in RBM20. These findings suggest that SR protein kinases may be potential targets for the treatment of RBM20 cardiomyopathy.

Patients with missense mutations in RNA binding motif protein 20 (RBM20), an RNA binding protein and splicing factor, develop a clinically aggressive form of dilated cardiomyopathy (DCM). Recent studies revealed that DCM-linked RBM20 missense mutations promote re-localization of the protein from the nucleus to the cytoplasm where it forms ribonucleoprotein (RNP) granules—non-membrane bound assemblies formed from RNAs and RNA regulatory proteins. The sequestration of RNAs and RNA regulatory proteins in these granules has the potential to dramatically alter the landscape of protein expression in the cardiomyocyte; yet, how RBM20 missense mutations impact global gene expression at the protein level has not been explored. Herein, we utilized mass spectrometry (MS)-based proteomics to identify global changes in protein expression in cardiac tissue harvested from mutation knock-in (KI) mice harboring the DCM-linked S637A and S639G RBM20 mutations. Our analysis revealed a total of 212 and 190 differentially expressed proteins (DEPs) in S637A and S639G KI mice, respectively, relative to wild-type. In general, identified DEPs were similar between KI mice with 81 and 38 proteins commonly up- and down-regulated, respectively. Consistent with mutant RBM20-mediated granule formation, 15 and 12 of the up-regulated proteins in S637A and S639G KI mice, respectively, have previously been implicated in granule assembly. Other up-regulated proteins in KI mice included chaperones and cytoskeletal proteins, as well as proteins involved in RNA metabolism and metabolite interconversion. Interestingly, proteins involved in the regulation of metabolite interconversion were also the most common protein class down-regulated in both KI mice, suggesting that broad changes in cellular metabolism may be a common feature associated with the RBM20 S637A and S639G missense mutations. Additionally, our proteomics analysis confirmed the differential expression of approximately 20-30% of previously identified differentially expressed genes based on RNAseq analyses from S637A and S639G KI mice. Our results provide the first glimpse of global changes in gene expression at the protein level associated with the RBM20 S637A and S639G missense mutations.

DNA methyltransferase 3β (DNMT3B) is a gene encoding a de novo methylation enzyme that is required for DNA methylation during mammalian embryo development. Previous genome-wide association analysis suggested DNMT3B is a candidate gene for goat fertility, but there is no study on the effect of DNMT3B on litter size in goat. The aim of this study was to identify possible insertion/deletion (indel) mutations associated with litter size. Seven putative indels were designed to study their association with litter size, but just one 11-bp insertion variant of intron 22 (the last intron) was found in healthy female Shaanbei white cashmere goats (SBWC goats) (n = 1534). Statistical analysis showed that the 11-bp insertion was related to the first-born litter size (P 0.01) and the goats with the deletion/deletion genotype had a higher average first-born litter size (P 0.01). In addition, the expression profile of the DNMT3B mRNA in goat was detected, which revealed significant differences in DNMT3B mRNA expression in the gonads. Additionally, the results of western blotting revealed that the ovaries of mothers of multi-lamb (MML) had a higher level of DNMT3B protein than the ovaries of mothers of single-lamb (MSL). Furthermore, the mRNA of DNMT3B was widely expressed in male goats. Differences in mRNA expression levels were observed in the ovaries of MSL and MML. These findings indicated that the 11-bp indel in DNMT3B was significantly associated with first-born litter size, which can be used for marker-assisted selection (MAS) of goats for breeding.

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 (Rbm(20S637A)) 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.

Gain-of-function mutations of a gene result in new molecular function. Genetic studies show that mutations in a gene named as RNA binding motif 20 (RBM20) are associated with dilated cardiomyopathy (DCM). To test whether Rbm20 mutations are responsible for cardiac remodelling and potential underlying mechanisms, we selected two mutations (S637A and S639G) located in arginine/serine-rich domain of RBM20 and generated two knock-in mouse models ( Rbm20 S637A and Rbm20 S639G ) respectively. Both mouse lines were subjected to histological and anatomical analysis as well as cardiac function assessment with echocardiography. The results revealed that heterozygous (HT) and homozygous (HM) male and female mice from both lines displayed DCM phenotype with enlarged left ventricular (LV) chamber, thinner LV walls and reduced ejection fraction. Interestingly, we observed that Rbm20 S639G mice manifested more severe phenotype than that in Rbm20 S637A mice. Rbm20 S639G HM mice exhibited higher mortality rate (~50%) than that (~34%) in Rbm20 S637A HM mice at an early age (TTN and calcium handling genes. Force measurements and Ca 2+ transient evaluation of intact myocytes showed that the reduced cellular diastolic stiffness and impaired contractility in HT and HM mice. Studies suggested that phosphorylation of RS domain is critical for protein trafficking. We then performed mass spectrometry on the in vitro expressed RBM20 and identified phosphorylation on S637 and S639. Phosphomimetic assay showed that phosphorylation was not responsible for RBM20 trafficking and condensates. Together, we reported a new mechanism by which cardiac genetic mutations causes protein trafficking and aggregation in cardiac remodelling.

RNA binding motif 20 (RBM20) cardiomyopathy has been detected in approximately 3% of populations afflicted with dilated cardiomyopathy (DCM). It is well conceived that RBM20 cardiomyopathy is provoked by titin isoform switching in combination with resting Ca2+ leaking. In this study, we characterized the cardiac function in Rbm20 knockout (KO) rats at 3‐, 6‐, 9‐, and 12‐months of age and examined the effect of the ryanodine receptor stabilizer S107 on resting intracellular levels and cardiomyocyte contractile properties. Our results revealed that even though Rbm20 depletion promoted expression of larger titin isoform and reduced myocardial stiffness in young rats (3 months of age), the established DCM phenotype required more time to embellish. S107 restored elevated intracellular Ca2+ to normal levels and ameliorated cardiomyocyte contractile properties in isolated cardiomyocytes from 6‐month‐old Rbm20 KO rats. However, S107 failed to preserve cardiac homeostasis in Rbm20 KO rats at 12 months of age, unexpectedly, likely due to the existence of multiple pathogenic mechanisms. Taken together, our data suggest the therapeutic promises of S107 in the management of RBM20 cardiomyopathy., Loss of Rbm20 results in reduced myocardial stiffness at younger age and overt RBM20 cardiomyopathy at older age in rats. The RyR2 stabilizer S107 improves contractility properties by restoring the elevated resting intracellular Ca2+ level in Rbm20‐depleted cardiomyocytes. S107 could be a component of a therapeutic option for RBM20 cardiomyopathy.

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.

Pre-mRNA splicing plays an important role in muscle function and diseases. The RNA binding motif 20 (RBM20) is a splicing factor that is predominantly expressed in muscle tissues and primarily regulates pre-mRNA splicing of Ttn, encoding a giant muscle protein titin that is responsible for muscle function and diseases. RBM20-mediated Ttn splicing has been mostly studied in heart muscle, but not in skeletal muscle. In this study, we investigated splicing specificity in different muscle types in Rbm20 knockout rats and hormonal effects on RBM20-mediated splicing both in cellulo and in vivo studies. The results revealed that RBM20 is differentially expressed across muscles and RBM20-mediated splicing is muscle-type specific. In the presence of RBM20, Ttn splicing responds to hormones in a muscle-type dependent manner, while in the absence of RBM20, Ttn splicing is not affected by hormones. In differentiated and undifferentiated C2C12 cells, RBM20-mediated splicing in response to hormonal effects is mainly through genomic signaling pathway. The knowledge gained from this study may help further understand muscle-specific gene splicing in response to hormone stimuli in different muscle types.

The body status of livestock affects their physiological function and productive performances. Microsatellites, one of the most used DNA markers, have been found to be associated with pig productive traits. However, their identifications and effects on body measurement traits of the Chinese Qinghai Bamei pig still uncovered. According to our previous sequencing data, in this study, three novel microsatellites were found in this breed. Using time of flight-mass spectrometry (TOF-MS) method, these microsatellites were further identified in a large Bamei pig population. TOF-MS spectra showed that there are three microsatellites loci, named P1, P2 and P3. These microsatellites were linkage equilibrium based on the values of D' and

Molecular and cellular mechanisms of mutations of splicing factors in heart function are not well understood. The splicing of precursor mRNA is dependent on an essential group of splicing factors containing serine-arginine (SR) domain(s) that are critical for protein-RNA and protein-protein interaction in the spliceosome assembly. Phosphorylation of SR domains plays a key role in splicing control and the distribution of splicing factors in the cell. RNA binding motif 20 (RBM20) is a splicing factor predominantly expressed in muscle tissues with the highest expression level in the heart. However, its phosphorylation status is completely unknown up-to-date. In this study, we identified sixteen amino acid residues that are phosphorylated by middle-down mass spectrometry. Four of them are located in the SR domain, and two out of the four residues, S638 and S640, play an essential role in splicing control and facilitate RBM20 shuttling from the nucleus to the cytoplasm. Re-localization of RBM20 promotes protein aggregation in the cytoplasm. We have also verified that SR-protein kinases (SRPKs), cdc2-like kinases (CLKs) and protein kinase B (PKB or AKT) phosphorylate S638 and S640. Mutations of S638A and S640G reduce RBM20 phosphorylation and disrupt the splicing. Taken together, we determine the phosphorylation status of RBM20 and provide the first evidence that phosphorylation on SR domain is crucial for pre-mRNA splicing and protein trafficking. Our findings reveal a new role of RBM20 via protein shuttling in cardiac function.

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 steroid-hormone-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 CPF-induced toxicity in testicular cells and a theoretical basis for future treatment of male infertility.

Lysine specific demethylase 5B gene (KDM5B, also known as JARID1B or PLU-1), encoding an enzyme of the lysine-specific histone demethylase family, has been reported to regulate androgen receptor transcriptional activity and male reproduction. To fully study the expression characteristics and genetic effects of pig KDM5B gene, the objective of this study was to investigate the mRNA expression profiles of KDM5B among different tissues and testicular cells (spermatogonia stem cells, SSCs; sertoli cells, SCs; leydig cells, LCs), as well as to explore the insertion/deletion (indel) variations of this gene. Expression analysis results revealed that the KDM5B gene was highly expressed in testis than other tissues in 7-day-old piglet (P 0.01). In particular, this gene was highest expressed in testis of adult boar (P 0.01). Furthermore, the KDM5B mRNA expression level in SSCs was significantly higher than those in LCs and SCs (P 0.01). Besides, a 35-bp indel was first verified in the intron 11 of pig KDM5B gene, and the animals with insertion/insertion genotype exhibited superior testicular weight and testicular short perimeter than those with other genotypes (P 0.05) in 40-day-old Landrace pigs. Together, these findings suggest that KDM5B plays a crucial role in male fertility and the 35-bp indel can be used for marker-assisted selection of boar.

The casein alpha s1 (CSN1S1) gene encodes &alpha, s1 casein, one of the proteins constituting milk, which affects milk performance, as well as improving the absorption of calcium and bone development in mammals. A previous study found that an 11-bp insertion/deletion (indel) of this gene strongly affected litter size in goats. However, to our knowledge, the relationships between this polymorphism and the milk performance and body measurement traits of goats have not been reported. In this paper, the previously identified indel has been recognized in three Chinese goat breeds, namely the Guanzhong dairy goat (GZDG, n = 235), Shaanbei white cashmere goat (SBWC, n = 1092), and Hainan black goat (HNBG, n = 278), and the following three genotypes have been studied for all of the breeds: insertion/insertion (II), deletion/deletion (DD), and insertion/deletion (ID). The allele frequencies analyzed signified that the frequencies of the &ldquo, D&rdquo, allele were higher (47.8%&ndash, 65.5%), similar to the previous report, which indicates that this polymorphism is genetically stable in different goat breeds. Further analysis showed that this indel was markedly associated with milk fat content, total solids content, solids-not-fat content, freezing point depression, and acidity in GZDG (p &lt, 0.05), and also affected different body measurement traits in all three breeds (p &lt, 0.05). The goats with II genotypes had superior milk performance, compared with the others, however, goats with DD genotypes had better body measurement sizes. Hence, it may be necessary to select goats with an II or DD genotype, based on the desired traits, while breeding. Our study provides information on the potential impact of the 11-bp indel polymorphism of the CSN1S1 gene for improving the milk performance and body measurement traits in goats.

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.

In mammals, POU1F1 is a key transcription factor that directly affects the secretion of pituitary hormones (GH, PRL, and TSH) and maintains growth and development. The aim of this study was to identify novel single nucleotide polymorphisms (SNPs) of the POU1F1 gene and evaluate the relations to litter size and growth performance in Shaanbei white cashmere (SBWC) goats. The ear tissues of female goats (n = 653) were collected, and the corresponding data were recorded. From direct DNA sequencing, a missense mutation (NC_030808.1:g.34236169A C) was first detected in exon 6 of POU1F1 in SBWC goats that transformed the amino acid leucine into valine (L280V). Further analyses showed that the number of lambs of female goats with the TT genotype was significantly greater than that of female goats with the TG genotype (P 0.01). In addition, a Chi-square test showed that the distributions of the two genotypes were remarkably different in the litters of two different sizes (P = 2.06E-06). Simultaneously, the frequency of the TT genotype for female goats with multiple lambs (≥2) was much greater than that of the TG genotype. When the relationships between growth traits and L280V were evaluated, the individuals with the TT genotype had superior growth traits compared with those of the TG genotype (P 0.05), including for body height, body length, chest circumference, chest depth, chest width, height at hip cross, and cannon circumference. Notably, a positive relationship indicated that TT genotype female goats with improved growth traits tended to give birth to more lambs. We hypothesized that the mutation was in linkage disequilibrium with other responsible SNPs or affected the post-transcriptional regulation levels and then affected growth and litter size. This novel SNP may provide a functional genetic marker for improving economically valuable traits in goat breeding.

Steroidogenic acute regulatory protein (StAR), primarily expressed in Leydig cells (LCs) in the mammalian testes, is essential for testosterone biosynthesis and male fertility. However, no previous reports have explored the expression profiles, alternative splicing and genetic variations of StAR gene in pig. The aim of current study was to explore the expression profiles in different tissues and different types of testicular cells (LCs; spermatogonial stem cells, SSCs; Sertoli cells, SCs), to identify different splice variants and their expression levels, as well as to detect the indel polymorphism in pig StAR gene. Expression analysis results revealed that StAR was widely expressed in all tested tissues and the expression level in testis was significantly higher than that in other tissues (P 0.01); among different types of testicular cells, the StAR mRNA expression level was significantly higher in LCs than others (P 0.05). Furthermore, three splice variants, StAR-a, StAR-b and StAR-c, were first found in pig. Further study showed StAR-a was highly expressed in both testis and LCs when compared with other variants (P 0.01), suggesting StAR-a was the primary variant at StAR gene post-transcription and may facilitate the combination and transportation of cholesterol with StAR. In addition, a 5-bp duplicated deletion (NC_010457.5:g.5524-5528 delACTTG) was verified in the porcine StAR gene, which was closely related to male testicular morphology traits (P 0.05), and we speculated that the allele "D" of StAR gene might be a positive allele. Briefly, the current findings suggest that StAR and StAR-a play imperative roles in male fertility and the 5-bp indel can be a potential DNA marker for the marker-assisted selection in boar.