A-kinase anchoring protein 12 (
AKAP12
) plays key roles in male germ cells and female ovarian granulosa cells, whereas its influence on livestock litter size remains unclear. Herein we detected the ...genetic variants of
AKAP12
gene and their effects on litter size as well as alternative splicing variants expression in Shaanbei white cashmere (SBWC) goats, aiming at exploring theoretical basis for goat molecular breeding. We identified two Insertion/deletions (Indels) (7- and 13-bp) within the
AKAP12
gene. Statistical analyses demonstrated that the 13-bp indel mutation in the 3′ UTR was significantly associated with litter size (
n
= 1,019), and the carriers with DD genotypes presented lower litter sizes compared with other carriers (
P
< 0.01). Bioinformatics analysis predicted that this 13-bp deletion sequence could bind to the seed region of miR-181, which has been documented to suppress porcine reproductive and respiratory syndrome virus (PRRSV) infection by targeting PRRSV receptor CD163 and affect the pig litter size. Therefore, luciferase assay for this 13-bp indel binding with miRNA-181 was performed, and the luciferase activity of pcDNA-miR-181-13bp-Deletion-allele vector was significantly lower than that of the pcDNA-miR-181-13bp-Insertion-allele vector (
P
< 0.05), suggesting the reduced binding capability with miR-181 in DD genotype. Given that alternative spliced variants and their expression considerably account for the Indel genetic effects on phenotypic traits, we therefore detected the expression of the alternative spliced variants in different tissues and identified that
AKAP12-AS2
exhibited the highest expression levels in testis tissues. Interestingly, the
AKAP12-AS2
expression levels of homozygote DD carriers were significantly lower than that of individuals with heterozygote ID, in both testis and ovarian tissues (
P
< 0.05), which is consistent with the effect of the 13-bp deletion on the reduced litter size. Taken together, our results here suggest that this 13-bp indel mutation within goat
AKAP12
might be utilized as a novel molecular marker for improving litter size in goat breeding.
Although many circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) have been discovered in adipocytes, their precise functions and molecular mechanisms remain poorly understood. Based on ...existing circRNA and lncRNA sequencing data of bovine adipocytes, we screened for the differential expression of circFLT1 and lncCCPG1 in preadipocytes and adipocytes and further analyzed their function and regulation during adipogenesis. The overexpression of circFLT1 and lncCCPG1 together facilitated adipocyte differentiation and suppressed proliferation. Computationally, the RNA hybrid showed that circFLT1 and lncCCPG1 had multiple potential binding sites with miR-93. Additionally, luciferase reporting experiments verified that circFLT1 and lncCCPG1 may interact with miR-93. We also demonstrated that overexpressed miR-93 effectively suppresses the expression of lncSLC30A9. Signaling pathway enrichment analysis, luciferase activity assay, and expression analysis revealed that lncSLC30A9 inhibits proliferation by inhibiting the expression of AKT protein and promotes differentiation by recruiting the FOS protein to the promoter of peroxisome proliferator-activated receptor gamma (PPARG). In sum, our results elucidate the regulatory mechanisms of circFLT1 and lncCCPG1 as miR-93 sponges in bovine adipocytes.
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Adipogenesis is a complex and delicate programmed regulatory process involving a series of non-coding RNAs. In this study, the authors elucidate the regulatory mechanisms of circFLT1 and lncCCPG1 as miR-93 sponges in bovine adipocytes.
Skeletal muscle is one of the three major muscle types in an organism and has key roles in the motor system, metabolism, and homeostasis. RNA-Seq analysis showed that novel lncRNA,
lncFAM200B
, was ...differentially expressed in embryonic, neonatal, and adult cattle skeletal muscles. The main aim of this study was to investigate the molecular and expression characteristics of
lncFAM200B
along with its crucial genetic variations. Our results showed that bovine
lncFAM200B
was a 472 nucleotide (nt) non-coding RNA containing two exons. The transcription factor binding site prediction analysis found that
lncFAM200B
promoter region was enriched with SP1 transcription factor, which promotes the binding of myogenic regulatory factor MyoD and DNA sequence. The mRNA expression analysis showed that
lncFAM200B
was differentially expressed in embryonic, neonatal, adult bovine muscle tissues, and the
lncFAM200B
expression trend positively correlated with that of
MyoG
and
Myf5
in myoblast proliferation and differential stages. To identify the promoter active region of
lncFAM200B
, we constructed promoter luciferase reporter gene vector pGL3-Basic plasmids containing
lncFAM200B
promoter sequences and transfected them into 293T, C2C12, and 3T3-L1 cells. Our results suggested that
lncFAM200B
promoter active region was from −403 to −139 (264 nt) of its transcription start site, covering 6 SP1 potential binding sites. Furthermore, we found a novel C-T variation, named as SNP2 (ERZ990081 in European Variation Archive) in the promoter active region, which was linked to the nearby SNP1 (rs456951291 in Ensembl database). The genotypes of SNP1 and combined genotypes of SNP1 and SNP2 were significantly associated with Jinnan cattle hip height. The luciferase activity analysis found that the SNP1-SNP2 haplotype CC had the highest luciferase activity, which was consistent with the association analysis result that the combined genotype CC-CC carriers had the highest hip height in Jinnan cattle. In conclusion, our data showed that
lncFAM200B
is a positive regulator of muscle development and that SNP1 and SNP2 could be used as genetic markers for marker-assisted selection (MAS) breeding of beef cattle.
The precise functions and molecular mechanisms of microRNAs (miRNAs) in adipocytes are primarily unknown. Studies have demonstrated that miR-193b plays a pivotal role in the differentiation of ...preadipocytes. Herein, we evaluated the effects of bta-miR-193b on the growth and development of adipocytes, using the EdU cell proliferation method, flow cytometry analysis, CCK-8 assay, RT-qPCR, Western blotting, and oil red O staining. We observed that the overexpression of bta-miR-193b significantly affected the differentiation, proliferation, and apoptosis of adipocytes. The results of the dual-fluorescent reporter vector experiments demonstrated that bta-miR-193b directly targeted Acyl-CoA synthetase short-chain family member 2 (ACSS2). Additionally, the effects of ACSS2 overexpression on the proliferation and apoptosis in adipose cells were the opposite of those induced by bta-miR-193b. We also demonstrated that ACSS2 can significantly promote the expression of AKT and pAKT proteins. Therefore, this study presents a novel mechanism by which bta-miR-193b regulates adipocyte development by targeting ACSS2.
The A-kinase anchoring protein 12 gene (AKAP12) is a scaffold protein, which can target multiple signal transduction effectors, can promote mitosis and cytokinesis and plays an important role in the ...regulation of growth and development. In our previous study, P1–7 bp (intron 3) and P2–13 bp (3′UTR) indels within the AKAP12 gene significantly influenced AKAP12 gene expression. Therefore, this study aimed to identify the association between these two genetic variations and growth-related traits in Shaanbei white cashmere goats (SBWC) (n = 1405). Herein, we identified two non-linkage insertions/deletions (indels). Notably, we found that the P1–7 bp indel mutation was related to the height at hip cross (HHC; p < 0.05) and the P2–13 bp indel was associated with body weight, body length, chest depth, chest width, hip width, chest circumference and cannon (bone) circumference in SBWC goats (p < 0.05). Overall, the two indels’ mutations of AKAP12 affected growth traits in goats. Compared to the P1–7 bp indel, the P2–13 bp indel is more suitable for the breeding of goat growth traits.
CKLF like MARVEL transmembrane domain containing 2 (
CMTM2
) plays crucial roles in spermiogenesis, skeletogenous, growth, and development through PI3K/Akt and other pathways. The purpose of this ...study was to explore the expression profile and variation of different spliced
CMTM2
gene in Shaanbei white cashmere goats, as well as to find the relationships between a
CMTM2
promoter region 14 bp genetic variant and growth traits in 1366 Shaanbei white cashmere goats. In this study, we identified alternative
CMTM2
splicing and detected the effects of the spliced variants on mRNA expression levels in tissues. Meanwhile, an unreported spliced variant of
CMTM2
in goat was identified using in CDS cloning and RT-PCR, namely,
CMTM2-AS2
. Compared with the normal transcript (
CMTM2-AS1
), the novel variant had the higher expression level in muscle and liver tissues, indicating that it plays an effective role in growth traits. Furthermore, a 14 bp deletion was detected within
CMTM2
promoter region, and the different genotypes were significantly associated with growth traits (e.g., body length, circumference of cannon bone) in the large group of 1366 individuals in Shaanbei white cashmere goats. We found that the body length of the individuals with II (
n
= 571) genotype had better phenotypes than those with DD (
n
= 118) and ID (
n
= 650) genotypes. These results have direct guiding significance for goat breeding in the future and provide a new idea for studying the characteristics and functions of
CMTM2
gene in goats.
Circular RNA (circRNA) is a form of endogenous RNA that can regulate gene expression and participate in the regulation of myogenesis. However, the molecular mechanisms and potential roles of circRNAs ...in bovine muscle development remain largely unknown. Nevertheless, the RNA splicing factors regulating the biogenesis of bovine circRNA have not yet been characterized. In this study, we identified a novel circRNA, circMEF2D, formed by back-splicing of constitutive exons (exons 5-7) of the bovine MEF2D gene. Functional assays showed that circMEF2D inhibited the proliferation and differentiation of bovine myoblasts. Importantly, we showed that circMEF2D regulated the PI3K-AKT signaling pathway through direct and competitive binding to miR-486. Furthermore, to explore the formation mechanism of circMEF2D, we explored the MEF2D gene alternative splicing progress. Four alternative linear variants of MEF2D were found. Due to its role in alternative splicing, the RNA-binding protein HNRNPA1 was selected for further study and the modulation of HNRNPA1 levels showed that it negatively regulated both back-splicing and linear splicing of MEF2D gene. Overall, in addition to the characterization of bovine circRNAs, these findings revealed the crucial role of HNRNPA1 in MEF2D gene alternative splicing and demonstrated a regulatory circMEF2D-miR-486-PI3K-AKT axis.
The down syndrome cell adhesion molecule like 1 (DSCAML1), is associated with the development of the nervous system and neurologic diseases. Previous Genome-wide association studies have shown that ...it is associated with sperm morphology, suggesting it has a critical role in fecundity. In this study, expression profiles of goat DSCAML1 mRNA were analyzed. The results showed that its expression in the testis differ significantly between the mitotic stage and meiotic stage. Three insertion/deletion (indel) variants of goat DSCAML1 were determined in the Shaanbei White Cashmere Goat (SWCG, n = 2162). Based on the association analysis, two indels (P2-16bp, P14-15bp) were significantly related to sperm quality (sperm motility and sperm density) in male goat and three loci were markedly related to the first-birth litter size in female goat (P = 4.0 × 10−6; P = 1.0 × 10−6; P = 4.7 × 10−2). In male goats, the different genotypes of P2-16bp and P14-15bp revealed a noticeable effect on the expression of DSCAML1. Moreover, the effects observed in the first-birth litter followed a similar trend, which may provide the basis for further research of DSCAML1 gene function and marker assisted selection (MAS) programs to improve reproductive traits.
•Goat DSCAML1 mRNA in testis was significantly different in mitotic stage and meiotic stage.•Two indels were related to the sperm motility and density in male goats.•Three loci were related to the first-birth litter size in female goats.•Different genotypes of two indels can impact on DSCAML1 expression.
The proliferation and differentiation of preadipocytes is an important factor determining bovine fat development, which is closely related to the feed conversion ratio, carcass traits, and beef ...quality. The purpose of this study was to identify the effects of candidate circRNA and miRNA on the proliferation and differentiation of bovine preadipocytes in order to provide basic materials for molecular breeding in cattle. circRNA sequencing was performed on bovine adipocyte samples at different differentiation time points, and a total of 1830 differentially expressed circRNAs were identified. Among them, circBDP1, derived from the bovine BDP1 gene, has potential binding sites for miR-204 (known as a regulator of bovine fat development) and miR-181b, which gives us a hint that circBDP1 may regulate bovine fat development by adsorbing miR-204 and miR-181b. Here, our results revealed that circBDP1 overexpression promoted the proliferation and differentiation of bovine preadipocytes. The miRNA profile of bovine adipocytes at different differentiation time points was also analyzed using the small RNA sequencing method, and a total of 89 differentially expressed miRNAs were identified, including miR-204 and miR-181b. As expected, dual-luciferase reporter results showed that circBDP1 competitively adsorbed miR-181b and miR-204. Overexpression and interference of miR-181b in bovine preadipocytes and 3T3-L1 showed that miR-181b promoted the proliferation and differentiation of preadipocytes. Further results displayed that miR-181b and miR-204 simultaneously targeted the SIRT1 gene, and miR-204 also targeted the 3’ UTR region of the TRARG1 gene. In summary, this study found that miR-181b and miR-204 were involved in fat development by targeting SIRT1 and TRARG1. The results of this study will lay a foundation for the research of fat development and beef cattle industry.
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