m6A-modified cenRNA stabilizes CENPA to ensure centromere integrity in cancer cells.

m6A modification is best known for its critical role in controlling multiple post-transcriptional processes of the mRNAs. Here, we discovered elevated levels of m6A modification on centromeric RNA (cenRNA) in cancerous cells compared with non-cancerous cells. We then identified CENPA, an H3 variant, as an m6A reader of cenRNA. CENPA is localized at centromeres and is essential in preserving centromere integrity and function during mitosis. The m6A-modified cenRNA stabilizes centromeric localization of CENPA in cancer cells during the S phase of the cell cycle. Mutations of CENPA at the Leu61 and the Arg63 or removal of cenRNA m6A modification lead to loss of centromere-bound CENPA during S phase. This in turn results in compromised centromere integrity and abnormal chromosome separation and hinders cancer cell proliferation and tumor growth. Our findings unveil an m6A reading mechanism by CENPA that epigenetically governs centromere integrity in cancer cells, providing potential targets for cancer therapy.

Identification of novel alternative splicing of bovine lncRNA lncFAM200B and its effects on preadipocyte proliferation.

Adipogenesis is closely related to human health, livestock growth, and meat quality. A previous study identified that bovine lncFAM200B promoter has high activity in 3T3-L1 mice preadipocytes. Thus, lncFAM200B was a candidate gene for regulating adipogenesis. This study aimed to uncover the role of lncFAM200B in bovine adipogenesis and identify novel genetic variations within the bovine lncFAM200B gene. An expression analysis found that lncFAM200B was expressed higher in fat than that in muscle, but the difference was not related to the total methylation level of the promoter active region. Moreover, the expression of lncFAM200B exhibited a significant positive correlation with the expression of C/EBPa during bovine adipocyte differentiation. To uncover the function of lncFAM200B, the full-length lncFAM200B was cloned, and four kinds of transcript variants were found. Protein-coding potential prediction and prokaryotic expression system analysis showed that these four transcript variants were noncoding RNAs. The quantitative reverse-transcription polymerase chain reaction and 5-ethynyl-2'-deoxyuridine assay showed that the transcript variants decreased the messenger RNA expression of Cyclin D1 and inhibited the proliferation of bovine preadipocytes. Considering the important role of lncFAM200B in adipogenesis, we identified genetic variations in lncFAM200B. Three single-nucleotide polymorphisms (SNPs) were revealed, and two of them (SNP1 and SNP3) were associated with Nanyang cattle body measurement traits. In conclusion, this study found that bovine lncFAM200B inhibited preadipocyte proliferation, and two genetic variations of lncFAM200B could be used in cattle breeding.

Detection of insertions/deletions (InDels) within the goat Runx2 gene and their association with litter size and growth traits.

Runt-related transcription factor 2 (Runx2) is characterized by its critical functions in osteoblastic and ovulatory processes. The goal of this study was to explore the insertion/deletion (indel) variants of this gene and to evaluate their association with productive traits. Herein, a 12 bp and 6 bp insertion within the Runx2 gene was uncovered in Shaanbei white cashmere goats (SBWC; n = 1200). Chi-square analysis revealed that the 12 bp insertion was related to litter size (p < 0.01). Further association analysis also found this insertion was significantly associated with litter size (p = 1.1E-5). Interestingly, this insertion was also significantly associated with chest circumference (p = 0.018). Additionally, the 6 bp insertion was associated with body length (p = 0.003), chest width (p = 0.011), and chest circumference (p = 0.005). Furthermore, diplotype associations also uncovered that the combined genotypes of these two indels also significantly affected litter size and growth traits (p < 0.05). These findings suggested that these two insertions within the Runx2 gene were significantly associated with reproduction and growth traits, which would make them beneficial functional DNA markers that can be used in goat breeding.

Sheep zinc finger proteins 395 (ZNF395): insertion/deletion variations, associations with growth traits, and mRNA expression.

Zinc finger protein 395 (ZNF395), belonging to the Krüppel C2H2-type zinc-finger protein family, is a common transcription factor, which is involved in several cellular responses such as cell proliferation, differentiation, metabolism, and immunity, especially in the process of adipogenesis. In order to explore the insertion/deletion (indel) genetic variations of ZNF395 and evaluate their effects on sheep growth traits, a total of 1934 samples from five Chinese and two Mongolian sheep breeds were investigated. Genetic variation analysis of ZNF395 revealed a novel 23 bp indel at the intron 3 with three genotypes. Association analysis results showed that this locus was significantly associated with 10 different growth traits, especially chest width (p = 0.003) and chest circumference (p = 0.003) of Small Tail Han sheep and cannon circumference (p = 0.007) of Hu sheep. In addition, the mRNA expression of ZNF395 was investigated in two sheep breeds of different tail types. The result indicated that the expression was significantly different between the tail fat of Lanzhou Fat-Tail sheep and Small Tail Han sheep (p < 0.05). This study showed that the 23 bp indel locus in the ZNF395 gene were associated with sheep growth traits, which could be beneficial for animal breeding.

circMEF2D Negatively Regulated by HNRNPA1 Inhibits Proliferation and Differentiation of Myoblasts via miR-486-PI3K/AKT Axis.

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.

CircRNA Profiling Reveals an Abundant circBDP1 that Regulates Bovine Fat Development by Sponging miR-181b/miR-204 Targeting Sirt1/TRARG1.

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.

Goat AKAP12: Indel Mutation Detection, Association Analysis With Litter Size and Alternative Splicing Variant Expression.

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.

Exploration of Genetic Variants within the Goat A-Kinase Anchoring Protein 12 (AKAP12) Gene and Their Effects on Growth Traits.

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.

Corrigendum: Novel lncRNA lncFAM200B: Molecular Characteristics and Effects of Genetic Variants on Promoter Activity and Cattle Body Measurement Traits.

[This corrects the article DOI: 10.3389/fgene.2019.00968.].

circFLT1 and lncCCPG1 Sponges miR-93 to Regulate the Proliferation and Differentiation of Adipocytes by Promoting lncSLC30A9 Expression.

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.

Mir-193b Regulates the Differentiation, Proliferation, and Apoptosis of Bovine Adipose Cells by Targeting the ACSS2/AKT Axis.

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.

Goat CMTM2: mRNA expression profiles of different alternative spliced variants and associations analyses with 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.

Genetic effects of DSCAML1 identified in genome-wide association study revealing strong associations with litter size and semen quality in goat (Capra hircus).

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 membrane associated ring-CH-type finger 1 (MARCH1) mRNA expression and association with litter size.

Membrane-associated ring-CH-type finger 1 (MARCH1), which mediates the ubiquitination of CD86 and MHC class II proteins, plays crucial roles in ruminant fecundity. To completely explore the functions of MARCH1 in goat reproduction, the mRNA expression of this gene were investigated in different tissues of goats. Moreover, we analyzed the association of three insertions and deletions (indels) within MARCH1 with litter size in a large population of Shaanbei white cashmere goats (n = 2844). It showed that MARCH1 was expressed in all examined tissues, including the spleen, brain, lungs, liver, muscles, and testis, of 14- and 56-day-old male goats and in the ovary tissues of female goats. However, MARCH1 expression levels in the testis and lungs were significantly different between the 14- and 56-day-old male goats. Therefore, we examined MARCH1 expression levels in the testis of male goats belonging to different developmental age groups, i.e., 0-, 14-, 28-, 42-, and 56-day-old male goats. Our results indicated a potential association between MARCH1 expression and mitosis-to-meiosis transition. Furthermore, we identified three novel 7-, 15-, and 18-bp indels in MARCH1 in the Shaanbei white cashmere goat population. The presence of the three indels significantly affected MARCH1 expression in the testis and ovary. Statistical analyses indicated that these indels were associated with first-born litter size (P < 0.05). A significant difference was observed in the genotype distribution of the three indels between single- and multi-lamb female goats (P < 0.05). Together, these findings suggest that MARCH1 plays a crucial role in fertility and that the three novel indels in the goat MARCH1 can be used as effective molecular markers for marker-assisted selection of goats for breeding in the future.

Novel lncRNA lncFAM200B: Molecular Characteristics and Effects of Genetic Variants on Promoter Activity and Cattle Body Measurement Traits.

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.

A 14-bp functional deletion within the CMTM2 gene is significantly associated with litter size in goat.

CKLF-like MARVEL transmembrane domain-containing 2 (CMTM2) plays an important role in animal reproduction, and abnormal CMTM2 expression leads to spermatogenesis disorders. This study was conducted to assess the role of CMTM2 in goat reproduction by investigating an insertion/deletion (indel) variation and its effect on litter size, and to reveal its functional mechanism. A 14-base pair (bp) indel was found at position -861 to -848 nt of the CMTM2 promoter in 1131 female Shaanbei white cashmere goats. Association analyses revealed that the 14-bp indel in dams was significantly correlated with the size of the first litter (P = 0.013), with the 14-bp deletion significantly decreasing litter size. Moreover, litter size at first kidding was significantly correlated with genotype frequencies (P = 0.019). Expression of the goat CMTM2 gene was detected in testes and ovaries. In male, CMTM2 was increased after the initiation of meiosis in the developing testis. In female, CMTM2 expression was higher in ovary from multiple prolific goats at first kidding compared to non-prolific goats. Moreover, both in testis after initiation of meiosis and in ovary, the homozygous 14-bp inserted genotype II was associated with increased CMTM2 expression compared to the heterozygous genotype ID (P < 0.05). To further explore whether the 14-bp indel was located in the core promoter activity region of the CMTM2 promoter, the five recombinant plasmids pGL3-pro1 (-1756 to +83), pGL3-pro2 (-1200 to +83), pGL3-pro3 (-763 to +83), pGL3-pro4 (-496 to +83), and pGL3-pro5 (-205 to +83) were co-transfected with pRL-TK into HEK293T and GC-1 cells. A luciferase reporter assay showed that the pGL3-pro2 and pGL3-pro5 vectors produced significantly stronger luminescence than the other vectors. Interestingly, the 14-bp indel locus was included in the pGL3-pro2 plasmid, suggesting that the indel was functional. Subsequently, the two recombinant plasmids pGL3-pro2-inserted-allele and pGL3-pro2-deleted-allele were co-transfected with pRL-TK into HEK293T and GC-1 cells, respectively. The luciferase reporter assay demonstrated that the deleted allele of CMTM2 showed significantly lower luminescence activity than the inserted allele (P < 0.05), which corresponded to a decrease in litter size in the deletion-deletion genotype. Therefore, our results suggest that a 14-bp deletion in the promoter region of CMTM2 is significantly correlated with litter size in goats; this marker seems promising for breeding selection for improving economically important traits in goats.

Bovine pituitary homeobox 2 (PITX2): mRNA expression profiles of different alternatively spliced variants and association analyses with growth traits.

Pituitary homeobox 2 (PITX2) plays crucial roles in embryogenesis, ontogenesis, growth, and development via the Wnt/beta-catenin and POU1F1 pathways. To better understand the characteristics and genetic effects of the cattle PITX2 gene, we identified alternative PITX2 splicings, examined the effects of the spliced variants on mRNA expression levels in tissues, and then used association analyses to explore the relationships between a PITX2 deletion genetic variant and growth traits in 750 native Chinese cattle. An unreported spliced variant of PITX2, designated here as PITX2-V1, was identified in cattle using in silico cloning and RT-PCR. The entire coding sequence of PITX2 is 978 bp, encoding 325 amino acids, whereas that of PITX2-V1 is 357 bp encoding 118 amino acids. Cattle PITX2 exhibited both a perfect homeodomain and an OAR domain, but PITX2-V1 lacked the homeodomain. Analyses with qRT-PCR showed that the expression level of PITX2 in cattle testis was very low, and PITX2-V1 was only very slightly expressed in the brain and testis. Furthermore, a 24 bp deletion was detected within PITX2 intron, and the different genotypes were significantly associated with growth traits (e.g., body height, body length, heart girth) in four cattle breeds (P < 0.05). These results are of direct benefit to future cattle breeding, and provide new insights into the characteristics and functions of cattle PITX2 gene.