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Alternative Splicing Events in Pituitary and Hypothalamus of Zhuanghe Dagu Chickens with Different Egg Production Levels: Analysis Based on RNA-Seq Technology
MAZhiyong, LIMeicheng, MAWei, WANGChunqiang
Alternative Splicing Events in Pituitary and Hypothalamus of Zhuanghe Dagu Chickens with Different Egg Production Levels: Analysis Based on RNA-Seq Technology
In recent years, there have been great differences in the number of eggs laid by individuals of Zhuanghe Dagu chickens. Alternative splicing (AS) is an important mechanism of post-transcriptional regulation of eukaryotic genes, but there is no research on the correlation between AS and egg production in avian pituitary and hypothalamus. Based on RNA-Seq technology, this experiment analyzed the AS events of Zhuanghe Dagu chickens. The results showed that 769 and 813 differential AS events were detected in the pituitary and hypothalamic libraries, respectively. Five types of AS events were identified in both libraries (pituitary, hypothalamus), with the highest proportion of exon skipping (SE) and the lowest proportion of alternative 5' splice site (A5SS); NRCAM, SLMAP and CIB1 gene had a higher frequency of AS; 644 and 680 differentially spliced genes (DSGs) were screened from the two tissue libraries, respectively, with the highest proportion of SE and the lowest proportion of A5SS. GO and KEGG enrichment analysis annotated 329 and 337 DSGs, respectively; RT-PCR detection found that ESR1 was present in different AS events; a total of 6758 new transcripts were obtained from 12 libraries, and 12486 gene were optimized. The findings not only expand the understanding of AS events in poultry pituitary and hypothalamic tissues, but also provide new ideas for improving chicken egg production in research and production.
chicken / alternative splicing / RNA-Seq / differentially spliced genes / egg production {{custom_keyword}} /
表1 测序分析 |
样品名称 | 原始读数 | 干净读数 | 总定位基因数目 | 外显子读数 | 映射正链读数 | 映射负链度数 |
---|---|---|---|---|---|---|
LP_1 | 68317482 | 66328810 | 57680205(86.96%) | 66.60% | 27816090(41.94%) | 27768078(41.86%) |
LP_2 | 72359294 | 69806998 | 60386063(86.5%) | 62.10% | 29087549(41.67%) | 29092824(41.68%) |
LP_3 | 63889132 | 62116040 | 5366 920(86.4%) | 64.40% | 25918157(41.73%) | 25814561(41.56%) |
NP_1 | 64341034 | 61029484 | 52964336(86.78%) | 64.10% | 25448135(41.7%) | 25489645(41.77%) |
NP_2 | 69054884 | 67531462 | 59190144(87.65%) | 65.20% | 28511031(42.22%) | 28463500(42.15%) |
NP_3 | 62110264 | 60924682 | 52608969(86.35%) | 64.60% | 25342586(41.6%) | 25363479(41.63%) |
LH_1 | 79744218 | 77900 688 | 68487901(87.92%) | 69.50% | 32899952(42.23%) | 32968715(42.32%) |
LH_2 | 65534460 | 62447384 | 54556009(87.36%) | 66.60% | 26194279(41.95%) | 26265470(42.06%) |
LH_3 | 79835258 | 78021862 | 67701041(86.77%) | 68.20% | 32517332(41.68%) | 32596024(41.78%) |
NH_1 | 76681332 | 74134976 | 64570880(87.1%) | 70.70% | 30962806(41.77%) | 31058491(41.89%) |
NH_2 | 49332078 | 46982632 | 41205550(87.7%) | 67.10% | 19804054(42.15%) | 19834071(42.22%) |
NH_3 | 73617140 | 71760796 | 61408353(85.57%) | 66.90% | 29489160(41.09%) | 29529056(41.15%) |
表2 AS分类、数量及DSGs |
基因文库 | 基因 | AS事件类型 | AS事件总数 | 差异事件总数 | 差异剪接基因 |
---|---|---|---|---|---|
垂体 | 8720 | SE | 13804 | 549(264:285) | 548 |
MXE | 1464 | 159(91:68) | 159 | ||
RI | 512 | 32(15:17) | 32 | ||
A3SS | 397 | 18(7:11) | 18 | ||
A5SS | 281 | 11(7:4) | 11 | ||
下丘脑 | 9448 | SE | 15933 | 610(312:298) | 613 |
MXE | 1913 | 142(71:71) | 142 | ||
RI | 504 | 31(19:12) | 31 | ||
A3SS | 394 | 24(12:12) | 24 | ||
A5SS | 277 | 6(4:2) | 6 |
表3 参与AS事件的基因(5例) |
基因文库 | 基因 | SE | MXE | RI | A3SS | A5SS |
---|---|---|---|---|---|---|
垂体 | SLMAP | 25 | 14 | 0 | 0 | 0 |
CIB1 | 19 | 9 | 0 | 0 | 0 | |
NRCAM | 20 | 4 | 0 | 0 | 0 | |
CADM1 | 20 | 12 | 0 | 0 | 0 | |
CARMIL3 | 18 | 3 | 1 | 0 | 0 | |
下丘脑 | SLMAP | 23 | 10 | 0 | 0 | 0 |
CIB1 | 19 | 9 | 0 | 0 | 1 | |
NRCAM | 25 | 13 | 0 | 0 | 0 | |
EPB4IL3 | 22 | 6 | 0 | 1 | 0 | |
ABI3BP | 19 | 4 | 0 | 0 | 0 |
表4 KEGG途径富集分析以注释DSGs |
基因文库 | 途径 | 描述 | P值 | DSGs |
---|---|---|---|---|
垂体 | gga04010 | MAPK signaling pathway | 0.029472063 | 12 |
gga04260 | Cardiac muscle contraction | 0.081935761 | 5 | |
gga04261 | Adrenergic signaling in cardiomyocytes | 0.086900369 | 7 | |
下丘脑 | gga04520 | Adherens junction | 0.011715804 | 7 |
gga04530 | Tight junction | 0.062176625 | 6 | |
gga04020 | Calcium signaling pathway | 0.083233215 | 9 |
表5 新转录本结构注释结果(7例) |
染色体编号 | 来源 | 长度 | 正负链 | 外显子数 | 功能预测 |
---|---|---|---|---|---|
4 | Novel Gene | 21214 | + | 1 | nucleic acid binding |
22 | Novel Gene | 11154 | - | 4 | histone acetyltransferase KAT2B isoform X2 |
2 | Novel Gene | 8241 | + | 6 | heat shock transcription factor, X-linked-like |
12 | Novel Gene | 1896 | - | 15 | hypothetical protein ENH_00063870 |
6 | Novel Gene | 34081 | + | 28 | purine ribonucleoside binding |
15 | Novel Gene | 7214 | - | 41 | nucleoside binding |
17 | Novel Gene | 5900 | + | 68 | extracellular matrix structural constituent |
表6 已知基因结构优化(10例) |
基因ID | 染色体编号 | 正负链 | 原始跨度 | 延伸跨度 |
---|---|---|---|---|
ENSGALG00000000003 | 1 | + | 20174067-20177667 | 20157937-20179163 |
ENSGALG00000000081 | 16 | - | 205312-210738 | 204525-211382 |
ENSGALG00000000129 | 11 | + | 19064511-19086448 | 19064481-19107184 |
ENSGALG00000000233 | 27 | - | 4203293-4208774 | 4201773-4212041 |
ENSGALG00000000309 | 26 | + | 789839-794797 | 787968-795597 |
ENSGALG00000000296 | 5 | - | 27201732-27283175 | 27195404-27283845 |
ENSGALG00000000853 | 17 | + | 10878731-10887869 | 10872754-10887869 |
ENSGALG00000000862 | 23 | - | 1766445-1795825 | 1765749-1795881 |
ENSGALG00000000978 | 21 | + | 905170-916809 | 898302-932294 |
ENSGALG00000001042 | 19 | - | 615601-657520 | 613509-657908 |
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Nowadays, high epidemic obesity-triggered hypertension and diabetes seriously damage social public health. There is now a general consensus that the body’s fat content exceeding a certain threshold can lead to obesity. Calcium ion is one of the most abundant ions in the human body. A large number of studies have shown that calcium signaling could play a major role in increasing energy consumption by enhancing the metabolism and the differentiation of adipocytes and reducing food intake through regulating neuronal excitability, thereby effectively decreasing the occurrence of obesity. In this paper, we review multiple calcium signaling pathways, including the IP3 (inositol 1,4,5-trisphosphate)-Ca2+ (calcium ion) pathway, the p38-MAPK (mitogen-activated protein kinase) pathway, and the calmodulin binding pathway, which are involved in biological clock, intestinal microbial activity, and nerve excitability to regulate food intake, metabolism, and differentiation of adipocytes in mammals, resulting in the improvement of obesity.
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Understanding the dynamics of eukaryotic transcriptome is essential for studying the complexity of transcriptional regulation and its impact on phenotype. However, comprehensive studies of transcriptomes at single base resolution are rare, even for modern organisms, and lacking for rice. Here, we present the first transcriptome atlas for eight organs of cultivated rice. Using high-throughput paired-end RNA-seq, we unambiguously detected transcripts expressing at an extremely low level, as well as a substantial number of novel transcripts, exons, and untranslated regions. An analysis of alternative splicing in the rice transcriptome revealed that alternative cis-splicing occurred in approximately 33% of all rice genes. This is far more than previously reported. In addition, we also identified 234 putative chimeric transcripts that seem to be produced by trans-splicing, indicating that transcript fusion events are more common than expected. In-depth analysis revealed a multitude of fusion transcripts that might be by-products of alternative splicing. Validation and chimeric transcript structural analysis provided evidence that some of these transcripts are likely to be functional in the cell. Taken together, our data provide extensive evidence that transcriptional regulation in rice is vastly more complex than previously believed.
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Our previous work demonstrated that the type I GnRH receptor (GnRHR) resides exclusively and constitutively within membrane rafts in αT3-1 gonadotropes and that this association was necessary for the ability of the receptor to couple to the ERK signaling pathway. Gαq, c-raf, and calmodulin have also been shown to reside in this compartment, implicating a raft-associated multiprotein signaling complex as a functional link between the GnRHR and ERK signaling. In the studies reported here, we used subcellular fractionation and coimmunoprecipitation to analyze the behavior of ERKs with respect to this putative signaling platform. ERK 2 associated partially and constitutively with low-density membranes both in αT3-1 cells and in whole mouse pituitary. Cholesterol depletion of αT3-1 cells reversibly blocked the association of both the GnRHR and ERKs with low-density membranes and uncoupled the ability of GnRH to activate ERK. Analysis of the kinetics of recovery of ERK inducibility after cholesterol normalization supported the conclusion that reestablishment of the association of the GnRHR and ERKs with the membrane raft compartment was not sufficient for reconstitution of signaling activity. In αT3-1 cells, the GnRHR and ERK2 coimmunoprecipitated from low-density membrane fractions prepared either in the presence or absence of detergent. The GnRHR also partitioned into low-density, detergent-resistant membrane fractions in mouse pituitary and coimmunoprecipitated with ERK2 from these fractions. Collectively, these data support a model in which coupling of the GnRHR to the ERK pathway in gonadotropes involves the assembly of a multiprotein signaling complex in association with specialized microdomains of the plasma membrane.
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In vertebrates, GnRH binds to its receptor and stimulates predominantly G(q/11)-mediated signal transduction in gonadotropes. However, little is known about the GnRH receptor and its signaling pathway in tunicates, a group that arose before the vertebrates. Although tunicates have had duplications of a few genes in the last 600 million years, the early vertebrates had duplications of the full genome. Also unknown is the nature of GnRH signaling in the tunicate, which lacks both a pituitary gland and sex steroids. However, we know that tunicates have GnRH peptides because we previously reported six GnRH peptides encoded within the tunicate genome of Ciona intestinalis. Here we clone and sequence cDNAs for four putative GnRH receptors from C. intestinalis. These are the only invertebrate GnRH receptors found to date. Each Ciona GnRH receptor was expressed in COS-7 cells, incubated with each of the six C. intestinalis GnRHs and assayed for a signaling response. GnRH receptors 1, 2, and 3 responded to Ciona GnRH peptides to stimulate intracellular cAMP accumulation. In contrast, only GnRH receptor 1 activated inositol phosphate turnover in response to one of the Ciona GnRHs. The green monkey type II GnRH receptor cDNA was tested as a comparison and a positive control. In conclusion, the four GnRH receptors encoded within the C. intestinalis genome were all transcribed into messenger RNA, but only three of the Ciona GnRH receptors were biologically active in our assays. The Ciona GnRH receptors almost exclusively activated the cAMP pathway.
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The lamprey, which are divided into three families, including the Petromyzonidae, Geotriidae, and Mordaciidae, have been shown to regulate the reproductive axis through a functional hypothalamic-pituitary-gonadal axis. To date, two forms of gonadotropin-releasing hormone (GnRH) have been identified in the sea lamprey (Petromyzon marinus), lamprey GnRH-I (decapeptide and cDNA) and lamprey GnRH-III (decapeptide), both of which have been shown to be expressed in the preoptic-anterior hypothalamic region and both forms have been demonstrated to regulate reproductive function (i.e. steroidogenesis and gametogenesis). The objective of this study was to isolate the cDNA encoding the prepro-lamprey GnRH-III from eight species of lamprey using a PCR based subcloning procedure. A degenerate primer designed to the lamprey GnRH-III decapeptide was used to amplify the 3' end of each transcript, while gene specific primers were used to amplify the 5' ends. Phylogenetic analysis using the prepro-lamprey GnRH-III amino acid sequences was performed, in which the lamprey GnRH-III sequences divided into three groups, supporting the current view of the lamprey lineage at the family level. Finally, a phylogenetic analysis of these newly identified deduced amino acid sequences together with 64 previously described GnRH sequences suggests that the lamprey GnRHs are unique, as they group together separately from the three previously described paralogous lineages of the GnRH family.
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GnRH and its analogs are used extensively for the treatment of hormone-dependent diseases and assisted reproductive techniques. They also have potential as novel contraceptives in men and women. A thorough delineation of the molecular mechanisms involved in ligand binding, receptor activation, and intracellular signal transduction is kernel to understanding disease processes and the development of specific interventions. Twenty-three structural variants of GnRH have been identified in protochordates and vertebrates. In many vertebrates, three GnRHs and three cognate receptors have been identified with distinct distributions and functions. In man, the hypothalamic GnRH regulates gonadotropin secretion through the pituitary GnRH type I receptor via activation of G(q). In-depth studies have identified amino acid residues in both the ligand and receptor involved in binding, receptor activation, and translation into intracellular signal transduction. Although the predominant coupling of the type I GnRH receptor in the gonadotrope is through productive G(q) stimulation, signal transduction can occur via other G proteins and potentially by G protein-independent means. The eventual selection of intracellular signaling may be specifically directed by variations in ligand structure. A second form of GnRH, GnRH II, conserved in all higher vertebrates, including man, is present in extrahypothalamic brain and many reproductive tissues. Its cognate receptor has been cloned from various vertebrate species, including New and Old World primates. The human gene homolog of this receptor, however, has a frame-shift and stop codon, and it appears that GnRH II signaling occurs through the type I GnRH receptor. There has been considerable plasticity in the use of different GnRHs, receptors, and signaling pathways for diverse functions. Delineation of the structural elements in GnRH and the receptor, which facilitate differential signaling, will contribute to the development of novel interventive GnRH analogs.
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Nerve growth factor (NGF) epitomizes a family of proteins known as the neurotrophins (NTs), which are required for the survival and differentiation of neurons within both the central and peripheral nervous system. Synthesis of NGF in tissues innervated by the peripheral nervous system is consistent with its function as a target-derived trophic factor. However, the presence of low- and high-affinity NGF receptors in the gonads suggests another function for the NTs within the reproductive endocrine system. We now report that NGF is required for the growth of primordial ovarian follicles, a process known to occur independently of pituitary gonadotropins. Both the NT receptor p75(NTR) and the NGF tyrosine kinase receptor trkA were found to be expressed in the ovaries of infantile normal mice and mice carrying a null mutation of the NGF gene. The ovaries from homozygote NGF-null (-/-) mutant animals, analyzed after completion of ovarian histogenesis, exhibited a markedly reduced population of primary and secondary follicles in the presence of normal serum gonadotropin levels, and an increased number of oocytes that failed to be incorporated into a follicular structure. Assessment of mitogenic activity using two complementary proliferation markers revealed a conspicuous reduction in somatic cell proliferation in the ovaries of NGF-deficient mice. These results suggest that the delay in follicular growth observed in NGF(-/-) mice may be related to the loss of a proliferative signal provided by NGF to the nonneural endocrine component of the ovary.
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The neurotrophin nerve growth factor (NGF) and its two membrane-anchored receptors are expressed in the developing ovary before the organization of the first primordial follicles. In the absence of NGF, the growth of primordial follicles is retarded, indicating that NGF contributes to facilitating early follicular development. The present experiments were undertaken to determine whether NGF can also be involved in the differentiation process by which ovarian follicles become responsive to gonadotropins. Treatment of 2-d-old rat ovaries in organ culture with NGF increased FSH receptor (FSHR) mRNA within 8 h of exposure. This effect was cAMP-independent but additive to the cAMP-mediated increase in FSHR gene expression induced by either forskolin or vasoactive intestinal peptide, a neurotransmitter previously shown to induce FSHR formation in neonatal rat ovaries. After NGF treatment, the ovary acquired the capacity of responding to FSH with cAMP formation and preantral follicular growth, indicating that exposure to the neurotrophin resulted in the formation of biologically active FSHRs. Quantitative measurement of FSHR mRNA demonstrated that the content of FSHR mRNA is reduced in the ovaries of mice carrying a null mutation of the NGF gene. These results indicate that one of the functions of NGF in the developing ovary is to facilitate the differentiation process by which early growing follicles become gonadotropin-dependent during postnatal life, and that it does so by increasing the synthesis of FSHRs.
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Oogenesis is the process by which ovarian germ cells undertake meiosis and differentiate to become eggs. In mice, Stra8 is required for the chromosomal events of meiosis to occur, but its role in differentiation remains unknown. Here we report Stra8-deficient ovarian germ cells that grow and differentiate into oocyte-like cells that synthesize zonae pellucidae, organize surrounding somatic cells into follicles, are ovulated in response to hormonal stimulation, undergo asymmetric cell division to produce a polar body and cleave to form two-cell embryos upon fertilization. These events occur without premeiotic chromosomal replication, sister chromatid cohesion, synapsis or recombination. Thus, oocyte growth and differentiation are genetically dissociable from the chromosomal events of meiosis. These findings open to study the independent contributions of meiosis and oocyte differentiation to the making of a functional egg.
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In addition to nuclear-initiated (genomic) responses, estrogen receptors (ERs) have the ability to facilitate rapid, membrane-initiated, estrogen-triggered signaling cascades via a plasma membrane-associated form of the receptor. These rapid responses are dependent on assembly of membrane ER-centered multimolecular complexes, which can transduce ligand-activated signals to affect a variety of enzymatic pathways, often occurring in a cell-type-specific fashion with tissue-specific physiological outcomes. In some instances, cross-talk occurs between these membrane-initiated and nuclear responses, ultimately regulating transcriptional activation. The role of splice variants in membrane-initiated estrogen responses has been described, notably those within the vascular endothelium. In this review, we describe the evidence for membrane ERs, the molecular components of the aforementioned signaling complexes and pathways, the relevance of ER splice variants, and ER-mediated responses in specific tissues. Our growing understanding of ER-mediated actions at a molecular level will provide insight into the controversies surrounding hormone replacement therapy in postmenopausal women.
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