Natural antisense transcript of MYOG regulates development and regeneration in skeletal muscle by shielding the binding sites of MicroRNAs of MYOG mRNA 3'UTR.

Natural antisense transcripts (NATs) are endogenous RNAs opposite to sense transcripts, and they can significantly contribute to regulating various biological processes through multiple epigenetic mechanisms. NATs can affect their sense transcripts to regulate the growth and development of skeletal muscle. Our analysis of third-generation full-length transcriptome sequencing data revealed that NATs represented a significant portion of the lncRNA, accounting for up to 30.19%-33.35%. The expression of NATs correlated with myoblast differentiation, and genes expressing NATs were mainly involved in RNA synthesis, protein transport, and cell cycle. We found a NAT of MYOG (MYOG-NAT) in the data. We found that the MYOG-NAT could promote the differentiation of myoblasts in vitro. Additionally, knockdown of MYOG-NAT in vivo led to muscle fiber atrophy and muscle regeneration retardation. Molecular biology experiments demonstrated that MYOG-NAT enhances the stability of MYOG mRNA by competing with miR-128-2-5p, miR-19a-5p, and miR-19b-5p for binding to MYOG mRNA 3'UTR. These findings suggest that MYOG-NAT plays a critical role in skeletal muscle development and provides insights into the post-transcriptional regulation of NATs.

The A1762T/G1764A mutations enhance HBV replication by alternating viral transcriptome.

The A1762T/G1764A mutations, one of the most common mutations in the hepatitis B virus basal core promoter, are associated with the progression of chronic HBV infection. However, effects of these mutations on HBV replication remains controversial. This study aimed to systematically investigate the effect of the mutations on HBV replication and its underlying mechanisms. Using the prcccDNA/pCMV-Cre recombinant plasmid system, a prcccDNA-A1762T/G1764A mutant plasmid was constructed. Compared with wild-type HBV, A1762T/G1764A mutant HBV showed enhanced replication ability with higher secreted HBV DNA and RNA levels, while Southern and Northern blot indicated higher intracellular levels of relaxed circular DNA, single-stranded DNA, and 3.5 kb RNA. Meanwhile, the mutations increased expression of intracellular core protein and decreased the production of HBeAg and HBsAg. In vitro infection based on HepG2-NTCP cells and mice hydrodynamic injection experiment also proved that these mutations promote HBV replication. 5'-RACE assays showed that these mutations upregulated transcription of pregenomic RNA (pgRNA) while downregulating that of preC RNA, which was further confirmed by full-length transcriptome sequencing. Moreover, a proportion of sub-pgRNAs with the potential to express polymerase were also upregulated by these mutations. The ChIP-qPCR assay showed that A1762T/G1764A mutations created a functional HNF1α binding site in the BCP region, and its overexpression enhanced the effect of A1762T/G1764A mutations on HBV. Our findings revealed the mechanism and importance of A1762T/G1764A mutations as an indicator for management of CHB patients, and provided HNF1α as a new target for curing HBV-infected patients.

Full-length transcriptome sequencing reveals extreme incomplete annotation of the goat genome.

Despite recent advances in generating high-quality reference genome assemblies, the genome sequences for most livestock species, including goats, are still poorly annotated. Single-molecule long-read sequencing has greatly facilitated gene annotation by obtaining full-length transcripts. In this study, we generated full-length transcriptome data for samples from abomasum (n = 2) and testicle (n = 1), using PacBio Iso-Seq technology. We further combined these data with published data from abomasum (5ZY, SRR8618141) to evaluate and improve the gene annotation of the goat genome. We identified 14.5-16.3% of novel genes per sample from the four Iso-Seq datasets. At the transcript level, 40.6% of them were novel, including 29.7% novel transcripts from known genes and 10.9% from novel genes. We further verified the expression of novel genes in four additional RNA-seq data and found that the expression level of novel genes was significantly lower than that of known genes, indicating that the lowly expressed genes tend to be missed in the current genome annotation. This study shows the superiority of full-length transcriptome data in gene annotation, and more such data are required to improve the gene annotation for goat genome and other species.

Anthocyanin regulatory networks in Solanum tuberosum L. leaves elucidated via integrated metabolomics, transcriptomics, and StAN1 overexpression.

BACKGROUND: Anthocyanins, which account for color variation and remove reactive oxygen species, are widely synthesized in plant tissues and organs. Using targeted metabolomics and nanopore full-length transcriptomics, including differential gene expression analysis, we aimed to reveal potato leaf anthocyanin biosynthetic pathways in different colored potato varieties. RESULTS: Metabolomics analysis revealed 17 anthocyanins. Their levels varied significantly between the different colored varieties, explaining the leaf color differences. The leaves of the Purple Rose2 (PurpleR2) variety contained more petunidin 3-O-glucoside and malvidin 3-O-glucoside than the leaves of other varieties, whereas leaves of Red Rose3 (RedR3) contained more pelargonidin 3-O-glucoside than the leaves of other varieties. In total, 114 genes with significantly different expression were identified in the leaves of the three potato varieties. These included structural anthocyanin synthesis-regulating genes such as F3H, CHS, CHI, DFR, and anthocyanidin synthase and transcription factors belonging to multiple families such as C3H, MYB, ERF, NAC, bHLH, and WRKY. We selected an MYB family transcription factor to construct overexpression tobacco plants; overexpression of this factor promoted anthocyanin accumulation, turning the leaves purple and increasing their malvidin 3-o-glucoside and petunidin 3-o-glucoside content. CONCLUSIONS: This study elucidates the effects of anthocyanin-related metabolites on potato leaves and identifies anthocyanin metabolic network candidate genes.

Genome-wide association study and transcriptome of olecranon-type traits in peach (Prunus persica L.) germplasm.

BACKGROUND: The top of the olecranon honey peach (Prunus persica L.) fruit appears similar to an eagle's beak. In this study, a single olecranon honey peach with a round-type fruit was observed in our fruit orchard. To explore the genetic mechanism of olecranon formation, we performed full-length transcriptome sequencing analysis of olecranon and round peaches as well as a genome-wide association study of the association of olecranon-type trait loci. RESULTS: The gene locus was 26,924,482 base pairs in NC_034014.1. Transcriptome sequencing showed that the clean sequencing data of each sample reached 7.10GB, with 14,360 genes and 23,167 transcripts expressed in both the olecranon honey peach and round peach. Among the 11 differentially expressed genes selected as candidate genes, six were highly expressed in olecranon peach and named as LOC18775282, LOC18772209, LOC18773929, LOC18772013, LOC18773401, and ONT.13798.5. Five genes were highly expressed in round peach and named as LOC18773079, LOC18773525, LOC18773067, LOC18775244, and LOC18772236. Notably, ONT.13798.5 was not previously identified. The genes were within 1 Mb up- or down-stream of the main genome-wide association study locus for olecranon-type traits. CONCLUSIONS: This study revealed loci associated with olecranon and provides useful information for analysis and breeding of olecranon honey peach.

Oxford Nanopore Technologies (ONT) Full-length Transcriptomics Shows Electroacupuncture ameliorates Lipid Metabolic Disorder through Suppressing Hepatic Pdia3/Perk/Qrich1 Expression in Rats.

BACKGROUND: The incidence of dyslipidemia increases after menopause. Electroacupuncture (EA) has been recommended for menopause-related disease. However, the positive effect on lipid metabolism disorders is still unclear. OBJECTIVES: To investigate the underlying mechanism of EA treatment on lipid metabolism disorders through ONT full-length transcriptome sequencing Methods: Adult female SD rats were randomly divided into Ctrl, sham operation+high-fat feed(Sham+HFD), Ovariectomized+high-fat feed (OVX+HFD), Ovariectomized+high-fat feed + Atorvastatin (OVX+HFD+ATO) and OVX+HFD+EA groups. Periovarian adipose tissue around the bilateral ovaries of rats in the Sham+HFD group was resected. Rats in the OVX+HFD, OVX+HFD+ATO and OVX+HFD+EA groups were subjected to bilateral oophorectomy to prepare the ovariectomized rat model. Treatment was applied to rats in the OVX+HFD+EA group. ST36, PC6, SP6, BL18 and ST40 were the selected acupoints. Daily food intake and body weights of rats were recorded. The samples were collected 30 days after treatment. The serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein (HDL-C) were detected to assess the improvement of lipid metabolism disorders. HE and oil red O staining were used to stain the liver tissues. Total RNA was extracted from liver tissues, and its transcriptional changes were determined by high-throughput sequencing. Additionally, RTÁqPCR and immunofluorescence staining were used to verify the crucial signal pathway screened by the ONT fullÁlength transcriptome sequencing. RESULTS: EA treatment resulted in a lowered weight of perirenal fat and liver and a significant improvement in the color of the liver. In addition, EA could improve the lipid profile and hepatic steatosis in OVX+HFD rats. According to fullÁlength transcriptome sequencing, 2292 genes showed differential expression in the OVX+HFD group; of these, 1121 were upregulated and 1171 down-regulated. 609 DEGs were found in the OVX+HFD+EA group compared to the OVX+HFD group; 235 up-regulated and 374 down-regulated. We also found that 77 genes are significantly upregulated after EA intervention through Venn map analysis (including Agtr1a, Pdia3, etc.), which may be the targeted genes for EA treatment of lipid metabolism disorders. Finally, we verified the expression of Pdia3, Perk and Qrich1 levels in liver tissues. HFD feeding could increase the expression of Pdia3 and its downstream signal pathways molecular Perk and Qrich1. But these effects were reversed by EA treatment, the results demonstrated that the expression of pdia3, Perk, as well as Qrich1 of OVX+HFD rats had a decreasing trend after EA treatment. CONCLUSIONS: EA could ameliorate lipid metabolic disorder in OVX+HFD rats. The Pdia3/Perk/Qrich1 signal pathway may play crucial roles in the improvement of lipid metabolism disorder of OVX+HFD rats after EA treatment.

Full-Length Transcriptional Analysis of the Same Soybean Genotype With Compatible and Incompatible Reactions to Heterodera glycines Reveals Nematode Infection Activating Plant Defense Response.

Full-length transcriptome sequencing with long reads is a powerful tool to analyze transcriptional and post-transcriptional events; however, it has not been applied on soybean (Glycine max). Here, a comparative full-length transcriptome analysis was performed on soybean genotype 09-138 infected with soybean cyst nematode (SCN, Heterodera glycines) race 4 (SCN4, incompatible reaction) and race 5 (SCN5, compatible reaction) using Oxford Nanopore Technology. Each of 9 full-length samples collected 8 days post inoculation with/without nematodes generated an average of 6.1 GB of clean data and a total of 65,038 transcript sequences. After redundant transcripts were removed, 1,117 novel genes and 41,096 novel transcripts were identified. By analyzing the sequence structure of the novel transcripts, a total of 28,759 complete open reading frame (ORF) sequences, 5,337 transcription factors, 288 long non-coding RNAs, and 40,090 novel transcripts with function annotation were predicted. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of differentially expressed genes (DEGs) revealed that growth hormone, auxin-activated signaling pathway and multidimensional cell growth, and phenylpropanoid biosynthesis pathway were enriched by infection with both nematode races. More DEGs associated with stress response elements, plant-hormone signaling transduction pathway, and plant-pathogen interaction pathway with more upregulation were found in the incompatible reaction with SCN4 infection, and more DEGs with more upregulation involved in cell wall modification and carbohydrate bioprocess were detected in the compatible reaction with SCN5 infection when compared with each other. Among them, overlapping DEGs with a quantitative difference was triggered. The combination of protein-protein interaction with DEGs for the first time indicated that nematode infection activated the interactions between transcription factor WRKY and VQ (valine-glutamine motif) to contribute to soybean defense. The knowledge of the SCN-soybean interaction mechanism as a model will present more understanding of other plant-nematode interactions.

Full-Length Transcriptome Sequencing: An Insight Into the Dog Model of Heart Failure.

Heart failure (HF) leads to a progressive increase in morbidity and mortality rates. This study aimed to explore the transcriptional landscape during HF and identify differentially expressed transcripts (DETs) and alternative splicing events associated with HF. We generated a dog model of HF (n = 3) using right ventricular pacemaker implantation. We performed full-length transcriptome sequencing (based on nanopore platform) on the myocardial tissues and analyzed the transcripts using differential expression analysis and functional annotation methods [Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses]. Additionally, we estimated the expression of the selected genes by quantitative real-time PCR (qRT-PCR) and detected the proportion of immune cells using flow cytometry. We found that increased B-type natriuretic peptide reduced ejection fraction, and apparent clinical signs were observed in the dog model of HF. We identified 67,458 transcripts using full-length transcriptome sequencing. A total of 785 DETs were obtained from the HF and control groups. These DETs were mainly enriched in the immune responses, especially Th1, Th2, and Th17 cell differentiation processes. Furthermore, flow cytometry results revealed that the proportion of Th1 and Th17 cells increased in patients with HF compared to controls, while the proportion of Th2 cells decreased. Differentially expressed genes in the HF and control groups associated with Th1, Th2, and Th17 cell differentiation were quantified using qRT-PCR. We also identified variable splicing events of sarcomere genes (e.g., MYBPC3, TNNT2, TTN, FLNC, and TTNI3). In addition, we detected 4,892 transcription factors and 406 lncRNAs associated with HF. Our analysis based on full-length transcript sequencing provided an analysis perspective in a dog model of HF, which is valuable for molecular research in an increasingly relevant large animal model of HF.

Screening and Identification of the First Non-CRISPR/Cas9-Treated Chinese Miniature Pig With Defective Porcine Endogenous Retrovirus pol Genes.

Pig to human xenotransplantation is considered to be a possible approach to alleviate the shortage of human allografts. Porcine endogenous retrovirus (PERV) is the most significant pathogen in xenotransplantation. We screened for pigs that consistently did not transmit human-tropic replication competent PERVs (HTRC PERVs), namely, non-transmitting pigs. Then, we conducted whole-genome resequencing and full-length transcriptome sequencing to further investigate the sequence characteristics of one non-transmitting pig. Using in vitro transmission assays, we found 5 (out of 105) pigs of the Chinese Wuzhishan minipig inbred line that did not transmit PERV to human cells, i.e., non-transmitting pigs. Whole-genome resequencing and full-length transcriptome sequencing of one non-transmitting pig showed that all of the pol genes were defective at both the genome and transcript levels. We speculate that the defective PERV pol genes in this pig might be attributable to the long-term inbreeding process. This discovery is promising for the development of a strain of highly homozygous and genetically stable pigs with defective PERV pol genes as a source animal species for xenotransplantation.

Screening of Potential Key Transcripts Involved in Planarian Regeneration and Analysis of Its Regeneration Patterns by PacBio Long-Read Sequencing.

Dugesia japonica is an excellent animal model for studying the regeneration mechanism due to its characteristics of rapid regeneration and easy breeding. PacBio sequencing was performed on the intact planarians (In) and regenerating planarians of 1 day (1d), 3 days (3d), and 5 days (5d) after amputation. The aim of this study is to deeply profile the transcriptome of D. japonica and to evaluate its regenerate changes. Using robust statistical analysis, we identified 5931, 5115, and 4669 transcripts differentially expressed between 1d and In, 3d and In, 5d and In, respectively. A total of 63 key transcripts were screened from these DETs. These key transcripts enhance the expression in different regenerate stages respectively to regulate specific processes including signal transduction, mitosis, protein synthesis, transport and degradation, apoptosis, neural development, and energy cycling. Finally, according to the biological processes involved in these potential key transcripts, we propose a hypothesis of head regeneration model about D. japonica. In addition, the weighted gene co-expression network analysis provides a new way to screen key transcripts from large amounts of data. Together, these analyses identify a number of potential key regulators controlling proliferation, differentiation, apoptosis, and signal transduction. What's more, this study provides a powerful data foundation for further research on planarians regeneration.

Transcriptome analysis of the molecular mechanism of Chrysanthemum flower color change under short-day photoperiods.

Chrysanthemum [Dendranthema morifolium Tzvel.] is an ornamental plant grown under long-term artificial cultivation conditions. In production, early Chrysanthemum blossoms are often promoted by artificial short-day treatment. However, we found that the flower colour of Chrysanthemum blossoms induced by artificial short-day treatment was lighter than those induced by the natural photoperiod. To explore the intrinsic mechanism of colour fading in flowers, we performed full-length transcriptome sequencing of Chrysanthemum morifolium cv. 'Jinbeidahong' using single-molecule real-time sequencing and RNA-sequencing under natural daylight (ND) and short daylight (SD) conditions. The clustered transcriptome sequences were assigned to various databases, such as NCBI, Swiss-Prot, Gene Ontology and so on. The comparative results of digital gene expression analysis revealed that there were differentially expressed transcripts (DETs) in the four stages under ND and SD conditions. In addition, the expression patterns of anthocyanin biosynthesis structural genes were verified by quantitative real-time PCR. The major regulators of the light signalling ELONGATED HYPOCOTYL5 genes were markedly upregulated under ND conditions. The patterns of anthocyanin accumulation were consistent with the expression patterns of CHI1 and 3GT1. The results showed that the anthocyanin synthesis is tightly regulated by the photoperiod, which will be useful for molecular breeding of Chrysanthemum.

Identification of Multigene Biomarker for Shrimp White Feces Syndrome by Full-Length Transcriptome Sequencing.

The pacific white shrimp, Litopenaeus vannamei, with the largest shrimp industry production in the world, is currently threatened by a severe disease, white feces syndrome (WFS), which cause devastating losses globally, while its causal agents remain largely unknown. Herein, compared to the Control shrimp by metagenomic analysis, we firstly investigated that the altered functions of intestinal microbial community in WFS shrimp were the enrichment of bacterial chemotaxis and flagellar assembly pathways, hinting at a potential role of pathogenic bacteria for growth and development, which might be related to WFS occurrence. Single-molecule real-time (SMRT) sequencing was to further identify the gene structure and gene regulation for more clues in WFS aetiology. Totally 50,049 high quality transcripts were obtained, capturing 39,995 previously mapped and 10,054 newly detected transcripts, which were annotated to 30,554 genes. A total of 158 differentially expressed genes (DEGs) were characterized in WFS shrimp. These DEGs were strongly associated with various immune related genes that regulated the expression of multiple antimicrobial peptides (e.g., antilipopolysaccharide factors, penaeidins, and crustin), which were further experimentally validated using quantitative PCR on transcript level. Collectively, multigene biomarkers were identified to be closely associated with WFS, especially those functional alterations in microbial community and the upregulated immune related gene with antibacterial activities. Our finding not only inspired our cogitation on WFS aetiology from both microbial and host immune response perspectives with combined metagenomic and full-length transcriptome sequencing, but also provided valuable information for enhancing shrimp aquaculture.

Full-Length Transcriptome Sequencing and the Discovery of New Transcripts in the Unfertilized Eggs of Zebrafish (Danio rerio).

Understanding early gene expression in zebrafish embryos is a prerequisite for developmental biology research. In this study, 1,629,447 polymerase reads were obtained from the unfertilized eggs of zebrafish via full-length transcriptome sequencing using the PacBio RS II platform first. Then, 102,920 unique isoforms were obtained by correction, clustering and comparison with the zebrafish genome. 12,782 genes in the genome were captured, accounting for 39.71% of the all annotated genes. Approximately 62.27% of the 12,782 genes have been alternatively spliced. GO and KEGG annotations revealed that the unfertilized eggs primarily stored genes that participate in RNA processing and nuclear protein complex composition. According to this PacBio data that aligned with the genome, 3,970 fusion genes, 819 ncRNAs, and 84 new transcripts were predicted. Illumina RNA-seq and RT-qPCR detection found that the expression of two new transcripts, PB.5289.1 and PB.10209.1, were significantly up-regulated at the 2-cell stage and down-regulated rapidly thereafter, suggesting their involvement in minor ZGA during early embryonic development. This study indicated that the unfertilized eggs of zebrafish may have retained genes directly related to cell division and development to initiate the subsequent development in a limited space and time. On the other hand, NTRs or new transcriptome regions in the genome were discovered, which provided new clues regarding ZGA of MZT during early embryonic development in fish.