Dysregulation of alternative splicing has been repeatedly associated with neurodevelopmental disorders, but the extent of cell-type-specific splicing in human neural development remains largely uncharted. Here, single-cell long-read sequencing in induced pluripotent stem cell (iPSC)-derived cerebral organoids identifies over 31,000 uncatalogued isoforms and 4,531 cell-type-specific splicing events. Long reads uncover coordinated splicing and cell-type-specific intron retention events, which are challenging to study with short reads. Retained neuronal introns are enriched in RNA splicing regulators, showing shorter lengths, higher GC contents, and weaker 5′ splice sites. We use this dataset to explore the biological processes underlying neurological disorders, focusing on autism. In comparison with prior transcriptomic data, we find that the splicing program in autistic brains is closer to the progenitor state than differentiated neurons. Furthermore, cell-type-specific exons harbor significantly more de novo mutations in autism probands than in siblings. Overall, these results highlight the importance of cell-type-specific splicing in autism and neuronal gene regulation.
Epigallocatechin gallate (EGCG) is the major ingredient of sinecatechins ointment, approved for the treatment of external genital and perianal warts. However, the molecular mechanism for EGCG's effect on warts resulting from the human papillomavirus (HPV) infection of keratinocytes is not well understood. HPV may survive in proliferative keratinocytes and may be involved in cell cycle regulation and progression. The objective of this study was to investigate the mechanism underlying EGCG's treatment on external genital warts of HPV infection through the cultured keratinocyte cells from the HaCaT cell line.MTT and flow cytometry assays were used to measure cell viability and the cell cycle profile, with and without EGCG treatment, for HaCaT keratinocyte cells cultured in a calcium-free medium and 1.8 mM calcium which induced proliferative and differentiated keratinocytes, respectively, for 24 h. The expression levels of cytotoxic proteins and factors were evaluated with the RT-PCR and western blotting analysis.EGCG influenced the proliferation stage but not the differentiation stage of keratinocytes. We suggest that apoptosis and autophagy might be the possible mechanism for the EGCG's effect on the proliferative HaCaT cells. Furthermore, we found that EGCG reduced the protein levels of cyclin D1 and Zac1 (a zinc-finger protein which regulates apoptosis and cell cycle arrest 1) dose-dependently in proliferative as compared to differentiated keratinocytes. It also induced the expression of p21 and DEC1 (differentiated embryo-chondrocyte expressed gene 1), and promoted G1 arrest of cell cycle in proliferative keratinocytes.These results help clarify the mechanisms of EGCG treatment of HPV-infected keratinocytes and may contribute to new targets, such as Zac1 and DEC1 for external genital and perianal warts.
Abstract Selection signatures that contribute to phenotypic diversity, especially morphogenesis in pigs, remain to be further elucidated. To reveal the regulatory role of genetic variations in phenotypic differences between Eastern and Western pig breeds, we performed a systematic analysis based on seven high-quality de novo assembled genomes, 1,081 resequencing data representing 78 domestic breeds, 162 methylomes, and 162 transcriptomes of skeletal muscle from Tongcheng (Eastern) and Landrace (Western) pigs at 27 developmental stages. Selective sweep uncovers different genetic architectures behind divergent selection directions for the Eastern and Western breeds. Notably, two loci showed functional alterations by almost fixed missense mutations. By integrating time-course transcriptome and methylome, we revealed differences in developmental timing during myogenesis between Eastern and Western breeds. Genetic variants under artificial selection have critical regulatory effects on progression patterns of heterochronic genes like GHSR and BDH1 , by the interaction of local DNA methylation status, particularly during embryonic development. Altogether, our work not only provides valuable resources for understanding pig complex traits, but also contributes to human biomedical research.
Acting as a sequence-specific transcription factor, p53 tumor suppressor involves in a variety of biological processes after being activated by cellular stresses such as DNA damage. In recent years, microRNAs (miRNAs) have been confirmed to be regulated by p53 in several cancer types. However, it is still unclear how miRNAs orchestrate their regulation and function in p53 network after p53 activation in hepatocellular carcinoma (HCC). In this study, we used small RNA sequencing and systematic bioinformatic analysis to characterize the regulatory networks of differentially expressed miRNAs after the p53 activation in HepG2. Here, 33 miRNAs significantly regulated by p53 (12 up-regulated and 21 down-regulated) were detected between the doxorubicin-treated and untreated HepG2 cells in two biological replicates for small RNA sequencing and 8 miRNAs have been reported previously to be associated with HCC. Gene ontology (GO) and KEGG pathway enrichment analysis showed that 87.9% (29 out of 33) and 90.9% (30 out of 33) p53-regulated miRNAs were involved in p53-related biological processes and pathways with significantly low p-value, respectively. Remarkably, 18 out of 33 p53-regulated miRNAs were identified to contain p53 binding sites around their transcription start sites (TSSs). Finally, comprehensive p53-miRNA regulatory networks were constructed and analyzed. These observations provide a new insight into p53-miRNA co-regulatory network in the context of HCC.
Quantifying reproducibility and compliance of HKU and PLOS's open data policies, and also exploring the data availability of Scientific Reports paper, in published papers from HKU. All 2018 papers in HKU scholarhub were assessed by students in the HKU data curation class (MLIM7350). To increase the numbers of papers, students also looked at HKU PeerJ publications, and CityU Scholars Hub PLOS One and Scientific Reports publications.
Adenosine-to-inosine (A-to-I) RNA editing meditated by adenosine deaminases acting on RNA (ADARs) enzymes is a widespread post-transcriptional event in mammals. However, A-to-I editing in skeletal muscle remains poorly understood. By integrating strand-specific RNA-seq, whole genome bisulphite sequencing, and genome sequencing data, we comprehensively profiled the A-to-I editome in developing skeletal muscles across 27 prenatal and postnatal stages in pig, an important farm animal and biomedical model. We detected 198,892 A-to-I editing sites and found that they occurred more frequently at prenatal stages and showed low conservation among pig, human, and mouse. Both the editing level and frequency decreased during development and were positively correlated with ADAR enzymes expression. The hyper-edited genes were functionally related to the cell cycle and cell division. A co-editing module associated with myogenesis was identified. The developmentally differential editing sites were functionally enriched in genes associated with muscle development, their editing levels were highly correlated with expression of their host mRNAs, and they potentially influenced the gain/loss of miRNA binding sites. Finally, we developed a database to visualize the Sus scrofa RNA editome. Our study presents the first profile of the dynamic A-to-I editome in developing animal skeletal muscle and provides evidences that RNA editing is a vital regulator of myogenesis.
Abstract Background Bone marrow (BM) and umbilical cord (UC) are the main sources of mesenchymal stem cells (MSCs). These two MSCs display significant differences in many biological characteristics, yet the underlying molecular mechanisms need to be explored. Results In this study, to better understanding the biological features of MSCs, we isolated BMMSCs and UCMSCs from inbred Wuzhishan miniature pigs and generated the first global DNA methylation and gene expression profiles of porcine MSCs. The results showed that the osteogenic and adipogenic differentiation ability of porcine BMMSCs is stronger than that of UCMSCs. Stem cell surface marker CD90 were positively detected in both BMMSCs and UCMSCs. 587 genes were differentially methylated (280 hypermethylated and 307 hypomethylated) at the promoter regions between BMMSCs and UCMSCs. Meanwhile, 1,979 differentially expressed genes (1,407 up-regulated and 572 down-regulated) were identified between BMMSCs and UCMSCs. Integrative analysis reveals that 120 genes displayed differences in both gene expression and promoter methylation. Gene Ontology enrichment analysis revealed that these differential genes were associated with cell differentiation, cell migration, and immunogenicity properties. Remarkably, skeletal system development related genes were significantly hypomethylated and up-regulated in UCMSCs, while cell cycle genes were significantly higher down-regulated and hypermethylated, implying UCMSCs have higher cell proliferative activity and lower osteogenic differentiation potential than BMMSCs. Conclusions Our results indicate that DNA methylation plays an important role in regulating the biological characteristics differences between BMMSCs and UCMSCs. The study might provide a molecular theory basis for the application of porcine MSCs in human.
The selection of suitable reference genes is crucial to accurately evaluate and normalize the relative expression level of target genes for gene function analysis. However, commonly used reference genes have variable expression levels in developing skeletal muscle. There are few reports that systematically evaluate the expression stability of reference genes across prenatal and postnatal developing skeletal muscle in mammals. Here, we used quantitative PCR to examine the expression levels of 15 candidate reference genes ( ACTB , GAPDH , RNF7 , RHOA , RPS18 , RPL32 , PPIA , H3F3 , API5 , B2M , AP1S1 , DRAP1 , TBP , WSB , and VAPB ) in porcine skeletal muscle at 26 different developmental stages (15 prenatal and 11 postnatal periods). We evaluated gene expression stability using the computer algorithms geNorm, NormFinder, and BestKeeper. Our results indicated that GAPDH and ACTB had the greatest variability among the candidate genes across prenatal and postnatal stages of skeletal muscle development. RPS18 , API5 , and VAPB had stable expression levels in prenatal stages, whereas API5 , RPS18 , RPL32 , and H3F3 had stable expression levels in postnatal stages. API5 and H3F3 expression levels had the greatest stability in all tested prenatal and postnatal stages, and were the most appropriate reference genes for gene expression normalization in developing skeletal muscle. Our data provide valuable information for gene expression analysis during different stages of skeletal muscle development in mammals. This information can provide a valuable guide for the analysis of human diseases.
Genetic improvement of meat production traits has always been the primary goal of pig breeding. Geographical isolation, natural and artificial selection led to significant differences in the phenotypes of meat production traits between Chinese local pigs and Western commercial pigs. Comparative genomics and transcriptomics analysis provided powerful tools to identify genetic variants and genes associated with skeletal muscle growth. However, the number of available genetic variants and genes are still limited. In this study, a comprehensive comparison of transcriptomes showed that ribosomal protein S27-like (RPS27L) gene was highly expressed in skeletal muscle and up-regulated in Chinese local pigs when compared with Western commercial pigs. Functional analysis revealed that overexpression of RPS27L promoted myoblast proliferation and repressed differentiation in pig skeletal muscle cells. Conversely, the knockdown of RPS27L led to the inhibition of myoblast proliferation and the promotion of differentiation. Notably, a 13-bp insertion-deletion (InDel) mutation was identified within the RPS27L promoter, inserted in Chinese local breeds and predominantly deleted in Western commercial breeds. Luciferase reporter assay suggested this InDel modulated RPS27L expression by influencing transcription factor 3 (TCF3) and myogenic differentiation antigen (MYOD) binding to promoter. Furthermore, a positive correlation was observed between the expression of RPS27L expression and backfat thickness. Association studies demonstrated this InDel was significantly associated with the body weight of pigs at the age of 240 days. Together, our results suggested that RPS27L was a regulator of skeletal muscle development and growth, and was a candidate marker for improving meat production traits in pigs. This study not only provided a biomarker for animal breeding, but also was helpful for understanding skeletal muscle development and muscle-related disease in humans.
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