SnRNA-seq reveals heterogeneity of mouse cholangiocyte in liver Mallory-Denk bodies formation
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA949782 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR23999300_1.fastq.gz | Fastqsanger.gz | |||
| SRR23999300_2.fastq.gz | Fastqsanger.gz | |||
| SRR23999301_1.fastq.gz | Fastqsanger.gz | |||
| SRR23999301_2.fastq.gz | Fastqsanger.gz | |||
| SRR23999302_1.fastq.gz | Fastqsanger.gz |
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Project description:Mallory-Denk bodies (MDBs) are misfolded protein aggregates that include hepatocytes present in liver diseases, and are considered a typical feature of chronic liver diseases. Cholangiocytes are an epithelial cell type constituting the hepatic parenchyma, and play a role in liver regeneration if hepatocyte regeneration is compromised. UbD, which is overexpressed in hepatocellular carcinoma (HCC) plays an important role in immune regulation and MDB pathogenesis. In this study, we conducted the first comprehensive study on cholangiocyte heterogeneity to demonstrate the pathogenesis of MDBs formation using single-nucleus RNA sequencing (snRNA-seq) from fractionations of normal and DDC-treated mouse livers. We found that cholangiocytes in the MDB-forming groups were significantly expanded, and the enrichment pathways of differentially expressed genes (DEGs) were markedly involved in metabolic processes, neurodegenerative diseases, autophagy, and ubiquitin-mediated proteolysis. We proposed a cellular-state landscape for cholangiocytes using uniform manifold approximation and projection (UMAP) analysis, which includes numerous cell subpopulations, referred to as Clusters0 through 7, with Cluster1 being the sole subpopulation made up entirely of DDC-treated cells. UbD was highly expressed in Cluster4 and colocalized with the cholangiocyte marker gene cytokeratin 19 (Krt19), implying that cholangiocytes function as facultative liver progenitor cells (LPCs) to supply damaged hepatocytes to promote MDBs formation. In summary, we revealed, for the first time, the heterogeneity of cholangiocytes and provided a novel viewpoint for understanding the mechanism of MDBs formation as well as chronic liver disease progression.
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Project description:Spatiotemporal hepatocyte heterogeneity impacts in vivo gene engineering [snRNA-Seq]
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Project description:Spliceosomal snRNA are key components of small nuclear ribonucleoprotein particles (snRNPs), the building blocks of the spliceosome. The biogenesis of snRNPs is a complex process involving multiple cellular and subcellular compartments, the details of which are yet to be described. In short, the snRNA is exported to the cytoplasm as 3‘-end extended precursor (pre-snRNA), where it acquires a heptameric Sm ring. The SMN complex which catalyses this step, recruits Sm proteins and assembles them around the pre-snRNA at the single stranded Sm site. After additional modification, the complex is re-imported into the nucleus where the final maturation step occurs. Our modeling suggests that during the cytoplasmic stage of maturation pre-snRNA assumes a compact secondary structure containing Near Sm site Stem (NSS) which is not compattible with the formation of the Sm ring. To validate our in silico predictions we employed selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) on U2 snRNA in vivo, ex vivo and in vitro, and U4 pre-snRNA in vitro. For the in vivo experiment HeLa cells were incubated for 10 min at 37°C with NAI or DMSO to final concentration 200 mM. RNA was isolated using Trizol (Sigma) and 200 µl chloroform and precipitated with ethanol at -20°C overnight. For the ex vivo experiment, RNA was isolated from HeLa cells after Protease K treatment at room temperature for 45 min. After incubation, RNA was isolated using equilibrated phenol/chloroform/isoamyl alcohol buffered by folding buffer (110 mM HEPES pH 8.0, 110 mM KCl, 11 mM MgCl2) and cleaned on a PD-10 column according to the manufacturer’s instructions. Isolated RNA was treated with 100mM NAI or DMSO for 10 min at 37°C. For the in vitro experiment, U2WT and U4 pre-snRNA were transcribed by T7 polymerase followed by DNase I (30 min at 37 °C) and Proteinase K (30 min at 37°C) treatments. U2 snRNA was purified on 30 kDa Amicon columns, folded for 30 min at 37°C in 57 mM MgCl2 and incubated with 100 mM NAI at 37°C for 10 min. DMSO was used as a negative control. U4 pre-snRNA was purified on Superdex 200 Increase 10/300GL, folded for 30 min at 37°C in 60 mM MgCl2 and incubated with 100 mM NAI at 37°C for 10 min. DMSO was used as a negative control. All prepared RNA samples (in vitro, ex vivo, in vivo) were used for reverse transcription with the gene-specific primer 5’-CGTTCCTGGAGGTACTGCAA for U2 snRNA and 5’- AAAAATTCAGTCTCCG for U4 pre-snRNA. We used SHAPE MaP buffer (50 mM Tris-HCl pH 8.0, 75 mM KCl, 10 mM DTT, 0.5 mM dNTP, 6 mM MnCl2) and SuperScript II (Invitrogen). Amplicons for snRNAs were generated using gene-specific forward and reverse primers. Importantly, the primers include Nextera adaptors required for downstream library construction. PCR reaction products were cleaned using Monarch PCR&DNA Clean-up Kits. Remaining Illumina adaptor sequences were added using the PCR MasterMix and index primers provided in the NexteraXT DNA Library Preparation Kit (Illumina) according to the manufacturer’s protocol. Libraries were quantified using Qubit (Invitrogen) and BioAnalyzer (Agilent). Amplicons were sequenced on a NextSeq 500/550 platform using a 150 cycle mid-output kit. All sequencing data was analyzed using the ShapeMapper 2 analysis pipeline1. The ‘—amplicon’ and ‘—primers’ flags were used, along with sequences of gene-specific handles PCR primers, to ensure primer binding sites are excluded from reactivity calculations. Default read-depth thresholds of 5000x were used. Analysis of statistically significant reactivity differences between ex vivo and in vivo-determined SHAPE reactivities was performed using the DeltaSHAPE automated analysis tool and default settings2. 1. Busan, S. & Weeks, K.M. Accurate detection of chemical modifications in RNA by mutational profiling (MaP) with ShapeMapper 2. RNA 24, 143-148 (2018). 2. Smola, M.J., Rice, G.M., Busan, S., Siegfried, N.A. & Weeks, K.M. Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis. Nat Protoc 10, 1643-69 (2015).
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