Project description:Full-length single-cell RNA-seq applied to a viral human cancer: Applications to HPV expression and splicing analysis in HeLa S3 cells
Project description:The goal of this study was to define how loss of full-length DEK reshapes global transcriptional programs in HeLa S3 cells by comparing gene-expression profiles of wild-type (WT) HeLa S3 and the 1E7 DEK-knockout cells generated by TALEN-mediated genomic disruption. The 1E7 clone lacks detectable full-length DEK, while retaining a low-abundance truncated DEK species, consistent with an alternative transcriptional start site/allelic expression.
Project description:Single-cell RNA-seq is one of the most important and widely used approaches to characterize cell types and to understand major parts of biological systems. As of today, most methods are only able to capture parts of the whole transcriptome, mainly the protein-coding genes, and therefore lack information about non-coding biotypes or full-length transcripts. Here, we present “Vast transcriptome Analysis of Single-cells by dA-tailing (VASA-seq)”, a method for highly-sensitive, full-length and total RNA-seq in single-cells. VASA-seq is compatible with plate-formats for sorting of rare cell populations, and with droplet microfluidics for high-throughput applications and atlasing. We applied VASA-seq to >30,000 single cells in the developing mouse embryo during gastrulation and early organogenesis. Our in-depth analysis revealed expression of novel cell-type specific non-coding RNA markers, enhanced cell-cycle characterization and alternative splicing patterns. We believe the method and mouse dataset will serve as a useful tool to further investigate the expression of different RNA biotypes and splicing patterns in large datasets.
Project description:We used bs-ATLAS-seq to comprehensively map the genomic location and assess the DNA methylation status of human full-length LINE-1 elements (L1). The approach is focused on the youngest family (L1HS), but it also catches a significant fraction of L1PA2 to L1PA8 elements. This was performed in a panel of 12 human primary or transformed cell lines (BJ, IMR90, MRC5, H1, K562, HCT116, HeLa S3, HepG2, MCF7, HEK-293, HEK-293T, 2102Ep).
Project description:Specific types of human papillomaviruses (HPVs) cause cervical cancer, the second most common tumor in females worldwide. Both cellular transformation and the maintenance of the oncogenic phenotype of HPV-positive tumor cells are linked to the expression of the viral E6 and E7 oncogenes. In order to identify downstream cellular target genes for the viral oncoproteins, we silenced endogenous E6 and E7 expression in HPV-positive HeLa cells by RNA interference (RNAi). Subsequently, we assessed changes of the cellular transcriptome by genome-wide microarray analysis. We identified, 648 genes wich were either downregulated (360 genes) or upregulated (288 genes) upon inhibition of E6/E7 expression. A large fraction of these genes is involved in tumour-relevant processes, such as apoptosis control, cell cycle regulation, or spindle formation. Others may represent novel cellular targets for the HPV oncogenes, such as a large group of C-MYC-associated genes involved in RNA splicing. Keywords: siRNA mediated knockdown
Project description:RBPMS regulates alternative splicing decision via recruiting other trans factors. This experiment aims to investigate the interactome of Strep-II tagged recombinant RBPMS in Hela nuclear extract. The interactome of the full length protein is compared to the construct lacking the far C-terminal 20 amino acids. The “R” series indicates full-length RBPMS pull-down. The “P” series indicates truncated RBPMS pull-down. The “BL” series indicates full length RBPMS pull-down in the absence of nuclear extract input but with bovine serum albumin beads blocking reagent. The “N” series indicates non-baited negative control pull-down.
Project description:We performed poly(A)+ stranded RNA-seq of a panel of human primary or transformed cell lines (BJ, IMR90, MRC5, K562, HCT116, HeLa S3, HepG2, MCF7, HEK-293, HEK-293T, 2102Ep). In parallel, we determined the genomic location and DNA methylation levels of human full-length LINE-1 elements (L1) from the same cell lines using bs-ATLAS-seq (E-MTAB-10895). To link DNA methylation and L1 expression, we used cell pellets from the same cell culture to perform both RNA-seq and bs-ATLAS-seq.
Project description:Nonsense-mediated mRNA decay (NMD) is a eukaryotic, translation-dependent degradation pathway that targets mRNAs with premature termination codons and also regulates the expression of some mRNAs that encode full-length proteins. Although many genes express NMD-sensitive transcripts, identifying them based on short-read sequencing data remains a challenge. To identify and analyze endogenous targets of NMD, we applied cDNA Nanopore sequencing and short-read sequencing to human cells with varying expression levels of NMD factors. Our approach detects full-length NMD substrates that are highly unstable and increase in levels or even only appear when NMD is inhibited. Among the many new NMD-targeted isoforms that our analysis identified, most derive from alternative exon usage. The isoform-aware analysis revealed many genes with significant changes in splicing but no significant changes in overall expression levels upon NMD knockdown. NMD-sensitive mRNAs have more exons in the 3΄UTR and for mRNAs with a termination codon in the last exon. The length of the 3΄UTR per se does not correlate with NMD sensitivity. Analysis of splicing signals reveals isoforms where NMD has been co-opted in the regulation of gene expression, but a main function is most likely to rid the transcriptome of isoforms resulting from spurious splicing events. Long-read sequencing enabled the identification of many novel NMD-sensitive mRNAs and revealed both known and unexpected features concerning their biogenesis and their biological role. Our data provide a highly valuable resource of human NMD transcript targets for future genomic and transcriptomic applications.
