Capture and sequence analysis of RNAs with 5'-hydroxyl termini in S. cerevisiae
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ABSTRACT: Data from 5OH-seq experiments to map RNAs with 5'-hydroxyl termini in S. cerevisiae 17 samples of yeast RNA analyzed by 5OH-seq. 2 Replicates: +/- Xrn1,+/- Tm, +/- SAP. 1 replicate: pre-fragmented RNA
Project description:By using NGS-derived retinal transcriptome profiling (RNA-seq) to compare the gene expression profiling between 4 differently treated NPC cells Examination of different gene expression in EBV-miRNA-BART1/3/7 lentivirus and their control infected nasopharyngeal carcinoma cells.
Project description:DNA double-strand breaks (DSBs) represent a threat to the genome because they can lead to loss of genetic information and chromosome rearrangements. The DNA repair protein p53 binding protein 1 (53BP1) protects the genome by limiting nucleolytic processing of DSBs by a mechanism that requires its phosphorylation, but whether it does so directly is not known. Here we identify Rapl-interacting factor 1 (Rif1) as an Ataxia-Telangiectasia Mutated (ATM) phosphorylation-dependent interactor of 53BP1, and show that absence of Rif1 results in 5M-bM-^@M-^Y-3M-bM-^@M-^Y DNA end resection in mice. Consistent with enhanced DNA resection, Rif1 deficiency impairs DNA repair in the G1 and S phases of the cell cycle, interferes with class switch recombination (CSR) in B lymphocytes, and leads to accumulation of chromosome DSBs. Study of Rif1 DNA-end protection activity against resection via analysis of single-stranded DNA binding protein RPA and Rad51 accumulation at sites of AID-induced DNA damage by ChIP-seq. All samples shown in Fig. 4 are included (controls and test samples, 7 samples in total).
Project description:The splicing factor SF3B1 is the most commonly mutated gene in the myelodysplastic syndromes (MDS), particularly in patients with refractory anemia with ring sideroblasts (RARS). MDS is a disorder of the hematopoietic stem cell and we thus studied the transcriptome of CD34+ cells from MDS patients with SF3B1 mutations using RNA-sequencing. Genes significantly differentially expressed at the transcript and/or exon level in SF3B1 mutant compared to wildtype cases include genes involved in MDS pathogenesis (ASXL1, CBL), iron homeostasis and mitochondrial metabolism (ALAS2, ABCB7, SLC25A37) and RNA splicing/processing (PRPF8, HNRNPD). Many genes regulated by a DNA damage-induced BRCA1-BCLAF1-SF3B1 protein complex showed differential expression/splicing in SF3B1 mutant cases. Our data indicate that SF3B1 plays a critical role in MDS by affecting the expression and splicing of genes involved in specific cellular processes/pathways, many of which are relevant to the known RARS pathophysiology, suggesting a causal link. RNA-Seq was performed to compare the transcriptome of bone marrow CD34+ cells from eight MDS patients with SF3B1 mutation, four MDS patients with no known splicing mutation and five healthy controls.
Project description:In this study we developed TRS, a computational approach to determine the 3’ termini of transcripts from total RNA data generated by the RNAtag-seq protocol. To show the applicability of our approach, we applied our algorithm to sequencing data generated by the RNAtag-seq and the term-seq protocols. In term-seq, a 3’ adaptor is ligated to the RNA 3' termini prior to the RNA fragmentation, and thus, the sequences adjacent to the adapter sequence in the library reads represent original 3' termini that were present in the RNA sample. The RNAtag-seq protocol involves ligation of a 3’ adaptor after the RNA fragmentation and thus, the sequences adjacent to the adapter sequence in the library reads represent genuine 3’ termini that were present in the RNA sample and artificial 3’ termini generated by the RNA fragmentation. We show that our computational approach can reliably identify genuine 3’ termini from RNAtag-seq data by comparing the set of identified 3’ termini based on the RNAtag-seq and the term-seq protocols.
Project description:Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here we show that the novel oncogene RASSF1C drives mesenchymal to amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that amoeboid cells display the cancer stem cell markers CD133, ALDH1 and the pluripotent marker Nanog; are accompanied by higher invasive potential in vitro and in vivo; and employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification.
Project description:The objective of this study is to investigate the changes of the breast milk proteome from four individual mothers over a six month lactation period by shotgun proteomic techniques, because a comprehensive understanding of the human milk proteome may lead to better understanding of the needs of infants. This may contribute to the improvement of infant formula.
Project description:The experiment intends to reveal the difference in gene expression profiles between the wild-type strain and the ∆cwp66 mutant of Clostridioides difficile. We first constructed the ∆cwp66 mutant, and the phenotypic changes of the ∆cwp66 mutant against the wild-type strain were studied. To further elucidate the mechanism of phenotypic changes of the ∆cwp66 mutant, RNA-sequencing experiments were carried out to reveal the underlying mechanism of phenotypic changes.
Project description:Methylation of cytosines (5meC) is a widespread heritable DNA modification. During mammalian development, two global demethylation events are followed by waves of de novo DNA methylation. In vivo mechanisms of DNA methylation establishment are largely uncharacterized. Here we use Saccharomyces cerevisiae as a system lacking DNA methylation to define the chromatin features influencing the activity of the murine DNMT3B. Our data demonstrate that DNMT3B and H3K4 methylation are mutually exclusive and that DNMT3B is co-localized with H3K36 methylated regions. In support of this observation, DNA methylation analysis in yeast strains without Set1 and Set2 show an increase of relative 5meC levels at the TSS and a decrease in the gene-body, respectively. We extend our observation to the murine male germline, where H3K4me3 is strongly anti-correlated while H3K36me3 correlates with accelerated DNA methylation. These results show the importance of H3K36 methylation for gene-body DNA methylation in vivo. Collecting Yeast Whole Genome Bisulfite Sequencing Data
Project description:Hypoxia is a feature of the microenvironment during P. aeruginosa infection in several disease states. We confirm that the pathogenicity of P. aeruginosa derived from sites of acute infection is higher than those derived from sites of chronic infection. Hypoxia attenuated the pathogenicity of acute but not chronic strains implicating a role for hypoxia in controlling virulence. Mass spectrometric analysis revealed reduced expression of multiple virulence factors in hypoxia including exotoxin A, alkaline protease and proteins important in the synthesis of pyoverdine. Inhibition of pyoverdine production by iron supplementation mimicked the effects of hypoxia. Finally, strains of P. aeruginosa which lacks a functional pseudomonas prolyl-hydroxylase domain containing protein (PPHD) do not respond to hypoxia implicating a possible role for PPHD as an oxygen-sensing determinant of pathogenicity. Understanding how hypoxia influences bacterial virulence will identify new targets for anti–infective therapy against P. aeruginosa.