Project description:Circadian variability in kidney function has long been recognized but is often ignored as a potential confounding variable in in vivo physiological experiments. To provide a guide for physiological studies on the kidney proximal tubule, we have now created a data resource consisting of expression levels for all measurable mRNA transcripts in microdissected proximal tubule segments from mice as a function of the time of day. This approach employs small-sample RNA-sequencing (RNA-seq) applied to microdissected renal proximal tubules including both S1 proximal convoluted tubules (PCTs) and S2 proximal straight tubules (PSTs). The data were analyzed using JTK-Cycle to detect periodicity. The data are provided as a user-friendly web page at https://esbl.nhlbi.nih.gov/Databases/Circadian-Prox/. In PCTs, 234 transcripts were found to vary in a circadian manner (3.7 % of total quantified). In PSTs, 334 transcripts were found to vary in a circadian manner (5.3 % of total quantified). Transcripts previously known to be associated with corticosteroid action and transcripts associated with increased flow were found to be overrepresented among circadian transcripts peaking during the “dark” portion of the day (Zeitgeber 14-22), corresponding to the peak levels of corticosterone and glomerular filtration rate in mice.
Project description:Nontransformed cells form heterotypic cadherin junctions with adjacent transformed cells to inhibit tumor cell growth and motility. Transformed cells must override this form of growth control, called contact normalization, to invade and metastasize during cancer progression. Heterocellular cadherin junctions between transformed and nontransformed cells are needed for this process. However, specific mechanisms downstream of cadherin signaling have not been clearly elucidated. Here, we utilized a b-catenin reporter construct to determine if contact normalization affects Wnt signaling in transformed cells. b-catenin driven GFP expression in Src transformed mouse embryonic cells was decreased when cultured with cadherin competent nontransformed cells compared to transformed cells cultured with themselves, but not when cultured with cadherin deficient nontransformed cells. We also utilized a layered culture system to investigate the effects of oncogenic transformation and contact normalization on gene expression and oncogenic Src kinase mediated phosphorylation events. RNA-Seq analysis found that the cadherin dependent contact normalization inhibited the expression of 22 transcripts that were induced by Src transformation, and increased the expression of 78 transcripts that were suppressed by Src transformation. Phosphoproteomic analysis of cells expressing a temperature sensitive Src kinase construct found that contact normalization decreased phosphorylation of 10 proteins on tyrosine residues that were phosphorylated within 1 hour of Src kinase activation in transformed cells. Taken together, these results indicate that cadherin dependent contact normalization inhibits Wnt signaling to regulate oncogenic kinase activity and gene expression, particularly PDPN expression, in transformed cells in order to control tumor progression.
Project description:Alternative splicing of mRNA diversifies the function of human proteins, with tissue- and cell-specific protein isoforms being the most difficult to validate. While transcriptomic experiments enable the detection of many alternatively spliced transcripts, it is not known if these transcripts have protein-coding potential. We recently published the PG Nexus pipeline, which facilitates high confidence validation of exons and exon-exon junctions of spliced transcripts by integrating transcriptomics and proteomics data. Using the PG Nexus, we analyzed undifferentiated human mesenchymal stem cells and compared the number of protein isoforms validated using different protein sequence database, including public online databases and RNA-seq derived databases. With significant overlaps with other databases, we identified 8,011 exons and 3,824 splice junctions with the Ensembl database. Both exonic and junction peptides were important for protein isoform validation. The Ensembl database consistently outperformed the other data sources, but predicted open reading frames from RNA-seq derived transcripts were comparable, with only 6 less splice junctions validated. Using proteotypic and isoform-specific peptides, we validated 462 protein isoforms. This number increases to 1,083 if multiple proteotypic peptides per protein are included. Multiplexing proteotypic peptides in SRM assays or similar experiments will increase the confidence and coverage of protein isoform validation experiments.
Project description:Comprehensive RNA-seq experiments to measure the expression of homoeologs across different developmental stages, as a part of the Xenopus laevis genome project. This work is funded by Agency Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT; "Genome Science" Grant ID 221S0002).
Project description:Data analysis is a critical part of quantitative proteomics studies in interpreting biological questions. Numerous computational tools including protein quantification, imputation, and differential expression (DE) analysis were generated in the past decade. However, searching optimized tools is still an unsolved issue. Moreover, due to the rapid development of RNA-Seq technology, a vast number of DE analysis methods are created. Applying these newly developed RNA-Seq-oriented tools to proteomics data is still a question that needs to be addressed. In order to benchmark these analysis methods, a proteomics dataset constituted the proteins derived from human, yeast, and drosophila with different ratios were generated. Based on this dataset, DE analysis tools (including array-based and RNA-Seq based), imputation algorithms, and protein quantification methods were compared and benchmarked. This study provided useful information on analyzing quantitative proteomics datasets. All the methods used in this study were integrated into Perseus which are available at https://www.maxquant.org/perseus.
Project description:Interventions: lesion tissues vs. adjacent tissues of colorectal cancer patients:nil
Primary outcome(s): RNA
Study Design: Factorial
Project description:<p>Despite the nuclear localization of the m6A machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively m6A-modified. However, these findings are mostly based on m6A-seq, an antibody-dependent technique with a high rate of false positives. Here, we addressed the presence of m6A in CHIKV and DENV RNAs. For this, we combined m6A-seq and the antibody-independent SELECT and nanopore direct RNA sequencing techniques with functional, molecular, and mutagenesis studies. Following this comprehensive analysis, we found no evidence of m6A modification in CHIKV or DENV transcripts. Furthermore, depletion of key components of the host m6A machinery did not affect CHIKV or DENV infection. Moreover, CHIKV or DENV infection had no effect on the m6A machinery’s localization. Our results challenge the prevailing notion that m6A modification is a general feature of cytoplasmic RNA viruses and underscore the importance of validating RNA modifications with orthogonal approaches.</p>
Project description:The fungal skin disease chytridiomycosis has caused the devastating decline and extinction of hundreds of amphibian species globally, yet the potential for evolving resistance, and the underlying pathophysiological mechanisms remain poorly understood. We exposed 406 naïve, captive-raised alpine tree frogs (Litoria verreauxii alpina) to the aetiological agent Batrachochytrium dendrobatidis in two concurrent and controlled infection experiments. We investigated (A) survival outcomes and clinical pathogen burdens between populations and clutches, and (B) individual host tissue responses to chytridiomycosis. Here we present multiple interrelated datasets associated with these exposure experiments, including animal signalment, survival and pathogen burden of 355 animals from Experiment A, and the following datasets related to 61 animals from Experiment B: animal signalment and pathogen burden; raw RNA-Seq reads from skin, liver and spleen tissues; de novo assembled transcriptomes for each tissue type; raw gene expression data; annotation data for each gene; and raw metabolite expression data from skin and liver tissues. These data provide an extensive baseline for future analyses.
Project description:Comprehensive RNA-seq experiments to measure the expression of homoeologs across different developmental stages, as a part of the Xenopus laevis genome project. This work is funded by Agency Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT; "Genome Science" Grant ID 221S0002). Collect mRNA from whole embryos; two clutches were used (Taira dataset for one pair, Ueno dataset for the other pair)