POS-1 protects posterior gut specification by blocking GLD-3/2 polyadenylation of anterior factor neg-1
ABSTRACT: The Poly(A)-Tail focused RNA-seq, or PAT-seq approach was utilised to determine the gene expression, poly(A)-site and polyadenylation state of the transcriptome of early C. elegans embryos having been depleated for a series of RNA binding proteins. Namely; pos-1 and mex-5 were independently knocked down using dsRNA expressing E. coli HT115(DE3) fed to ~100,000 starved/synchronized L1 larvae. When half of the population reached adulthood and half were still in the L4 stage, worms were bleached and embryos harvested and stored in Trizol. This ensured an enrichment of early embryos between 1 and 24 cell stage. mex-6(pk440) were synchronized and grown on OP50 until the same time point and embryos harvested in the same manner. Since prolonged gld-3 and gld-2 RNAi result in sterility, starved/synchronized L1 larvae were fed diluted OP50 (200uL of concentrated OP50 diluted in 2mL of M9 and starved L1s) for 16 hours. At that point the OP50 had been mostly consumed by the larvae and concentrated gld-3 or gld-2 dsRNA expressing E. coli were added to the plates. When half of the population reached adulthood and half were still in the L4 stage, worms were bleached and embryos harvested and stored in Trizol. Total RNA was isolated using standard procedures. Analysis of poly(A) dynamics in early C. elegans embryos reponding to depletion of specific RNA binding proteins and adneylation state regulators
Project description:Single-cell polyadenylation site quantification 48 single cells each of murine embryonic stem cells maintained in FCS+LIF medium ("ESC"), embryonic stem cells maintained in serum free, 2i containing medium ("2i") and neural stem cells ("NSC") were sequenced by BATSeq, a single cell transcriptomic protocol for mapping polyadenylation sites in single cells.
Project description:The post-transcriptional fate of messenger RNAs (mRNAs) is largely dictated by their 3' untranslated regions (3'UTRs), which are defined by cleavage and polyadenylation (CPA) of pre-mRNAs. We used poly(A)-position profiling by sequencing (3P-Seq) to map poly(A) sites at eight developmental stages and tissues in the zebrafish. Analysis of over 60 million 3P-Seq reads substantially increased and improved existing 3'UTR annotations, resulting in confidently identified 3'UTRs for more than 78.79% of the annotated protein-coding genes in zebrafish. Most zebrafish genes undergo alternative CPA with more than a thousand genes using different dominant 3'UTRs at different stages. 3'UTRs tend to be shortest in the ovaries and longest in the brain. Isoforms with some of the shortest 3'UTRs are highly expressed in the ovary yet absent in the maternally contributed RNAs of the embryo, perhaps because their 3'UTRs are too short to accommodate a uridine-rich motif required for stability of the maternal mRNA. At two hours post-fertilization, thousands of unique poly(A) sites appear at locations lacking a typical polyadenylation signal, which suggests a wave of widespread cytoplasmic polyadenylation of mRNA degradation intermediates. Our insights into the identities, formation, and evolution of zebrafish 3'UTRs provide a resource for studying gene regulation during vertebrate development. 3P-Seq was used to map the 3' ends of protein-coding genes in the zebrafish genome
Project description:To obtain a global view of mRNA uridylation in Arabidopsis, we generated TAIL-seq libraries from WT plants, urt1 and xrn4 single mutants, and urt1 xrn4 double mutant. The TAIL-seq protocol was recently developed to deep-sequence the 3' ends of RNAs (Chang et al., 2014). We generated TAIL-seq libraries from WT plants, urt1 and xrn4 single mutants, and urt1 xrn4 double mutant.
Project description:This dataset is part of a study that investigated how the hematopoietic system coordinates the rapid and efficient regeneration of the megakaryocytic lineage during stress scenarios. We found that the phenotypic hematopoietic stem cell (HSC) compartment contains stem-like megakaryocyte-committed progenitors (SL-MkPs), a cell population that shares many features with multipotent HSCs and serves as a lineage-restricted emergency pool for inflammatory insults. This dataset contains single-cell RNA sequencing data of 30 hematopoietic stem and progenitor cells which, in the context of our study, confirmed that MK-specfic transcripts are of highly variable expression in HSCs. The dataset further showed that variations in MK transcript expression in HSCs is not correlated with global transcriptomic rearrangements. Murine bone marrow cells were sorted by Lin-cKit+CD150+CD48- (referred to as cd150+ in the following) and Lin-cKit+CD150- (referred to as cd150- in the following). Transcriptomes of 11 cd150- and 9 cd150+ HSCs were determined using QUARTZ, a single-cell RNASeq protocol
Project description:The use of alternative polyadenylation sites is common and affects the post-transcriptional fate of mRNA, including its stability, localization, and translation. Here we present a method for genome-wide and strand-specific mapping of poly(A) sites and quantification of RNA levels at unprecedented efficiency by using an on-cluster dark T-fill procedure on the Illumina sequencing platform. Our method outperforms former protocols in quality and throughput, and reveals new insights into polyadenylation in Saccharomyces cerevisiae. Experimental benchmark of five different protocols (3tfill, bpmI, internal, rnaseq and yoon) for genome-wide identification of polyadenylation sites in Saccharomyces cerevisiae and transcript quantification. RNA was extracted from WT cells grown in glucose (ypd) or galactose (ypgal) as carbon source. The same RNA was used for 3 independent library constructions (technical replicates, rep).
Project description:We obtained global measurements of decay and translation rates for mammalian mRNAs with alternative 3' untranslated regions (3' UTRs). 1 3P-Seq sample from 3T3 cells and 1 3P-Seq sample from mouse ES cells; 2 2P-Seq steady state and 4 2P-Seq with actinomycin D; 6 polysome fraction 2P-Seq
Project description:This project aims to investigate the metabolic pathways expressed by the active microbial community occurring at the deep continental subsurface. Subsurface chemoLithoautotrophic Microbial Ecosystems (SLiMEs) under oligotrophic conditions are supported by H2; however, the overall ecological trophic structures of these communities are poorly understood. Some deep, fluid-filled fractures in the Witwatersrand Basin, South Africa appear to support inverted trophic pyramids wherein methanogens contributing <5% of the total DNA apparently produce CH4 that supports the rest of the community. Here we show the active metabolic relationships of one such trophic structure by combining metatranscriptomic assemblies, metaproteomic and stable isotopic data, and thermodynamic modeling. Four autotrophic β-proteobacteria genera that are capable of oxidizing sulfur by denitrification dominate. They co-occur with sulfate reducers, anaerobic methane oxidizers and methanogens, which each comprises <5% of the total community. Defining trophic levels of microbial chemolithoautotrophs by the number of transfers from the initial abiotic H2-driven CO2 fixation, we propose a top-down cascade influence of the metabolic consumers that enhances the fitness of the metabolic producers to explain the inverted biomass pyramid of a multitrophic SLiME. Symbiotic partnerships are pivotal in the deep biosphere on and potentially beyond the Earth.
Project description:Transcription factors direct gene expression, and so there is much interest in mapping their genome-wide binding locations. Current methods do not allow for the multiplexed analysis of TF binding, and this limits their throughput. We describe a novel method for determining the genomic target genes of multiple transcription factors simultaneously. DNA-binding proteins are endowed with the ability to direct transposon insertions into the genome near to where they bind. The transposon becomes a “Calling Card” marking the visit of the DNA-binding protein to that location. A unique sequence “barcode” in the transposon matches it to the DNA-binding protein that directed its insertion. The sequences of the DNA flanking the transposon (which reveal where in the genome the transposon landed) and the barcode within the transposon (which identifies the TF that put it there) are determined by massively-parallel DNA sequencing. To demonstrate the method’s feasibility, we determined the genomic targets of eight transcription factors in a single experiment. The Calling Card method promises to significantly reduce the cost and labor needed to determine the genomic targets of many transcription factors in different environmental conditions and genetic backgrounds. These data contain Ty5 insertion sites mapped by an Illumina GAII analyzer in the S. cerevisiae genome for the background strain without any Sir4 present (1 run), in strains expressing Sir4-tagged copies of three well-characterized TFs: Gal4, Leu3, and Gcn4 (1 run each), and a multiplex of eight Sir4-tagged TFs pooled in a single experiment (2 biological replicates), and insertions from the Thi2-Sir4 fusion expressed from its native locus in two conditions (1 run each). The format of each insertions file is [chromosome number] [position of genomic base] [direction of insertion] [number of reads at that position]. Raw sequencing data comes in two varieties. Paired-end data contains a 5 bp barcode at the beginning of read #2. Single-end data contains a 2 bp barcode on the beggining of read #1.
Project description:The ability to chronicle transcription factor binding events throughout the development of an organism would facilitate mapping of transcriptional networks that control cell fate decisions. We describe a method for permanently recording protein-DNA interactions in mammalian cells. We endow transcription factors with the ability to deposit a transposon into the genome near to where they bind. The transposon becomes a “Calling Card” the transcription factor leaves behind to record its visit to the genome. The locations of the Calling Cards can be determined by massively-parallel DNA sequencing. We show that the transcription factor SP1 fused to the piggyBac transposase directs insertion of the piggyBac transposon near SP1 binding sites. The locations of transposon insertions are highly reproducible, and agree with sites of SP1-binding determined by ChIP-seq. Genes bound by SP1 are more likely to be expressed in the HCT116 cell line we used, and SP1-bound CpG islands show a strong preference to be unmethylated. This method has the potential to trace transcription factor binding throughout cellular and organismal development in a way that has heretofore not been possible. These data contain mapped insertion sites from the piggyBac (PB) transposon into HCT116 cell line and sequenced using an Illumina GAII analyzer. The first set contains the insertion sites of transposons mapped from the wild-type PB transposase and the second set contains the insertion sites of transposons mapped with the PB transposase fused to the transcription factor SP1. Other sequencing data present are ChIP-seq data for SP1 in the HCT116 cell line and RNA-seq data.
Project description:The PAF complex (Paf1C) has been shown to regulate chromatin modifications, gene transcription, and PolII elongation. Here, we provide the first genome-wide analysis of chromatin occupancy by the entire PAF complex in mammalian cells. We show that Paf1C is recruited not only to promoters and gene bodies, but also to regions downstream of cleavage/polyadenylation (pA) sites at 3’ ends, a profile that sharply contrasted with the yeast complex. Remarkably, our studies identified novel, subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation (APA) and upstream antisense transcription. Moreover, we found that depletion of Paf1C subunits also resulted in the accumulation of RNA polymerase II (PolII) over gene bodies, which coincided with APA. Depletion of specific Paf1C subunits leads to global loss of histone H2B ubiquitylation, but surprisingly, there is little impact of Paf1C depletion on other histone modifications, including the tri-methylation of histone H3 on lysines 4 and 36 (H3K4me3 and H3K36me3), previously associated with this complex. Our results provide surprising differences with yeast, while unifying observations that link Paf1C with PolII elongation and RNA processing, and suggest that Paf1C could play a role in protecting transcripts from premature cleavage by preventing PolII accumulation at TSS-proximal pA sites. ChIP-seq, RNA-seq and 3'READS of Paf1C factors in mouse C2C12 myoblast cells