Browse
Submit Data
Databases
API
Help

Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

43 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Saccharomyces cerevisiae 3' poly(A) site mapping

ABSTRACT: 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).

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Aino Järvelin

PROVIDER: E-GEOD-40110 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Json Xml

Publications

An efficient method for genome-wide polyadenylation site mapping and RNA quantification.

Wilkening Stefan S Pelechano Vicent V Järvelin Aino I AI Tekkedil Manu M MM Anders Simon S Benes Vladimir V Steinmetz Lars M LM

Nucleic acids research 20130107 5

The use of alternative poly(A) sites is common and affects the post-transcriptional fate of mRNA, including its stability, subcellular localization and translation. Here, we present a method to identify poly(A) sites in a genome-wide and strand-specific manner. This method, termed 3'T-fill, initially fills in the poly(A) stretch with unlabeled dTTPs, allowing sequencing to start directly after the poly(A) tail into the 3'-untranslated regions (UTR). Our comparative analysis demonstrates that it ...[more]

PMID: 23295673

Similar Datasets

Cleavage Factor Im as a key regulator of 3’ UTR length

Project description:In eukaryotes, the 3' ends of RNA polymerase II-generated transcripts are made in the majority of cases by site-specific endonucleolytic cleavage, followed by the addition of a poly(A) tail. By alternative polyadenylation, a gene can give rise to multiple mRNA isoforms that differ in the length of their 3' UTRs and hence in their susceptibility to post-transcriptional regulatory factors such as microRNAs. A series of recently conducted high-throughput studies of poly(A) site usage revealed an extensive tissue-specific control of 3’ UTR length and drastic changes in 3’ UTR length of mRNAs upon induction of proliferation in resting cells. To understand the dynamics of polyadenylation site usage, we recently identified binding sites of the major pre-mRNA 3’ end processing factors - cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and cleavage factor Im (CF Im) - and mapped cleaved polyadenylation sites in HEK293 cells. Our present study extends previous findings on the role of CF Im in alternative polyadenylation and reveals that subunits of the CF Im complex generally control 3’ UTR length. More specifically, we demonstrate that the loss-of-function of CF Im68 and CF Im25 but not of CF Im59 leads to a transcriptome-wide increase of the use of proximal polyadenylation sites. 3' ends of transcripts were profiled by high-throughput sequencing in HEK 293 cells under normal conditions, and in HEK 293 cells depleted of 3' end processing factors CF Im25, CF Im59, and CF Im68.

2012-10-30 | E-GEOD-40137 | biostudies-arrayexpress

Genome?wide analysis of pre?mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length: A-seq

Project description:Through alternative polyadenylation, human mRNAs acquire longer or shorter 3' untranslated regions, the latter typically associated with higher transcript stability and increased protein production. To understand the dynamics of polyadenylation site usage, we mapped transcriptome?wide both binding sites of 3' end processing factors CPSF?160, CPSF?100, CPSF?73, CPSF?30, Fip1, CstF?64, CstF-64tau, CF Im25, CF Im59, and CF Im68 and 3' end processing sites in HEK293 cells. We found that although binding sites of these factors generally cluster around the poly(A) sites most frequently used in cleavage, CstF?64/CstF-64tau and CF Im proteins have much higher positional specificity compared to CPSF components. Knockdown of CF Im68 induced a systematic use of proximal polyadenylation sites, indicating that changes in relative abundance of a single 3' end processing factor can modulate the length of 3' untranslated regions transcriptome-wide, and suggesting a mechanism behind the previously observed increase in tumor cell invasiveness upon CF Im68 knockdown. 3' ends of transcripts were profiled by high-throughput sequencing in HEK 293 cells under normal conditions, and in HEK 293 cells depleted of 3' end processing factors CF Im 68 and CstF-64.

2012-05-15 | E-GEOD-37037 | biostudies-arrayexpress

Gene expression profiling in allele replacement panel strains containing all combinations of four sporulation QTN in S. cerevisiae

Project description:We study the effect of four QTN in RME1, IME1 & RSF1 that are causative for variation in sporulation efficiency. We investigate the relationship between genotype, gene expression and phenotype and whether the amount of gene expression variation explained by the sporulation QTN is predictive of the amount of phenotypic variation explained by them. RNA-Seq analysis of 4 replicates each of 16 allele replacement panel strains containing all combinations of the four sporulation QTN after 2 hours in sporulation medium.

2014-05-30 | E-GEOD-55409 | biostudies-arrayexpress

POS-1 protects posterior gut specification by blocking GLD-3/2 polyadenylation of anterior factor neg-1

Project description: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

2015-05-25 | E-GEOD-57993 | biostudies-arrayexpress

Global analyses of how 3' UTR-isoform choice influences mRNA stability and translational efficiency

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

2013-02-28 | E-GEOD-44698 | biostudies-arrayexpress

Ribosome profiling of early zebrafish embryos -- miRNA-mediated regulation during embryogenesis causes translational repression before mRNA decay

Project description:MicroRNAs regulate gene expression through deadenylation, repression and mRNA decay. However, the contribution of each mechanism in non-steady-state situations remains unclear. We monitored the impact of miR-430 on ribosome occupancy of endogenous mRNAs in wild type and dicer mutants lacking mature miR-430. Our results indicate that miR-430 reduces the number of ribosomes on target mRNAs before causing mRNA decay. Translational repression occurs before complete deadenylation, and disrupting deadenylation using an internal poly(A) tail did not block target repression. Finally, we observe that ribosome density along the length of the target mRNA remains constant, suggesting that translational repression occurs by reducing the initiation rate rather than reducing elongation or causing ribosomal drop-off. In summary, our results show that miR-430 regulates translation initiation before inducing mRNA decay. Time course parallel ribosome profiling and input mRNA quantification in wildtype and MZdicer mutant embryos

2012-03-15 | E-GEOD-34743 | biostudies-arrayexpress

BATSeq of ESC-FCS/ESC-2i/NSC

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.

2015-06-09 | E-GEOD-60768 | biostudies-arrayexpress

Poly(A) tail length profiling of mRNAs from fission yeast

Project description:To obtain global data on polyadenylation of mRNAs, we fractionated the mRNAs according to their poly(A) tail length using a poly-U sepharose column followed by differential elution at five temperatures. Five mRNA fractions with distinct ranges of poly(A) tail length were then hybridized to microarrays using total eluate as a reference.

2007-05-16 | E-TABM-217 | biostudies-arrayexpress

The systematic shortening of 3’ UTRs has limited influence on the relative change in protein abundance in proliferating cells

Project description:Alternative polyadenylation is an important cellular mechanism that enables generation of mRNA isoforms that differ in their 3' untranslated regions (3' UTRs) and consequently in their susceptibility to miRNA and RNA binding protein mediated regulation. A dramatic change in polyadenylation site usage, leading to the systematic expression of short 3’ UTR isoforms is known to occur upon induction of proliferation in resting cells. To understand the functional consequences of short 3’ UTR isoform expression we used 3' end sequencing and quantitative mass spectroscopy to determine polyadenylation site use, mRNA and protein levels in murine and human naive and activated T cells. We found that while the process and its impact on the susceptibility to miRNA and RNA binding protein mediated regulation are evolutionarily conserved, the conservation is poor at the level of individual orthologous genes. Contrary to the common belief, we did not find that transcriptome-wide 3' UTR shortening leads to a matched increase in mRNA and protein levels of genes with tandem polyadenylation sites. 3' ends of transcripts were profiled by high-throughput sequencing in murine and human naive and activated T cells.

2014-09-25 | E-GEOD-54950 | biostudies-arrayexpress

Transcription profiling of a S96xSK1 yeast cross and its pool of spores.

Project description:To dissect local regulation into cis and trans contribution, we measured allelic differential expression in a hybrid cross of two yeast strains and compared it against allelic differential expression in a pool of spores of the same cross. RNA- and DNA-seq of a diploid S. cerevisiae hybrid and its haploid spores.

2015-02-04 | E-GEOD-61553 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data