Browse
Submit Data
Databases
API
Help

Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

36 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

RNA-biotin based pull down assay with polyA site (PAS) RNA substrates in Hela Nuclear Extract followed by RNA-seq to discover trans-acting RNA molecules involved in mRNA 3 processing

ABSTRACT: We aimed to discover trans-acting RNA molecules involved in mRNA 3 processing. We reasoned that, if there exist such functional RNAs, they must directly associate with the key machinery responsible for mRNA 3 processing. Therefore, it would be of great value to comprehensively identify RNAs interacting with pre-mRNA 3 processing complex. To this goal, we took advantage of previously well-characterized system combined with high-throughput sequencing to investigate the target RNAs at the transcriptomic level. Briefly, we used two polyA site (PAS) RNA substrates, SV40 late (SVL) and adenovirus L3 pre-mRNAs, and corresponding control RNAs with point mutation (U to C) at the highly conserved cis-element AAUAAA. RNA substrates were first biotinylated at the 3 end, and then bound to the streptavidin magnetic beads. After incubation with Hela Nuclear Extract (NE) under polyadenylation condition, the two wild type RNA substrates could specifically recruit mRNA 3 processing factors in NE for complex assembly. The purification of the protein complex and its interacting RNAs were performed using biotin-streptavidin pull-down. we extracted RNAs from the pull-down sample, prepared the strand-specific RNA-Seq libraries and submitted them for deep-sequencing. See related experiments: E-MTAB-5384; E-MTAB-5389

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: chengguo yao

PROVIDER: E-MTAB-5383 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Json Xml

Similar Datasets

RNA-biotin based pull down assay with Hela Nuclear Extract followed by RNA-seq

Project description:We aimed to discover trans-acting RNA molecules involved in mRNA 3’ processing. We reasoned that, if there exist such functional RNAs, they must directly associate with the key machinery responsible for mRNA 3’ processing. Therefore, it would be of great value to comprehensively identify RNAs interacting with pre-mRNA 3’ processing complex. To this goal, we took advantage of previously well-characterized system combined with high-throughput sequencing to investigate the target RNAs at the transcriptomic level. Briefly, we used polyA site (PAS) RNA substrates, SV40 late (SVL), and corresponding control RNAs with point mutation (U to C) at the highly conserved cis-element AAUAAA. RNA substrates were first biotinylated at the 3’ end, and then bound to the streptavidin magnetic beads. After incubation with Hela Nuclear Extract (NE) under polyadenylation condition, the two wild type RNA substrates could specifically recruit mRNA 3’ processing factors in NE for complex assembly. The purification of the protein complex and its interacting RNAs were performed using biotin-streptavidin pull-down. We extracted RNAs from the pull-down sample, prepared the strand-specific RNA-Seq libraries and submitted them for deep-sequencing.

2017-07-01 | GSE98218 | GEO

Proteomic and Genomic Analysis of transcription factor TFII-I reveals insight into the response to cellular stress

Project description:We expressed biotin-tagged transcription factor TFII-I (GTF2I) in K562 cells and subjected crosslinked chromatin to streptavidin mediated pull down. We subjected the isolated DNA to Illumina sequencing. We identified 18920 TFII-I binding peaks. Nearest gene analysis revealed 5886 genes associated with a TFII-I binding peak in proliferating K562 cells. We generated two K562 single cell clones stably expressing bio-tagged TFII-I, and K562 cells expressing only the BirA protein biotin ligase from E.coli. These three clones were subjected to streptavidin mediated pull down and Illumina sequencing.

2014-05-14 | E-GEOD-51065 | biostudies-arrayexpress

Protein interactome of small RNA fragments: identification and functional implications

Project description:Populations of small eukaryotic RNAs, in addition to relatively well recognized molecules (such as miRNAs or siRNAs), also contain fragments derived from all classes of constitutively expressed non-coding RNAs. It has been recently demonstrated that the formation and accumulation of RNA fragments (RFs) is cell-/tissue-specific and depends on internal and external stimuli. Unfortunately, the mechanisms underlying RF biogenesis and function remain unclear. To better understand them, we employed RNA pull-down and mass spectrometry methods to characterize the interaction networks of seven RFs originating from tRNA, snoRNA and snRNA. In addition, we performed an in silico screen of the selected RFs against publicly available cross-linking and immunoprecipitation datasets. We determined that the RF interactome comprises a large number of proteins, which were generally different from those that interact with their parental full length RNAs. Proteins that were differentially bound by the RFs were involved in mRNA splicing, tRNA processing, DNA recombination/replication, protein biosynthesis and carbocyclic acid metabolism. Our data suggest that RFs can be endogenous aptamer-like molecules and potential players in emerging RNA-protein regulatory networks.

2018-10-24 | PXD010075 | Pride

Determination of the RNA linked to the paraspeckle in the GH4C1 cell line

Project description:The goal of this study was to determine the identity of the RNA linked to the paraspeckles in the rat GH4C1 cell line. GH4C1 cell were fixed with 1% paraformaldehyde in PBS. Then nuclei were purified, lysed and sonicated. RNA pull-down was performed using two antisense DNA biotinylated oligonucleotide probes that target the lncRNA Neat1. Streptavidin-magnetic beads were added to hybridization reaction and complexes were captured by magnets. RNA was isolated using NucleoSpinÂ®RNA XS (Macherey-Nagel).

2016-07-21 | E-GEOD-81972 | biostudies-arrayexpress

Biotin-modified cardiac-hypertrophy-associated piRNA (CHAPIR) pulldown proteins

Project description:PIWI-interacting RNAs (piRNAs) are abundantly expressed during cardiac hypertrophy development, but their influence on pathological hypertrophy and the underlying mechanisms remains to be elucidated. Here, we identified a cardiac-hypertrophy-associated piRNA (CHAPIR), and we found that it regulates pathological hypertrophy. To investigate the molecular mechanisms by which CHAPIR regulates cardiomyocyte hypertrophy, we transfected the biotinylated CHAPIR into cardiomyocytes and performed streptavidin bead pull down assay. The CHAPIR pull-down materials and its control were resolved using SDS-PAGE gel and stained with coomassie blue. Then the entire gel lanes of the CHAPIR pull-down materials and its control were excised and send for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

2024-07-04 | PXD020098 | Pride

Genome-Wide Study Based On Abasic Site Revealed Selective RNA Oxidation In Neuro 2a Cells

Project description:To investigate selectivity of mRNA oxidation, total RNA and oxidized RNA isolated from Neuro 2a cells before and after H2O2 treatment were employed for microarray analysis. It was found that selective oxidation of mRNA already occurs under normal culture conditions but was increased by H2O2, especially in a subset of mRNAs related to certain functions. Moreover, mRNA oxidation level is also related to its abundance or stability (half-life time). This shows for the first time that mRNA oxidation is associated with RNA homeostasis including function, stability and abundance depending on cellular redox status in a genome-wide scale. Neuro 2a cells received hydrogen peroxide treatment or no treatment as a control. Samples were applied for RNA extraction and ARP labeling, which could bind with apurinic/apyrimidinic sites, and then a pull-down process to isolate oxidized RNA. Total RNA and oxidized RNA were used for subsequent transcriptomic profiling. 4 types of samples were analyzed: Basal-total: untreated N2a cells labeled with ARP, but not processed for the pull-down assay. Ox-total: hydrogen peroxide-treated N2a cells labeled with ARP, but not processed for the pull-down assay. Basal-ARP: untreated N2a cells labeled with ARP, and processed for the pull-down assay. ARP-derivatized RNA, which is also oxidized RNA, was concentrated and used for the microarray analysis. Ox-ARP: hydrogen peroxide-treated N2a cells labeled with ARP, and processed for the pull-down assay. ARP-derivatized RNA, which is also oxidized RNA, was concentrated and used for the microarray analysis.

2012-10-01 | E-GEOD-39641 | biostudies-arrayexpress

HeLa mRNA interactome

Project description:Pull-down of poly(A)-mRNA cross linked proteins using two cross-linking methods (conventional cross-linking and PAR-cross-linking) to identify all mRNA-binding proteins (GO:0003729). The provided data is quantitative proteomic data for comparison of cross-linking and control samples.

2012-08-14 | E-MTAB-869 | biostudies-arrayexpress

NSun2-mediated cytosine-5 methylation in Vault non-coding RNA determines its processing into small RNAs [RNA-seq]

Project description:Autosomal-recessive loss of the NSUN2 gene has been recently identified as a causative link to intellectual disability disorders in humans. NSun2 is an RNA methyltransferase modifying cytosine-5 in transfer RNAs (tRNA). Whether NSun2 methylates additional RNA species is currently debated. Here, we adapted the individual-nucleotide resolution UV cross-linking and immunoprecipitation method (iCLIP) to identify NSun2-mediated methylation in RNA transcriptome. We confirm site-specific methylation in tRNA and identify messenger and non-coding RNAs as potential methylation targets for NSun2. Using RNA bisulfite sequencing we establish Vault non-coding RNAs as novel substrates for NSun2 and identified six cytosine-5 methylated sites. Furthermore, we show that loss of cytosine-5 methylation in Vault RNAs causes aberrant processing into argonaute-associating small RNA fragments (svRNA). Thus, impaired Vault non-coding RNA processing may be an important contributor to the etiology of NSUN2-deficieny human disorders. mRNA-seq in Embryonic kidney (HEK293) cells transfected with siRNA against Nsun2 vs control

2013-07-23 | E-GEOD-44384 | biostudies-arrayexpress

RNA MS2-tag based affinity method was used to purify 3' processing factors associated with intronic PAS processing

Project description:three copies of the MS2 hairpin were fused to the intronic PAS of the nr3c1 gene. To make a negative control RNA substrate, the core hexamer AAUAAA within PAS was mutated to AACAAA. Subsequently, the RNA substrate was incubated with HeLa NE, and the mRNA 3' processing complex was purified using an MS2 tag

2023-02-27 | PXD040405 |

The miRNA miR-139-5p is a regulator of metastatic pathways in breast cancer.

Project description:Whole transcriptome Identification of direct targets of miR-139-5p using biotinylated pull-downs found that this miRNA has roles in breast cancer invasion and migration. MCF7 cells were transfected with biotinylated miR-139-5p. The miRNAs and target mRNA were pulled down with streptavidin and compared to the input control.

2013-12-01 | E-GEOD-40411 | 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