Changes in axonal and somatic transcriptomes in response to Pum2 knockdown
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA557265 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9858320_1.fastq.gz | Fastqsanger.gz | |||
| SRR9858320_2.fastq.gz | Fastqsanger.gz | |||
| SRR9858321_1.fastq.gz | Fastqsanger.gz | |||
| SRR9858321_2.fastq.gz | Fastqsanger.gz | |||
| SRR9858322_1.fastq.gz | Fastqsanger.gz |
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Project description:We report that knockdown of Pum2 results in an increase of PBE containing mRNAs in axons, without changing mRNA levels in the cell bodies.
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2015-07-14 | E-GEOD-70860 | biostudies-arrayexpress
Project description:We grew neurons in microchambers to separate axons and soma in different compartments which could be independently harvested for proteomic analysis. Using this model system, we studied the effect of the knock-out of hnRNP R on the axonal and somatic proteome.
2024-08-22 | PXD038368 | Pride
Project description:Purpose: PUMILIO proteins are known to repress target genes by binding to PUMILIO response elements (PREs) in target mRNAs. The goal of this study was to demonstrate binding of endogenous PUM2 to the noncoding RNA NORAD and to identify PUM2 target genes in NORAD wild-type and knockout HCT116 cells. Methods: PAR-CLIP was performed with endogenous PUM2 in HCT116 cells and isogenic NORAD knockout cells. Results: Endogenous PUM2 binds to NORAD in HCT116 cells. In addition, PUM2 target genes were identified in HCT116 cells. Conclusions: PUM2 binds to NORAD through multiple PREs on NORAD. Compared to all other PUM2 target genes in HCT116, NORAD is the preferred binding partner of endogenous PUM2.
2015-12-03 | GSE75439 | GEO
Project description:The functions of human PUM1 and PUM2 are considered to be redundant given that both PUF1 and PUF2 recognize the same PBE motif UGUANAUA. However pools of mRNAs published so far for PUM1 and PUM2 do not overlap. Therefore we sought to investigate the issue of redundancy in human cells. The both PUM proteins are less conserved in the region that is outside the PUM domain. We sought that interactors could be different. We identified mRNA pools binding separately PUM1 and PUM2 by RIP-Seq approach, normalized them using the whole TCam-2 cells transcriptome and aligned them with mRNA pools activated or repressed as tested by siRNA PUM1 and PUM2 knockdown.
2018-11-28 | GSE123016 | GEO
Project description:This SuperSeries is composed of the following subset Series: GSE12351: mRNAs associated with human PUM1 protein GSE12352: mRNAs associated with human PUM2 protein Refer to individual Series
2008-09-08 | E-GEOD-12357 | biostudies-arrayexpress
Project description:The presence of the PUF (Pumilio/FBF) domain defines a conserved family of RNA-binding proteins involved in repressing gene expression. It has been suggested that a conserved function of PUF proteins is to repress differentiation and sustain the mitotic proliferation of stem cells. In humans, Pumilio2 (PUM2) is expressed in embryonic stem cells and adult germ cells. To identify mRNAs associated with human PUM2 protein in adipose tissue stem cells (ADSC), we used a modified Ribonucleoprotein-ImmunoPrecipitation Microarray (RIP-Chip). PUM2 ribonucleoprotein (RNP) complexes were performed with 2 µg of anti-Pum2 antibody (goat polyclonal, Santa Cruz Biotechnology, CA, USA) bound to protein G-agarose beads (Sigma, Deisenhofen, Germany). ADSCs were lysed in polysome lysis buffer (Tris-HCl pH 7.4 15mM, MgCl2 15 mM, NaCl 0,3 M, 1% Triton X-100, 1 mM DTT, 100 U/ml RNase Out, PMSF 1mM and E64 10uM) for one hour at 4°C. Beads were washed, then buffer and cell lysate were added, and the reaction mixtures were tumbled for 2 hours at 4°C. After this incubation, the beads were thoroughly washed again with polysome lysis buffer and then either RNA extracted for microarray and RT-PCR experiments using the RNeasy mini kit (Qiagen). To control for non-specifically enriched RNAs, identical IPs were performed with beads precoated with preimmune goat serum as a negative control. RNA was processed for hybridization with GeneChip 3’ IVT Express (Affymetrix - Santa Clara, USA), according to the manufacturers instruction. Briefly, cDNA was synthesized from immunoprecipitated RNA using reverse transcriptase followed by second strand synthesis to generate double-stranded cDNA. An in vitro transcription reaction was used to generate biotinylated cRNA. After purification and fragmentation, cRNA was hybridized onto GeneChip Affymetrix Human Genome U133 Plus 2.0 arrays. Post hybridization washes were preformed on an Affymetrix GeneChip Fluidics Station 450. Arrays were scanned on an Affymetrix GeneChip Scanner 3000. Scanned arrays were normalized using GCRMA in Partek software (Partek Incorporated. St. Louis, MO). Differentially enriched RNAs were found after performing One-way ANOVA analysis comparing immunoprecipitated samples against control samples. Final lists of genes were obtained by filtering the data from the statistical results according to fold enrichment more than 2 and a p value associated of less than 0.05 for features with signal well above the background. Pum2 IP and control IP samples were analyzed for each of 2 biological replicates.
2011-07-31 | E-GEOD-26626 | biostudies-arrayexpress
Project description:To identify mRNAs associated with human PUM2 protein, we used a modified Ribonucleoprotein-ImmunoPrecipitation Microarray (RIP-Chip) approach on HeLa S3 cancer cells that express PUM2. PUM ribonucleoprotein (RNP) complexes were captured from cell-free extracts with specific anti PUM2 antibody coupled to protein A sepharose beads, and then eluted with SDS-EDTA. To control for non-specifically enriched RNAs, the same procedure was performed with beads that were not coupled with immunoprecipitating antibodies (mock samples). Total RNA was isolated from cell extracts and immunopurified samples with the mirVanaTM PARISTM kit (Ambion). RNA was quantified with a NanoDrop device (Witeg AG). Poly-adenylated RNAs were amplified in the presence of aminoallyl-UTP with Amino Allyl MessageAmp II aRNA kit (Ambion). For this purpose, 500 ng total RNA from extracts and half (50-100 ng) of the immunopurified RNAs were used for amplification. 8 ug of the amplified RNAs (aaRNA) were fluorescently labeled with NHS-monoester Cy3 and Cy5 dyes (GE HealthSciences), except for mock RNA samples, where an aaRNA amount proportional to the yield obtained from corresponding PUM affinity isolates was used.For PUM2 RIPs, we performed four biological replicates but omitted the dye swaps due to the lower aaRNA obtained after amplification (~10 ug aaRNA from PUM2 RIPs, 9 ug aaRNA from mock RIPs). The Cy3- and Cy5-labeled aaRNA samples were mixed and hybridized to human cDNA microarrays. Detailed methods for microarray experiments are available at http://cmgm.stanford.edu/pbrown/protocols/index.html. cDNA microarrays were produced by the Stanford Functional Genomic Facility and contained 43,197 human probes representing 26,524 Unigene cluster IDs (12,466 ENSEMBL annotated genes) spotted on Corning Ultra GAPS slides. Spotted cDNAs were cross-linked with 65 mJ of UV irradiation on slides, which were then post-processed for 1 hour at 42°C in pre-hybridization solution (5x SSC, 0.1% SDS, 0.1 mg/ml BSA), washed twice in 400 ml of 0.1x SSC for 5 min, dunked in 400 ml ultrapure water for 30 sec, and dried by centrifugation at 550 rpm for 5 min. Slides were used the same day. Cy3- and Cy5-labeled aaRNA probes were mixed and applied to arrays in hybridization solution (3x SSC, 20 ug poly(A) RNA [Invitrogen], 20 ug yeast tRNA [Invitrogen], 20 ug Human Cot-1 DNA [Invitrogen], 20 mM HEPES [pH 7.0] and 0.3% SDS) for 18 h at 65°C. The arrays were then washed sequentially in 400 ml of 2x SSC with 0.1% SDS, 1x SSC, and 0.2x SSC. The first wash was performed for 5 min at 65°C, the subsequent washes were performed for 5 min at RT. The arrays were dried by centrifugation and immediately scanned with an AxonScanner 4200A (Molecular Devices). Data were collected using GENEPIX 5.1 (Molecular Devices). Arrays were normalized computationally by the Stanford Microarray Database (SMD). The data were filtered for signal over background of greater than 1.5 in the channel measuring aaRNA from extract, and only features that met these criteria in > 50% of the arrays were included for further analysis. Log2 median ratios were retrieved and exported into Microsoft Excel. To identify transcripts that were specifically enriched by association with PUM2, we performed two class Significance Analysis of Microarrays (SAM) on median centered arrays. A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments. Antibody used in IP: 50 ug rabbit anti-PUMILIO 2 (Bethyl Laboratories, #300-202A) Keywords: replicate_design Computed
2008-09-08 | E-GEOD-12352 | biostudies-arrayexpress
Project description:Expression analysis in HeLa stable cell clones expressing a control shRNA (SCR) or two independent shRNAs targeting PUM2
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Project description:Local protein synthesis in sensory neuron axons is necessary for axonal regeneration with the efficiency of regeneration decreasing with age. Because the full repertoire of transcripts in embryonic and adult rat sensory axons is unknown we asked how the pool of mRNAs dynamically changes during ageing. We isolated mRNA from pure axons and growth cones devoid of non-neuronal or cell body contamination. Genome-wide microarray analysis reveals that a previously unappreciated number of transcripts are localised in sensory axons and that this repertoire changes during development toward adulthood. Embryonic sensory axons are enriched in transcripts encoding cytoskeletal-related proteins with a role in axonal outgrowth. Surprisingly, adult axons are highly enriched in mRNAs encoding immune molecules with a role in nociception. To validate our experimental approach we show that Tubulin-beta3 mRNA is present only in embryonic axons where it is locally synthesised. In summary, we show that the population of axonal mRNAs dynamically changes during development, which may partly contribute to the intrinsic capacity of axons at different ages to regenerate after injury and to modulate pain. Pure axonal RNA were extracted from the axons of embryonic and adult dorsal root ganglion neurons, each with 5 biological replicates. The axonal transcriptomes were analysed using Affymetrix Rat Genome 230 2.0 Arrays.
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