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Effect of Nop53 depletion on the exosome interactome

ABSTRACT: In order to evaluate how Nop53 modulates the interaction of Rrp6 and Rrp43 with other factors, a coimmunoprecipitation assay was performed both in the presence (-doxicycline) and upon depletion (+ doxicycline) of Nop53 using the condional strain Δnop53/tetOff::GFP-NOP53 carrying the Rrp6-TAP or Rrp43-TAP fusion. As a negative control, the same strain carrying only the TAP tag was employed. For each condition, the elution of biological triplicates was subjected to label free quantitative analysis.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Felipe Bagatelli

LAB HEAD: Carla C. Oliveira

PROVIDER: PXD014187 | Pride | 2021-09-08

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	MyPatternLabProject-TechnicalReplicateRRP43.plp	Other
	MyPatternLabProject-TechnicalReplicateRRP6.plp	Other
	NC_1.sepr	Other
	NC_2.sepr	Other
	NC_3.sepr	Other

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Publications

The ribosome assembly factor Nop53 controls association of the RNA exosome with pre-60S particles in yeast.

Cepeda Leidy Paola P LPP Bagatelli Felipe F M FFM Santos Renata M RM Santos Marlon D M MDM Nogueira Fabio C S FCS Oliveira Carla C CC

The Journal of biological chemistry 20191029 50

Eukaryotic ribosomal biogenesis is a high-energy-demanding and complex process that requires hundreds of <i>trans</i>-acting factors to dynamically build the highly-organized 40S and 60S subunits. Each ribonucleoprotein complex comprises specific rRNAs and ribosomal proteins that are organized into functional domains. The RNA exosome complex plays a crucial role as one of the pre-60S-processing factors, because it is the RNase responsible for processing the 7S pre-rRNA to the mature 5.8S rRNA. T ...[more]

PMID: 31662437

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CLIP-seq of S. cerevisiae Drn1-TAP and Dbr1-TAP

Project description:Data for replicate Drn1-TAP and Dbr1-TAP CLIP-seq experiments to identify RNA-protein interactions Drn1-TAP and Dbr1-TAP CLIP-seq

2013-06-28 | E-GEOD-44959 | biostudies-arrayexpress

Whi3 RIP-Chip

Project description:Whi3 associated mRNAs were identified by immunoprecipitation of TAP-tagged Whi3 followed by microarray analysis RNA IP of Whi3-TAP and Whi3-dRRM-TAP with total RNA as reference. Each sample has 2 replicates

2013-10-31 | E-GEOD-51784 | biostudies-arrayexpress

ChIP-nexus analysis of fission yeast transcription factors Cbf11, Cbf11DBM, Cbf12, Cbf12DBM, Mga2 and Fkh2

Project description:Genome-wide identification of transcription factor (TF) binding sites in the genome of the fission yeast Schizosaccharomyces pombe. The ChIP-nexus method was used. TFs included were: Cbf11-TAP and Cbf12-TAP (and their DBM mutants with impaired DNA binding), TAP-Mga2, and Fkh2-TAP (as an irrelevant control TF). IPs from an untagged WT strain were also analyzed. Cbf11-related IPs were performed from exponential cultures, while Cbf12-related IPs were performed from stationary cultures. YES complex medium was used for all cultivations.

2024-04-05 | E-MTAB-13258 | biostudies-arrayexpress

Transcription profiling by array of human MCF-7 breast cell clones expressing ER-beta tagged with TAP-tag at the C-terminal or N-terminal

Project description:MCF-7 TET Off cells (MCF-7 wt) were used to produce stable clones expressing ER-beta tagged with TAP-tag respectively at the C-term and at the N-term (C-TAP-ER-beta and N-TAP-ER-beta) or expressing ER-alpha tagged (C-TAP-ER-alpha) as previously described.Cells were cultured in standard growth conditions, then were lysed and RNA was extracted using TRIzol method.Total RNA were fluorescently labelled, amplified and hybridized in triplicate (MCF7-TAP and C-TAP-ER-alpha) and in duplicate (C-TAP-ER-beta_A, C-TAP-ER-beta_B, N-TAP-ER-beta) for 18 hours on Illumina v2 MicroRNA Expression BeadChips and after scanning, analysis was performed with GenomeStudio v.2010.1 software, for quality control and miRNA expression analysis.

2011-10-11 | E-TABM-1053 | biostudies-arrayexpress

Identification of protein-protein interactions involving the eIF4E binding proteins Caf20p and Eap1p on yeast via affinity purification and LC-MSMS.

Project description:There are multiple translational control pathways. These include pathways involving eIF4E binding proteins (4E-BPs), which inhibit translation by binding and sequestering the 5’ cap binding protein eIF4E away from its partner eIF4G. Saccharomyces cerevisiae has two 4E-BPs: Caf20p and Eap1p. Previous analyses had shown that each 4E-BP regulates different subsets of mRNAs. In order to assess whether binding different proteins caused their different regulatory role, we used tandem affinity purification followed by label-free mass spectrometry to compare the proteomes pulled-down with the TAP-tagged 4E-BPs, and the global proteome. These analyses point out that Caf20p and Eap1p share most interaction partners, including ribosomes, and that both bind several other RNA-binding proteins.

2016-06-29 | PXD001407 | Pride

Genomic targets sites of the Candida glabrata transcription factor Pdr1

Project description:Pdr1 is the major regulator of azole resistance in the fungal pathogen Candida glabrata. Earlier experiments demonstrated that expression of Pdr1 itself is increased when cells lose their mitochondrial genome (rho0). Here we use chromatin immunoprecipitation coupled with highthroughput sequencing (ChIP-seq) to map the genomic binding sites for Pdr1 in both normal and rho0 cells. These data provide the first look at genes that are likely to represent the direct targets of Pdr1 in this important pathogen. Examination of Pdr1 binding sites using two different antibodies in five different strains. Strains labeled TAP carry the TAP-tagged allele and capture was done with anti-TAP. The control was performed by carrying out a ChIP reaction on rho0 TAP-PDR1 cells but with no primary anti-TAP antibody. Since Pdr1 expression is the highest in this background, this was selected as the best control for antibody specificity. We also performed ChIP reactions on rho+, rho0 and cells lacking the PDR1 gene (pdr1D) cells using the anti-Pdr1 antibody. The control for these reactions was a ChIP experiment using anti-Pdr1 antibody on cells lacking the endogenous Pdr1 protein.

2014-09-12 | E-GEOD-59839 | biostudies-arrayexpress

Yeast exosome: immunoprecipitation 2

Project description:Nuclear RNA degradation pathways are highly conserved across eukaryotes and play important roles in RNA quality control. Key substrates for exosomal degradation include aberrant functional RNAs and cryptic unstable transcripts (CUTs). It has recently been reported that the nuclear exosome is inactivated during meiosis in budding yeast through degradation of the subunit Rrp6, leading to the stabilisation of a subset of meiotic unstable transcripts (MUTs) of unknown function. We have analysed the activity of the nuclear exosome during meiosis by deletion of TRF4, which encodes a key component of the exosome targeting complex TRAMP. We find that TRAMP mutants produce high levels of CUTs during meiosis which are undetectable in wild-type cells, suggesting that the nuclear exosome remains functional, and we further show that the meiotic exosome complex still contains Rrp6. Indeed Rrp6 over-expression is insufficient to suppress MUT transcripts, showing that the reduced amount of Rrp6 in meiotic cells does not directly cause MUT accumulation. Lack of TRAMP activity stabilises ~1600 CUTs in meiotic cells, which occupy 40% of the binding capacity of the nuclear cap binding complex (CBC). CBC mutants display defects in the formation of meiotic double strand breaks (DSBs), and we see similar defects in TRAMP mutants, suggesting that a key function of the nuclear exosome is to prevent saturation of the CBC complex by CUTs. Together, our results show that the nuclear exosome remains active in meiosis and has an important role in facilitating meiotic recombination.

2015-05-15 | PXD001239 | Pride

Yeast exosome: immunoprecipitation 1

2015-05-15 | PXD001223 | Pride

MicroRNA profiling by array of human MCF-7 breast cancer cells with ER-beta tagged at the C-terminal or N-terminal and treated with 17-beta-estradiol

Project description:Human estrogen-responsive breast cancer cell line MCF-7 TET Off (MCF-7 wt) were used to produce stable clones expressing ER-beta tagged with TAP-tag respectively at the C-term and at the N-term (C-TAP-ER-beta and N-TAP-ER-beta) or expressing ER-alpha tagged (C-TAP-ER-alpha) as previously described.(C-TAP-ER-beta, N-TAP-ER-beta, C-TAP-ER-alpha) were treated with 10nM of17-beta-estradiol, or vehicle (ETOH). miRNA expression was analyzed on total RNA extracted before or after 6, 12, 24, and 72 hours hormonal stimulation. Total RNA was fluorescently labelled, amplified in triplicate, to be, than, pooled for the Hybridization.Each pool, were hybridized for 18 hours on Illumina v2 MicroRNA Expression BeadChips, and after scanning, analysis was performed with GenomeStudio v.2010.1 software, for quality control and miRNA expression analysis.

2011-07-29 | E-TABM-1055 | biostudies-arrayexpress

Microarray analysis of TAP::ALG-1 associated RNAs isolated from synchronized 'wild-type' animals and 'mir-58' mutants

Project description:To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein (C. elegans strain WS4303). We crossed the TAP::ALG-1 transgene into the mir-58(n4640) mutant background to generate the strain WS5041. For simplicity, we will hereafter term the TAP::ALG-1 transgenic animals as wild typeand the transgenic WS5041 animals as mir-58. We compared the mRNA population that coimmunopurified with TAP::ALG-1 from synchronized L4 stage wild-type animals with that from synchronized L4 stage mir-58 mutant animals by one-color Affymetrix gene arrays. miR-58 target mRNAs should be specifically underrepresented in the latter samples. Strains WS4303 (wt) and WS5041 (mir-58) were used for TAP::ALG-1 IPs. All experiments were conducted in three independent replicates. For each replicate, WS4303 and WS5041 were grown in parallel. 150 ng of TAP::ALG-1 associated RNA isolated from synchronized late L4 animals were sent to the GeneCore facilty in Heidelberg, Germany (http://www.genecore.embl.de/index.cfm), and the microarray data were generated according to their standard protocol (Weinmann et al. 2009).

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