PXD000342

PXD000342 is an original dataset announced via ProteomeXchange.

Title	SILAC dat for two TERRA pull-down experiments using different salt conditions (1 M NaCl and 250 mM NaCl)
Description	ABSTRACT: Telomeres are actively transcribed into telomeric repeat-containing RNA (TERRA), which has been implicated in the regulation of telomere length and heterochromatin formation. Here, we applied quantitative mass spectrometry (MS)-based proteomics to obtain a high confidence interactome of TERRA. Using SILAC-labeled nuclear cell lysates in an RNA pull-down experiment, and two different salt conditions, we distinguished 115 proteins binding specifically to TERRA out of a large set of background binders. While TERRA binders identified in two previous studies showed little overlap, using quantitative mass spectrometry we obtain good overlap to both studies. To test whether novel candidates found here are involved in TERRA regulation, we performed an esiRNA-based interference analysis for 15 of them. Knock-down of ten proteins significantly affected total cellular levels of TERRA and RNAi of five candidates perturbed TERRA recruitment to telomeres. Notably, depletion of SRRT/Ars2, involved in miRNA processing, up-regulated both total and telomere-bound TERRA. Conversely, knock-down of MORF4L2, a component of the NuA4 histone acetyltransferase complex, reduced TERRA levels both globally and for telomere-bound TERRA. We thus identified new proteins involved in the homeostasis and telomeric abundance of TERRA, extending our knowledge of TERRA regulation. MS SECTION: Pull-downs were separated on a 4-12% NOVEX gradient SDS gel (Life Technologies) for 1 hour at 170V in 1x MOPS buffer (Life Technologies). Proteins were fixated and stained with the Colloidal Blue staining Kit (Life Technologies). The gel lane was cut into 8 slices, minced and de-stained with a 50 percent Ethanol/50 mM ABC solution. Proteins were reduced in 10 mM DTT for 1 hour at 56 °C and then alkylated with 55 mM iodoacetamide for 1 hours at room temperature. Proteins were digested with trypsin (Promega) at 37 °C overnight. Peptides were extracted from the gel using acetonitrile which was subsequently removed in a concentrator (Eppendorf) and loaded on stage tips for storage. For mass spectrometric analysis, peptides were separated on a C18 column with 75 µm diameter packed with 3 µm Reprosil beads (Dr. Maisch) mounted to a EASY HPLC (Thermo Fisher) and sprayed online into an Orbitrap-XL mass spectrometer (Thermo Fisher). We used a 120 minutes gradient from 2% to 60% acetonitrile in 0.5% acetic acid at a flow of 200 nl/minutes. The mass spectrometer was operated with CID fragmentation for top10 data-dependent MS/MS per full scan. Mass spectrometry raw data was searched using the Andromeda search engine (Cox et al. 2011) integrated into MaxQuant suite 1.2.2.0 (Cox and Mann 2008) using the IPI human database v3.68. Carbamidomethylation at cysteine was set as fixed modification while methionine oxidation and protein N-acetylation were considered as variable modifications. The search was performed with an initial mass tolerance of 7 ppm mass accuracy for the precursor ion and 0.5 Da (CID) for the MS/MS spectra. Search results were processed with MaxQuant and identification up to a false discovery rate of 0.01 were accepted.
HostingRepository	PRIDE
AnnounceDate	2013-08-02
AnnouncementXML	Submission_2013-08-02_01:06:33.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	F Butter
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	iodoacetamide derivatized residue; monohydroxylated residue; acetylated residue
Instrument	LTQ Orbitrap

Revision	Datetime	Status	ChangeLog Entry
0	2013-07-15 01:49:54	ID requested
⏵ 1	2013-08-02 01:06:33	announced
⏵ 1	2014-07-25 01:23:03	announced

Dataset with its publication pending

submitter keyword: SILAC, RNA pull-down

F Butter
contact affiliation	Dept. of Proteomics and Signal Transduction
contact email	f.butter@imb-mainz.de

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