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Translational specialization in pluripotency by RBPMS poises future lineage-decisions

ABSTRACT: Labeling active ribosome associated proteins by L-Azidohomoalanine (AHA)- pulldown. Humane embryonic stem cells were labeled via AHA in methionine-free medium for 40 min. AHA labeled nascent proteins together with ribosomes and associated proteins were isolated with beads. Samples were divided into ribosome plus associated protein fraction and nascent protein fraction by 8M UREA wash.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Prerana Wagle

LAB HEAD: Leo Kurian

PROVIDER: PXD032046 | Pride | 2023-10-26

REPOSITORIES: Pride

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Translational specialization in pluripotency by RBPMS poises future lineage-decisions

Project description:Immunoprecipitation for RBPMS in human embryonic stem cells. Triplicates of sheep anti-rabbit beads coupled with rabbit anti-RBPMS antibody were compared with and triplicates sheep anti-rabbit beads, which served as IgG control. Bands above heavy IgG, between heavy IgG and light IgG, below IgG and both IgG bands combined were excised from a gel and labeled via dimethyl labeling, before LC-MS run to increase detection resolution.

2023-10-26 | PXD032040 | Pride

Translational specialization in pluripotency by RBPMS poises future lineage-decisions

Project description:Total proteome for triplicates of WT and RBPMS KO hESCs to identify differences in proteins abundances due to a loss of RBPMS. Cells were lysed and prepared according to the in solution digest protocol and 50 µg proteins were reduced, alkylated and digested with Lys-C and trypsind.

2023-05-10 | PXD032045 | Pride

RNA-seq of total and nascent RNA after depletion of Mpe1

Project description:To test the role of Mpe1 in transcription termination we inserted a mini-auxin induced degron (mAID) at the C-terminal end of the endogenous MPE1 locus in the yeast Saccharomyces cerevisiae. We treated Mpe1-mAID cells (JRY101) or wild type cells (WT, YMK728) with 1 mM auxin for 30 minutes and labeled the nascent RNA with 4-thiouracil (4tU) for 6 minutes. The nascent RNA fraction was prepared by biotinylating the 4tU-labeled RNA followed purification with streptavidin beads. Nascent and total RNA fractions were depleted of ribosomal RNA and strand-specific libraries prepared. Libraries were single-end sequenced using an Ilummina HiSeq 4000 instrument.

2021-12-31 | E-MTAB-10820 | biostudies-arrayexpress

Analysis of the nascent proteome during murine norovirus infection

Project description:SILAC- and AHA-labelling analysis of Raw264.7 cells infected with MNV-1 at MOI=10TCID50/cell and harvested at 6.5 and 10.5 hours post infection. AP-MS enrichment of AHA-labelled nascent polypeptides represents the first quantitative neoproteomics analysis of norovirus infected cells.

2021-07-02 | PXD025169 | Pride

AATF/Che-1 binds ribosome-associated RNAs and regulates ribosome biogenesis

Project description:AATF is a predominantly nuclear protein that has been shown to be an apoptosis-inhibiting factor essential for both embryonic development and tumor growth. Several studies have shown a role of AATF in the modulation of pivotal cellular signal transduction pathways such as p53-, mTOR- and HIF-signaling. However, the exact molecular functions underlying its essential nature to cell proliferation and survival have remained elusive. Interestingly, several lines of evidence point towards a pivotal role of this protein in ribosome biogenesis and the maturation of ribosomal RNA. The function of AATF in this process is not clear, especially since the identity of the RNA-binding protein that links the AATF-containing protein complex (ANN) to rRNA precursors is unknown. In this study, we identify AATF in a screen for RNA-binding proteins. Importantly, AATF does not only bind to polyA-tailed RNA but CLIP-experiments reveal its association with ribosomal RNA. The RNA-binding domain of AATF is essential to maintain its positive effect on cellular rRNA levels. Furthermore, AATF binds to both mRNAs encoding for ribosome biogenesis factors as well as proteins involved with this process indicating AATF to be a central hub for the coordination of ribosome maturation. Consequently, our data for the first time identify the molecular basis to the role of AATF in ribosome biosynthesis and corroborate the link between tumorigenesis and ribosome biology.

2019-08-13 | PXD011055 | Pride

Folding stabilities of ribosome-bound nascent polypeptides probed by mass spectrometry

Project description:The folding of most proteins occurs during the course of their translation while their tRNA-bound C-termini are embedded in the ribosome. How the close proximity of nascent proteins to the ribosome influences their folding thermodynamics remains poorly understood. Here, we have developed a mass spectrometry-based approach for determining the stabilities of nascent polypeptide chains using methionine oxidation as a folding probe. This approach enables quantitative measurements sub-global folding stabilities of ribosome nascent chains (RNCs) within complex protein mixtures and extracts. To validate the methodology, we analyzed the folding thermodynamics of three model proteins (DHFR, CheY and DinB) in soluble and ribosome-bound states. The data indicated that the ribosome can significantly alter the stability of nascent polypeptides. Ribosome-induced stability modulations were highly variable among different folding domains and were dependent on localized charge distributions within nascent polypeptides. The results implicated electrostatic interactions between the ribosome surface and nascent polypeptides as the cause of ribosome-induced stability modulations. The study establishes a robust proteomic methodology for analyzing localized stabilities within ribosome-bound nascent polypeptides and sheds light on how the ribosome influences the thermodynamics of protein folding.

2023-08-09 | PXD037995 | Pride

BrU-labeled nascent RNA sequencing and m6A-IP from BrU-labeled nascent RNA

Project description:BrU-labeled nascent RNA sequencing and m6A-IP from BrU-labeled nascent RNA

| PRJNA326350 | ENA

Long-read nascent RNA sequencing from S. pombe

Project description:Long-read SMRT cDNA sequencing of nascent RNA from exponentially growing S. pombe cells was employed to obtain transcription elongation and splicing information from single transcripts. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010). The nascent 3’ end was labeled with a 3’ DNA adaptor through ligation. The adaptor sequence served as template for full-length reverse transcription and double-stranded cDNA was obtained in a transcriptome-wide PCR. SMRT DNA sequencing libraries were prepared subsequently.

2018-05-18 | GSE104679 | GEO

Long read sequencing of nascent RNA from S. cerevisiae and S. pombe

Project description:Long read SMRT cDNA sequencing of nascent RNA from exponentially growing S. cerevisiae and S. pombe cells was employed to obtain transcription elongation and splicing information from single transcripts. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010). The nascent 3’ end was labeled with a 3’ DNA adaptor through ligation. The adaptor sequence served as template for full-length reverse transcription and double-stranded cDNA was obtained in a PCR (gene-specific or transcriptome-wide). SMRT DNA sequencing libraries were prepared subsequently.

2016-03-24 | GSE70906 | GEO

Long read sequencing of nascent RNA from S. cerevisiae and S. pombe

Project description:Long read SMRT cDNA sequencing of nascent RNA from exponentially growing S. cerevisiae and S. pombe cells was employed to obtain transcription elongation and splicing information from single transcripts. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010). The nascent 3â?? end was labeled with a 3â?? DNA adaptor through ligation. The adaptor sequence served as template for full-length reverse transcription and double-stranded cDNA was obtained in a PCR (gene-specific or transcriptome-wide). SMRT DNA sequencing libraries were prepared subsequently. Nascent RNA profiles for mainly intron-containing genes were generated with long-read SMRT cDNA sequencing.

2016-03-24 | E-GEOD-70906 | biostudies-arrayexpress

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