Dataset Information

LC-MS/MS of co-immunoprecipitation of 3xFLAG-UFM1 from ribosomal pellet in control and upon anisomycin treatment

ABSTRACT: Clearance of arrested nascent polypeptides resulting from ribosomal stalling is essential for proteostasis. Stalled endoplasmic reticulum (ER)-bound ribosomes are marked by ubiquitin-fold modifier 1 (UFM1) on large ribosomal subunit protein RPL26, but the precise role of this process in ribosome-associated quality control (RQC) remains poorly understood. To identify proteins associated with UFMylated ribosomes, we isolated ribosomes from HCT116 cells expressing 3xFLAG-UFM1 upon stalling induction with anisomycin and in control conditions and preformed immunoprecipitation of 3xFLAG-UFM1.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Colorectal Cancer Cell Line

SUBMITTER: Richard Imre

LAB HEAD: Guelsuen Elif Karagoez

PROVIDER: PXD071028 | Pride | 2026-03-23

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20251003_07_A3_A2_Grujic_Dagdas_GMI_ID2317_2315_human_fullDB_UFM_Ratios.pdResult	Other
	20251003_A3_N8_Grujic_Dagdas_GMI_ID2317_S01_Lysate-control-1.raw	Raw
	20251003_A3_N8_Grujic_Dagdas_GMI_ID2317_S02_Lysate-control-2.raw	Raw
	20251003_A3_N8_Grujic_Dagdas_GMI_ID2317_S03_Lysate-control-3.raw	Raw
	20251003_A3_N8_Grujic_Dagdas_GMI_ID2317_S04_Lysate-ANS-1.raw	Raw

Items per page:

1 - 5 of 26

Similar Datasets

Project description:We use mRNA-seq in combination with polysome profiling to determine translational status for all mRNAs in Drosophila mature oocytes and activated eggs. Puromycin-treated lysates are used as a negative control in polysome profiling experiments. Additionally, we use ribosome footprinting to globally measure translational efficiency of mRNAs in wild type mature oocytes as well as wild type and png mutant activated eggs. Lysates of hand-dissected Drosophila mature oocytes (containing ~540 M-NM-<g of total RNA) were subjected to separation by velocity sedimentation through sucrose gradients. In this way, free mRNAs (present in RNPs fraction) or those comigrating with ribosomal subunits (40S or 60S+80S fractions) or with varying numbers of bound ribosomes (low polysomes (2-4 ribosomes), medium polysomes (5-9 ribosomes), and heavy polysomes (more than 10 ribosomes) can be separated based on their size and collected as sucrose gradient fractions. To compare quantitatively the levels of every mRNA across the polysome gradient fractions, we added 5ng of S. cerevisiae mRNA as an exogenous spike-in to each of the six fractions of interest: RNPs, 40S, 60S+80S, low polysomes, medium polysomes and heavy polysomes. RNA was extraced from these fractions, follwing proteinase K treatment, by hot acid phenol method. In case of unfractionated lysates, RNA was extracted using TRIzol (Invitrogen) according to manufacturerM-bM-^@M-^Ys instructions. mRNA-seq samples were prepared from 1 M-NM-<g of total RNA (in case of sucrose gradient fractions and unfractionated lysates) and subject to Illumina based sequencing. Puromycin-treated lysates of mature oocytes or 0-2h Drosophila activated eggs (containing ~540 M-NM-<g of total RNA) were also subjected to separation by velocity sedimentation through sucrose gradients. Puromycin causes premature termination of elongating ribosomes and thus it can be used to determine whether the mRNAs co-sedimenting with the polysomal peaks (defined here as M-bM-^IM-%5 ribosomes) were actively engaged in translation. As an independent approach to assess translation and obtain information on the position of ribosomes on mRNAs, we employed ribosome footprinting. In addition to analyzing the same samples, as by polysome profiling, we also analyzed png mutant activated eggs by ribosome footprinting. Ribosome footprint profiling measures the number of ribosome-protected fragments (RPFs) derived from the mRNAs of each gene, resulting in a singular value of translational efficiency (TE) for each gene (TE=RPF/RNA).

Dataset Information

LC-MS/MS of co-immunoprecipitation of 3xFLAG-UFM1 from ribosomal pellet in control and upon anisomycin treatment

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets