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PRM-based quantitative proteomic analysis of ribosomes in GC-1 cell line

ABSTRACT: Relative protein quantification of RPL4 and RPL39L in non-ribosome, 40S, 60S and ribosome fractions by PRM;Absolute quantification of RPL39 and RPL39L levels in ribosomes of GC-1 wild type cell, and RPL39L-IRES-eGFP-overexpressed RibosomeCore-/Y GC-1 cells with or without dox-induced expression of RPL39 by PRM. Bovine serum albumin (BSA) as a negative control.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: yue wang

LAB HEAD: Xuejiang Guo

PROVIDER: PXD037257 | Pride | 2022-10-19

REPOSITORIES: Pride

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A male germ-cell-specific ribosome controls male fertility.

Li Huiling H Huo Yangao Y He Xi X Yao Liping L Zhang Hao H Cui Yiqiang Y Xiao Huijuan H Xie Wenxiu W Zhang Dejiu D Wang Yue Y Zhang Shu S Tu Haixia H Cheng Yiwei Y Guo Yueshuai Y Cao Xintao X Zhu Yunfei Y Jiang Tao T Guo Xuejiang X Qin Yan Y Sha Jiahao J

Nature 20221214 7941

Ribosomes are highly sophisticated translation machines that have been demonstrated to be heterogeneous in the regulation of protein synthesis1,2. Male germ cell development involves complex translational regulation during sperm formation3. However, it remains unclear whether translation during sperm formation is performed by a specific ribosome. Here we report a ribosome with a specialized nascent polypeptide exit tunnel, RibosomeST, that is assembled with the m ...[more]

PMID: 36517592

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PRM-based quantitative proteomic analysis of mouse tissues, testicular cells and cell lines

Project description:PRM analysis of RPL39L, RPL10L and RPL3L in 9 mouse tissues, including testis, fat, brain, liver, kidney, lung, skeletal muscle, heart and spleen; PRM analysis of RPL39 and RPL39L, RPL10 and RPL10L, and RPL22 and RPL22L1 in testicular cells; PRM analysis of RPL39L and RPL39 in RibosomeRPL39L-/- and wild type mouse spermatocytes and spermatids; PRM analysis of 17 down-regulated proteins and 1 unchanged protein (DYNC1LI2) in RibosomeRPL39L-/- testes; PRM analysis of RPL39 and RPL39L in GC-1 ribosomes, and RibosomeRPL39L-/- and wild type N2a ribosomes, Consistency between the observed and expected signal ratios of the light and heavy peptides by PRM in serial dilution experiments. Bovine serum albumin (BSA) as a negative control.

2022-09-30 | PXD020922 | Pride

ETV4 mediates dosage-dependent prostate tumor initiation and cooperates with p53 loss to generate prostate cancer.

Project description:Comparision of transcriptome of PC3 and 22Rv1 prostate cell lines after 1 week overexpression of mutated ETV4: ETV4AAA-IRES-eGFP, wild type ETV4: ETV4WT-IRES-eGFP, and control vector IRES-eGFP by MSCV-IRES-GFP vector ((Addgene #20672)) and Lentivirus transduction.

2022-10-16 | GSE215437 | GEO

Transcriptional and polysomal profile of poliovirus-infected HUVECs

Project description:Translation initiation of eukaryotic mRNAs mostly occurs by the cap-dependent ribosome scanning mechanism. However, certain mRNAs are translated by ribosome assembly at internal ribosome entry sites (IRES), a mechanism that allows the synthesis of certain proteins when cap-dependent translation is inhibited by cellular stress. Whether IRES-mediated translation occurs in stressed human endothelial cells (EC) is unknown. We performed whole genome microarray analysis of polyribosomal mRNA (43,203 sequences) from virus-infected EC to identify IRES-containing mRNAs.

2009-03-27 | GSE15356 | GEO

Sox18 regulated genes

Project description:Study of Sox18 regulated genes: Human umbilical vein endothelial cells (HUVEC) were either transduced with adenoviral vectors expressing SOX18 from an IRES-EGFP casette, or IRES-EGFP alone, or left untreated. After 16 hours, mRNA was isolated and analyzed by hybridization to Affymetrix HG-U133A arrays. Human umbilical vein endothelial cells (HUVEC) were either transduced with adenoviral vectors expressing SOX18 from an IRES-EGFP casette, or IRES-EGFP alone, or left untreated. After 16 hours, mRNA was isolated and analyzed by hybridization to Affymetrix HG-U133A arrays.

2012-01-23 | E-GEOD-8952 | biostudies-arrayexpress

Evidence for rRNA 2'-O-methylation plasticity: control of intrinsic translational capabilities of human ribosomes

Project description:Ribosomal RNAs (rRNAs) are main effectors of mRNA decoding, peptide-bond formation and ribosome dynamics during translation. Ribose 2'-O-methylation (2'-O-Me) is the most abundant rRNA chemical modification, and display a complex pattern in rRNA. 2'-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2'-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2'-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin (FBL) in human cells. Using RiboMethSeq, a non-biased quantitative mapping of 2'-O-Me, we identified a repertoire of 2'-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2'-O-Me plasticity. Using the cricket paralysis virus (CrPV) IRES element, as a model, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through 2'-O-Me pattern and not by non-ribosomal actors of the translational machinery. Our results establish rRNA 2'-O-methylation plasticity as a mechanism providing functional specificity to human ribosomes.

2018-01-19 | GSE105248 | GEO

Structured elements drive extensive circular RNA translation

Project description:The human genome encodes tens of thousands circular RNAs (circRNAs) with mostly unknown functions. Circular RNAs requires internal ribosome entry sites (IRES) if they are to undergo translation without 5’ cap. Here, we develop a high-throughput screen to systematically discover RNA sequences that can direct circRNA translation in human cells. We identify over 17,000 endogenous and synthetic sequences as candidate circRNA IRES. 18S rRNA complementarity and a structured RNA element positioned on the IRES are important for driving circRNA translation. Ribosome profiling and peptidomic analyses show extensive IRES-ribosome association, hundreds of circRNA-encoded proteins with tissue -specific distribution, and antigen presentation. We find that circFGFR1p, a protein encoded by circFGFR1 that is down regulated in cancer, functions as a negative regulator of FGFR1 oncoprotein to suppress cell growth during stress. Systematic identification of circRNA IRES elements may provide important links among circRNA regulation, biological function, and disease.

2021-08-25 | GSE178718 | GEO

Sox18 regulated genes

2012-01-23 | GSE8952 | GEO

Gene expression changes in Apc-mutant mouse intestinal organoids 7 and 20 days after the induction of the Apc deletion

Project description:We isolated and selected intestinal adenoma organoids from Lgr5-EGFP-IRES-CreER; Apcflox/flox mice and added tamoxifen to induce the deletion of the Apc gene in the intestinal stem cells. Gene expressions on day7 and day20 after the addition of tamoxifen were compared, representing two stages with different colorectal cancer stem cell content. Total RNA obtained from Lgr5-EGFP-IRES-CreER; Apcflox/flox organoids were compared 7 days and 20 days after the addition of tamoxifen, cultured without the Wnt-agonist R-Spondin1.

2015-12-31 | E-GEOD-47569 | biostudies-arrayexpress

Identification of ribosome proteins bound to IRES in hypoxia

Project description:Hypoxic conditions prompt internal ribosome entry site (IRES)-mediated translation of some of the hallmark cancer genes, such as VEGF. This translational switch is extremely vital for cell survival and tumor progression. Heterogeneity in ribosomes due to the diversity of ribosomal RNA (rRNA) and protein composition has been postulated to generate ‘specialized ribosomes’ that differentially regulate translation. A VEGF IRES sequence was used as bait to identify unique proteins bound at the IRES in breast cancer cells grown in hypoxia.

2021-01-06 | PXD019238 | Pride

Transcription profiling analysis of human global and individual mRNA translational efficiencies

Project description:A cell synchronization protocol was established in which global and individual mRNA translational efficiencies could be examined. While global translational efficiency was reduced in mitotic cells, approximately 3% of mRNAs remained predominantly associated with large polysomes during mitosis, as determined by cDNA microarray analyses. The 5 noncoding regions of six mRNAs were shown to contain internal ribosome entry sites (IRES). However, not all known mRNAs that contain IRES elements were actively translated during mitosis, arguing that specific IRES sequences are differentially regulated during mitosis.

2005-10-31 | E-SMDB-2546 | biostudies-arrayexpress

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