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Proteomics of MCF7 cells (LC-MS/MS Orbitrap Fusion Tribrid)

ABSTRACT: We developed RiboLace, a novel method based on a new puromycin-containing molecule, for the isolation of active ribosomes and associate RNAs by means of an antibody-free and tag-free pull-down approach. We compared riboLace translation estimates with protein levels determined by proteomics in MCF7 cells. RiboLace can be easily and rapidly used to obtain a transcriptome-wide snapshot of active translation from eukaryotic in vitro and in vivo systems.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Epithelial Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Toma Tebaldi

LAB HEAD: Gabriella Viero

PROVIDER: PXD009417 | Pride | 2018-11-21

REPOSITORIES: Pride

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			Action	DRS
	MCF7_tot_1.raw	Raw
	MCF7_tot_2.raw	Raw
	MCF7_tot_3.raw	Raw
	MaxQuant_andromeda.zip	Other
	MaxQuant_txt.zip	Other

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Publications

Active Ribosome Profiling with RiboLace.

Clamer Massimiliano M Tebaldi Toma T Lauria Fabio F Bernabò Paola P Gómez-Biagi Rodolfo F RF Marchioretto Marta M Kandala Divya T DT Minati Luca L Perenthaler Elena E Gubert Daniele D Pasquardini Laura L Guella Graziano G Groen Ewout J N EJN Gillingwater Thomas H TH Quattrone Alessandro A Viero Gabriella G

Cell reports 20181001 4

Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possi ...[more]

PMID: 30355487

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Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3M-bM-^@M-^Y UTRs

Project description:The RNA helicase UPF1 is best known for its key function in mRNA nonsense-mediated mRNA decay (NMD), but has also been implicated in additional mRNA turnover mechanisms, telomere homeostasis, and DNA replication. In NMD, UPF1 recruitment to target mRNAs is thought to occur through interaction with release factors at terminating ribosomes, but evidence for translation-independent interaction of UPF1 with the 3M-bM-^@M-^Y untranslated region (UTR) of mRNAs has also been reported. To map UPF1 binding sites transcriptome-wide, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) in human cells, untreated or after inhibiting translation by puromycin. We found a strong association of UPF1 with 3M-bM-^@M-^Y UTRs in undisturbed, translationally active cells and a significant increase in UPF1 binding to coding sequence (CDS) after translation inhibition. These results indicate that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. This evidence for translation-independent UPF1-RNA interaction, which is corroborated by RNA immunoprecipitations experiments and by our observation that UPF1 also crosslinks to long non-coding RNAs, suggests that the decision to trigger NMD occurs after association of UPF1 with the mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination. Examination of Upf1 binding preferences via iCLIP in untreated HeLa cells and HeLa cells, where translation is blocked by puromycin treatment in vivo crosslinking and immunoprecipitation strategy (iCLIP)

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Active translatome profiling with RiboLace in MCF7 cells

Project description:We developed RiboLace, a novel method based on a new puromycin-containing molecule, for the isolation of active ribosomes and associate RNAs by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, it works with very low input material and can be easily and rapidly used to obtain a transcriptome-wide snapshot of active translation from eukaryotic in vitro and in vivo systems. Keywords: translation, RiboLace, polysome profiling, RNA-seq, POL-seq, active translation

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Active ribosome profiling from mouse brains with RiboLace

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Puromycin acetyltransferase-mediated cell-specific labeling of de novo synthesized proteins, Part II

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