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Absolute quantification analysis of ubiquitin chains conjugated to GP8 using an in vitro ubiquitination reaction.

ABSTRACT: To examine whether ARIH1-UBE2L3 is involved in the atypical ubiquitination of Nrf1 by SCFFBS2, we compared the ubiquitin linkage types between three reactions using GP8: E1/UBE2D1/SCFFBS2, the complete reaction including E1/UBE2D1/SCFFBS2/Nedd8 sys/ARIH1/UBE2L3, and the complete reaction in the absence of UBE2D1.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Hikaru Tsuchiya

LAB HEAD: Yukiko Yoshida

PROVIDER: PXD053707 | Pride | 2024-08-06

REPOSITORIES: Pride

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Source:

			Action	DRS
	20240123_Yoshida_Ub_PRM_01_No_Nedd8sysARIH1UBE2L3_FBS2_01.raw	Raw
	20240123_Yoshida_Ub_PRM_02_No_Nedd8sysARIH1UBE2L3_FBS2_02.raw	Raw
	20240123_Yoshida_Ub_PRM_03_No_Nedd8sysARIH1UBE2L3_FBS2_03.raw	Raw
	20240123_Yoshida_Ub_PRM_04_No_Nedd8sysARIH1UBE2L3_mut_01.raw	Raw
	20240123_Yoshida_Ub_PRM_05_No_Nedd8sysARIH1UBE2L3_mut_02.raw	Raw

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Project description:Regulation of gene expression at the post-transcriptional level plays an indispensable role during TGFbeta-induced EMT and metastasis. This regulation involves a transcript-selective translational regulatory pathway in which a ribonucleoprotein (mRNP) complex, consisting of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) and eukaryotic elongation factor 1A1 (eEF1A1), binds to a 3M-bM-^@M-^Y-UTR regulatory BAT (TGFM-NM-2 activated translation) element and silences translation of Dab2 and ILEI mRNAs, two transcripts which are involved in mediating EMT. TGFbeta activates a kinase cascade terminating in the phosphorylation of hnRNP E1, by isoform-specific stimulation of protein kinase B/Akt2, inducing the release of the mRNP complex from the 3M-bM-^@M-^Y-UTR element, resulting in the reversal of translational silencing and increased expression of Dab2 and ILEI transcripts. We adopted a combinatorial approach involving polysome profiling and RIP-Chip analyses using hnRNP E1 and filtered the array data based on the regulatory mechanism of Dab2 and ILEI. This led to the identification and validation of a cohort of target mRNAs that follow the same pattern of regulation as Dab2 and ILEI. To identify potential target mRNA transcripts that are translationally regulated by hnRNP E1 in a TGF-beta-dependent manner, we adopted a combinatorial approach involving expression profiling analyses and RNA immunoprecipitation analysis (RIP-Chip). We performed a screen using: 1) total mRNA and 2) RNA isolated from monosomal (non-translating) versus polysomal (translating) fractions from TGF-beta-treated (24 h) and non-treated NMuMG cells and from the hnRNP E1 knockdown derivative (E1KD), that undergo constitutive EMT even in the absence of TGF-beta. In addition, we screened for transcripts that selectively interact with hnRNP E1 in NMuMG cells under unstimulated conditions and subsequently lose their temporal association following TGF-beta stimulation. The samples were individually hybridized to Affymetrix GeneChipM-BM-. Mouse Genome 430 2.0 arrays.

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Absolute quantification analysis of ubiquitin chains conjugated to GP8 using an in vitro ubiquitination reaction.

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