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EIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5’UTR.

ABSTRACT: Regulation of the mRNA life-cycle is central to gene expression control and determination of cell fate. miRNAs represent a critical mRNA regulatory mechanism, but despite decades of research, their mode of action is still not fully understood. Here we show eIF4A2 is a major effector of the repressive miRNA pathway functioning via the Ccr4-Not complex. We demonstrate that while DDX6 interacts with Ccr4-Not, its effects in the mechanism are not as pronounced. Through its interaction with the Ccr4-Not complex eIF4A2 represses mRNAs at translation initiation. We show evidence that native eIF4A2 has similar properties to chemically inhibited eIF4A1. We demonstrate that eIF4A2 exerts it repressive effect by binding purine-rich motifs which are enriched in the 5’UTR directly upstream of the AUG start codon. The data supports a model whereby purine motifs towards the 3’ end of the 5’UTR are associated with increased ribosome occupancy and possible uORF activation similar to that observed for mRNAs affected by inhibited eIF4A1.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

DISEASE(S): Disease Free

SUBMITTER: Kelly Hodge

LAB HEAD: Sara Rossana Zanivan

PROVIDER: PXD014764 | Pride | 2019-10-08

REPOSITORIES: Pride

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			Action	DRS
	20190412_KEH_HF_SA_ANW_R11_100419_CNOT_F1_f001.raw	Raw
	20190412_KEH_HF_SA_ANW_R11_100419_CNOT_F1_f002.raw	Raw
	20190412_KEH_HF_SA_ANW_R11_100419_CNOT_F1_f003.raw	Raw
	20190412_KEH_HF_SA_ANW_R11_100419_CNOT_F1_f004.raw	Raw
	20190412_KEH_HF_SA_ANW_R11_100419_CNOT_F1_f005.raw	Raw

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eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5’UTR

Project description:Background: Regulation of the mRNA life cycle is central to gene expression control and determination of cell fate. miRNAs represent a critical mRNA regulatory mechanism, but despite decades of research, their mode of action is still not fully understood. Results: Here, we show that eIF4A2 is a major effector of the repressive miRNA pathway functioning via the Ccr4-Not complex. We demonstrate that while DDX6 interacts with Ccr4-Not, its effects in the mechanism are not as pronounced. Through its interaction with the Ccr4- Not complex, eIF4A2 represses mRNAs at translation initiation. We show evidence that native eIF4A2 has similar RNA-selectivity to chemically inhibited eIF4A1. eIF4A2 exerts its repressive effect by binding purine-rich motifs which are enriched in the 5’UTR of target mRNAs directly upstream of the AUG start codon. Conclusions: Our data support a model whereby purine motifs towards the 3’ end of the 5’UTR are associated with increased ribosome occupancy and possible uORF activation upon eIF4A2 binding.

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eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine rich motifs in th 5'UTR

Project description:Here we show eIF4A2 is a major effector of the represive miRNA pathway functioning via the Ccr4_Not complex; through its interaction with the Ccr4-Not complex eIF4A2 represses mRNAs at translation initiation. We show evidence that native eIF4A2 has similar properties to chemically inhibited eIF4A1. eIF4A2 exerts its repressive effect by binding purine-rich motifs which are enriched in the 5'UTR directly upstream of the AUG codon.

2019-10-10 | PXD015772 | Pride

Transcriptomic and proteomic profiling of KEAP1 disrupted and sulforaphane treated human breast epithelial cells

Project description:Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is a potent inhibitor of experimental mammary carcinogenesis and may be an effective, safe chemopreventive agent for use in humans. SFN acts in part on the Keap1/Nrf2 pathway to regulate a battery of cytoprotective genes. In this study transcriptomic and proteomic changes in the estrogen receptor negative, non tumorigenic human breast epithelial MCF10A cell line were analyzed following SFN treatment or KEAP1 knockdown with siRNA using microarray and stable isotopic labeling with amino acids in culture (SILAC), respectively. Changes in selected transcripts and proteins were confirmed by PCR and Western blot in MCF10A and MCF12A cells. There was strong correlation between the transcriptomic and proteomic responses in both the SFN treatment (R=0.679, P<0.05) and KEAP1 knockdown (R=0.853, P<0.05) experiments. Common pathways for SFN treatment and KEAP1 knockdown were xenobiotic metabolism and antioxidants, glutathione metabolism, carbohydrate metabolism and NADH/NADPH regeneration. Moreover, these pathways were most prominent in both the transcriptomic and proteomic analyses. The aldo-keto reductase family members, AKR1B10, AKR1C1, AKR1C2 and AKR1C3, as well as NQO1 and ALDH3A1, were highly upregulated at both the transcriptomic and proteomic level. Collectively, these studies served to identify potential biomarkers that can be used in clinical trials to investigate the initial pharmacodynamic action of SFN in the breast. MCF10A cells were treated with SFN or had KEAP1 knocked down by siRNA.

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eIF4A1 unwinding activity is RNA sequence-specifically deployed through eIF4A1-multimerisation to facilitate translation of mRNAs with local repressive RNA structure

Project description:Oncogenic translational programmes are an emerging hallmark of cancer and often driven by dysregulation of signaling pathways including KRAS and mTORC that converge on the eukaryotic translation initiation (eIF) 4F complex. Altered eIF4F activity promotes translation of oncogene mRNAs that typically contain highly structured 5’UTRs rendering their translation strongly dependent on RNA unwinding by DEAD-box helicase eIF4A1 subunit of the eIF4F complex. In addition, eIF4A1 separately functions to load mRNA into the 43S pre-initiation complex (PIC), an essential step for the translation of cellular mRNA. While eIF4A1-dependent mRNAs have been widely investigated, it is still unclear if highly structured mRNAs recruit and activate eIF4A1 unwinding specifically. Here, we uncover that unwinding by eIF4A1 is activated in an RNA sequence-dependent manner in cells. Our data demonstrate that eIF4A1-dependent mRNAs contain specific RNA sequences, particularly enriched for polypurine-motifs, in their 5’UTR which recruit and specifically stimulate unwinding of local repressive RNA structure by eIF4A1 in an RNA sequence-dependent manner to facilitate translation. Mechanistically, we show that polypurine-rich sequences trigger the formation of RNA sequence-specific multimeric eIF4A1-complexes, assembled of catalytically distinct eIF4A1 subunits, the joint activity of which enhances RNA unwinding activity. Together with our structural data, we describe a model in which conformational changes within eIF4A1 and the RNA through the process of eIF4A1 multimerisation, lead to an optimal interaction of eIF4A1-unwinding subunits with the structured RNA region which enhances unwinding. Hence, we conclude that RNA sequences in addition to protein cofactors contribute to the regulation of cellular eIF4A1 function and promotion of translation of eIF4A1-unwinding dependent mRNAs.

2023-01-17 | PXD034343 | Pride

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