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Dynamic O-GlcNAcylation of eIF3a controls translation reinitiation during integrated stress response

ABSTRACT: In eukaryotic cells, many mRNAs possess upstream open reading frames (uORFs) in addition to the main coding region. After uORF translation, the ribosome could either recycle at the stop codon or resume scanning for downstream start codons in a process known as reinitiation. Accumulating evidence suggests that some initiation factors, including eIF3, linger on the early elongating ribosome, forming an eIF3-80S complex. Very little is known how the eIF3 is carried along with the 80S during elongation and its implications in subsequent translation reinitiation. Here we report that eIF3a undergoes dynamic O-GlcNAc modification in response to nutrient starvation. Stress-induced de-O-GlcNAcylation promotes eIF3 retention on the elongating ribosome and facilitates reinitiation at downstream start codons. Eliminating the modification site from eIF3a via genome editing induces ATF4 reinitiation under the nutrient rich condition. Our findings illustrate a mechanism in balancing ribosome recycling and reinitiation, thereby linking integrated stress response and translational reprogramming.

ORGANISM(S): Mus musculus

PROVIDER: GSE181040 | GEO | 2021/11/01

REPOSITORIES: GEO

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Project description:The eukaryotic translation initiation factor (eIF) 3a is described in various tumor entities as potential tumor marker involved in development and progression of cancer. eIF3a is the largest subunit of the eIF3 complex, a key functional entity in 80S establishment and translation initiation. We hypothesize that eIF3a is more a specific than global translation initiator and involved in signalling pathways that are frequently targeted in UBC therapy. Methods: In FFPE samples of UBC patients eIF3a expression was analysed together with overall survival. In cell culture we investigated proliferation, migration, clonogenicity and tumorgenicity of UBC cell lines with and without knockdown of eIF3a and compared the cytotoxicity of eIF3a knockdown with other translational inhibitors, including the chemotherapeutic Rapamycine. Detailed information on changed gene expression and global translation initiation were gathered by mRNA expresssion and polysomal profiling. Results: and Conclusion eIF3a is upregulated in UBC and high expression of eIF3a corresponds with longer overall survival in low grade tumors. Knockdown of eIF3a in UBC cell lines reduces their malignant phenotype, including reduced tumor growth in xenotransplanted mice. eIF3a regulates DNA damage response (ATM, ATR, CDC25A) on a translational level and we show that reduction in eIF3a expression does not hamper global translation initiation. The knockdown of eIF3a strongly influences proliferation of UBC cell lines without dramatically disturbing general translation as depicted in polysomal profiles. We therefore analysed potential changes on the mRNA level in eIF3a knockdown compared to control HT1197 cells. Differential expression of genes between eIF3a knock-down and control samples was assessed with the moderated t-test (Bioconductor's limma package). Raw p-values were adjusted for multiple hypothesis testing using the method from Benjamini and Hochberg for a strong control of the false discovery rate.

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Dynamic O-GlcNAcylation of eIF3a controls translation reinitiation during integrated stress response

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