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Study of the eIFG1-eIF1 interaction by TCP-seq on translation initiation in HEK 293T cells

ABSTRACT: Study of the eIFG1-eIF1 interaction by TCP-seq on translation initiation in HEK 293T cells

PROVIDER: PRJNA938913 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR23622634.fastq.gz	Fastqsanger.gz
	SRR23622635.fastq.gz	Fastqsanger.gz
	SRR23622636.fastq.gz	Fastqsanger.gz
	SRR23622637.fastq.gz	Fastqsanger.gz
	SRR23622638.fastq.gz	Fastqsanger.gz

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Study of the eIFG1-eIF1 interaction by TCP-seq on translation initiation in HEK 293T cells

Project description:Start codon recognition by the 48S complex is a critical step in translation. However, understanding the in vivo initiation and its regulation at a global scale is limited. Here, we analyzed translation complex profiling (TCP-seq) data to determine the impact of eIF4G1-eIF1 inhibition and the 48S organization. Our analysis provides the first global view of leaky scanning and reveal the central roles of mRNA features and eIF4G1-eIF1 in its regulation. Specifically, non-leaky genes are enriched with a Kozak bearing a C at -1 position while those with short 5’ UTR with TISU. Additionally, the stability of the 48S complex and its integrity during scanning are impaired upon eIF4G1-eIF1 inhibition. Detailed analysis of initiation site footprints revealed three main classes conserved from yeast to human. Our analysis provides a general overview of AUG selection and evidence for conformational rearrangements in vivo.

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Study of the translation initiation by Selective TCP-seq in the context of eIF1-eIF4G1 inhibitor

Project description:Start codon recognition by the 48S complex is a critical step in translation. However, understanding the in vivo initiation and its regulation at a global scale is limited. To better understand the mechanism in vivo, we have screened for small molecules that specifically inhibit the function of eIF1-eIF4G1 interaction. We performed Selective-48S footprinting against eIF1 (HA-tagged), eIF2a (HA-tagged), eIF4G1 and eIF3c to answer questions regarding the function of that interaction in the context of the scanning and initiating 48S ribosome.

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nsp1 acts on HEK-293T

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Study of the translation initiation by Selective TCP-seq in the context of eIF1-eIF4G1 inhibitor

Project description:Study of the translation initiation by Selective TCP-seq in the context of eIF1-eIF4G1 inhibitor

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eIF1 discriminates against suboptimal initiation sites to prevent excessive uORF translation genome-wide

Project description:The translation pre-initiation complex (PIC) scans the mRNA for an AUG codon in favorable context. Previous findings suggest that the factor eIF1 discriminates against non-AUG start codons by impeding full accommodation of Met-tRNAi in the P site of the 40S ribosomal subunit, necessitating eIF1 dissociation for start codon selection. Consistent with this, yeast eIF1 substitutions that weaken its binding to the PIC increase initiation at UUG codons on a mutant his4 mRNA and particular synthetic mRNA reporters; and also at the AUG start codon of the mRNA for eIF1 itself owing to its poor Kozak context. It was not known however whether such eIF1 mutants increase initiation at suboptimal start codons genome-wide. By ribosome profiling, we show that the eIF1-L96P variant confers increased translation of numerous upstream open reading frames (uORFs) initiating with either near-cognate codons (NCCs) or AUGs in poor context. The increased uORF translation is frequently associated with reduced translation of the downstream main coding sequences (CDS). Initiation is also elevated at the NCCs initiating N-terminal extensions on GRS1 and ALA1 mRNAs, and at a small set of main CDS AUG codons with especially poor context, including that of eIF1 itself. Thus, eIF1 acts throughout the yeast translatome to discriminate against NCC start codons and AUGs in poor context; and impairing this function enhances the repressive effects of uORFs on CDS translation and alters the ratios of protein isoforms translated from near-cognate versus AUG start codons.

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