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Identifying DAP5 mRNA translation and direct-binding targets in human embryonic stem cells (hESCs) using Ribo-seq and CLIP-seq.

ABSTRACT: Identifying DAP5 mRNA translation and direct-binding targets in human embryonic stem cells (hESCs) using Ribo-seq and CLIP-seq.

PROVIDER: PRJNA794846 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR17454400.fastq.gz	Fastqsanger.gz
	SRR17454401.fastq.gz	Fastqsanger.gz
	SRR17454402.fastq.gz	Fastqsanger.gz
	SRR17454403.fastq.gz	Fastqsanger.gz
	SRR17454404.fastq.gz	Fastqsanger.gz

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Identifying DAP5 mRNA translation and direct-binding targets in human embryonic stem cells (hESCs) using Ribo-seq and CLIP-seq.

Project description:The eIF4G1 DAP5 homolog is involved in several non-canonical translation initiation mechanisms. We applied ribosome profiling and found that DAP5 translationally activated mRNAs are enriched with translated uORFs. We also performed a genome-wide UV-cross linking immunoprecipitation screen (CLIP-seq) for identifying mRNAs that directly interact with DAP5 in hESCs. We found 959 bound mRNAs common to both Abs (out of 1073 and 2152 enriched mRNAs identified by CS and MBL antibodies, respectively).

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Project description:Crosslinking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins (RBPs). We developed a method for CLIP data analysis and applied it to compare 254 nm CLIP with PAR-CLIP, which involves crosslinking of photoreactive nucleotides with 365 nm UV light. We found small differences in the accuracy of these methods in identifying binding sites of HuR, a protein that binds low-complexity sequences and Argonaute 2, which has a complex binding specificity. We show that crosslink-induced mutations lead to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect sufficiently their sites under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific ribonucleases strongly biases the set of recovered binding sites. We finally show that this bias can be substantially reduced by milder nuclease digestion conditions. We performed duplicate mRNA-Seq experiments for cells grown in the presence of modified nucleotides or in normal medium, and following crosslinking at 365nm, 254nm or without crosslinking. In addition, we performed single mRNA-Seq experiments of cells transfected with anti-GFP or anti-HuR siRNA.

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