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Illumina sequencing of CRAC data obtained with Ylr419w

ABSTRACT: Illumina sequencing of CRAC data obtained with Ylr419w

PROVIDER: PRJNA1057187 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR27352186.fastq.gz	Fastqsanger.gz
	SRR27352187.fastq.gz	Fastqsanger.gz
	SRR27352188.fastq.gz	Fastqsanger.gz
	SRR27352189.fastq.gz	Fastqsanger.gz
	SRR27352190.fastq.gz	Fastqsanger.gz

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Illumina sequencing of CRAC data obtained with Ylr419w

Project description:27 years after the yeast genome sequencing, the function of many ORFs remain unknown. Despite the evolutionary distance between human and yeast, homology with the conserved DEAD-box helicase domains allowed us to list DHX29, DHX36 and DHX57 as three putative homologs of the yeast Ylr419w. Functional studies first linked the Ylr419w protein to the translating ribosome, and cross-linking and analysis of cDNA (CRAC) experiments determined the precise region of Ylr419w in contact with the ribosome. It corresponds to the loop of the h16 helix in the 18S rRNA designing the translation initiation factor DHX29, as the functional homolog of Ylr419w.

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Project description:The nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic cytoplasmic surveillance pathway that degrades aberrant mRNAs carrying premature termination codons (PTC). In yeast, the long distance between the stop codon and the poly(A) tail dictates mRNA detection by NMD factors Upf1, Upf2 and Upf3, and trigger mRNA decay, independent of deadenylation. While Upf1 binds a wide range of mRNAs beyond PTC-containing substrates, the mechanism of its recruitment on non-PTC-containing mRNAs is unknown. Using Nanopore sequencing of Upf1-bound mRNAs, we discovered that known PTC-containing NMD targets have long poly(A) tails, confirming that Upf1 binding occurs rapidly prior to deadenylation. Strikingly, the largest category specifically bound to Upf1 consisted of mRNAs without PTC harboring short poly(A) tails, corresponding to well-translated and stable transcripts. We showed that the degradation of this subpopulation depends on the three Upf factors, revealing a hitherto unknown function of Upf1 and NMD cofactors in mRNA decay.

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Project description:UV-crosslinking and high througput sequencing of cDNAs (CRAC) was used to map the binding sites Hfq in enterohaemorhaggic E. coli (EHEC). Hfq was tagged with a His-FLAG dual affintiy tag and UV crosslinked after growth in the MEM-HEPES media essentailly as per Granneman et al (2009) PNAS. We additionally crosslinked Hfq in non-pathogenic E. coli K12 str. MG1655 grown in LE media. Hfq-RNA complexes were purified and trimmed using RNase A/T1. RNA fragments were isolated and converted to cDNA, PCR amplified and sequenced using Illumina Solexa GAxII and HiSeq2000 platforms. His-FLAG tagged Hfq and untagged controls were cultured to an OD of 0.8 and crosslinked with UV-C. Five replicates of tagged EHEC Hfq and 2 replicates of untagged Hfq were crosslinked. We have additionally crosslinked 2 replicates each of E. coli K12 tagged and untagged Hfq.

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