Detection of cryptic start codons in set2D by 5PSeq
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ABSTRACT:
PROVIDER: PRJNA486741 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7721041.fastq.gz | Fastqsanger.gz | |||
| SRR7721042.fastq.gz | Fastqsanger.gz | |||
| SRR7721043.fastq.gz | Fastqsanger.gz | |||
| SRR7721044.fastq.gz | Fastqsanger.gz |
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Project description:We investigated cryptic transcription start site usage, chromatin organization and post-transcriptional consequences in Saccharomyces cerevisiae. We used 5PSeq approach, which measures ribosome dynamics by sequencing the presence of co-translation mRNA degradation intermediates, to assess if cryptic transcripts are engaged in active translation. We show that chromatin-dependent cryptic transcripts can be recognized by ribosomes and have the potential to produce truncated polypeptides by using downs-stream, in-frame start codons. Our work suggests that a significant fraction of chromatin-dependent internal cryptic promoters are in fact alternative truncated mRNA isoforms.
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Project description:Cryptic transcription is widespread and generates a heterogeneous group of RNA molecules of unknown function. To improve our understanding of cryptic transcription, we investigated their transcription start site usage, chromatin organization and post-transcriptional consequences in Saccharomyces cerevisiae. We show that transcription start sites of chromatin-dependent internal cryptic transcripts resemble those of protein coding genes in terms of DNA sequence, directionality and chromatin accessibility. We define the 5’ and 3’ boundaries of cryptic transcripts and show that, contrary to RNA degradation-dependent ones , they often overlap with the end of the gene thereby using the canonical polyadenylation site and associate to polyribosomes. In fact, we show that chromatin-dependent cryptic transcripts can be recognized by ribosomes and may produce truncated polypeptides from downstream, in-frame start codons. Our work suggests that a significant fraction of chromatin-dependent internal cryptic promoters are in fact alternative truncated mRNA isoforms. The expression of these chromatin-dependent isoforms is conserved from yeast to human expanding the functional consequences of cryptic transcription and proteome complexity.
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Project description:we used N-terminomics approach to identify the N-terminal sequences of SEPs in Hep3B cells. Three SEPs enrichment methods (HCl precipitation, heating precipitation and ACN precipitation), three chemical derivatization methods (dimethylation, acetylation and guanidylation) and three N-terminal peptides enrichment materials (hexadecaldehyde, CNBr activated agarose and NHS activated agarose) were tested in this experiment. We also determined the start codons of SEPs with their N-terminal information.
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Project description:We investigated the association of cryptic unstable transcripts (CUTs) to ribosomes in Saccharomyces cerevisiae. We used 5’cap-sequence followed by sucrose gradient fractionation of polyribosome fractions after ultracentrifugation to assess the relative association of transcripts to the ribosomes. We used 5PSeq approach, which measures ribosome dynamics by sequencing the presence of co-translation mRNA degradation intermediates, to assess if cryptic transcripts are engaged in active translation. We performed 5PSeq after glucose depletion to increase the number of ribosomes stop at the 5’UTR and start codon.
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Project description:The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian ortholog DDX3 are critical for translation initiation. Mutations in DDX3 are linked to tumorigenesis and intellectual disability, and the enzyme is targeted by diverse viruses. How Ded1p and its orthologs engage RNAs to impact translation initiation has been a longstanding, unresolved question. Here we show that Ded1p associates with the pre-initiation complex at the mRNA entry channel of the small ribosomal subunit and that the helicase unwinds mRNA structure ahead of the scanning pre-initiation complex. Defective Ded1p causes pervasive translation in 5’UTRs, starting from near-cognate initiation codons located 5' of mRNA structures and concomitant decrease of protein synthesis from of the main ORFs. The data indicate that Ded1p functions to suppress translation initiation on near-cognate codons proximal to mRNA structure and show how the helicase is targeted to specific RNA sites without common sequence signatures. Our results reveal a straightforward mechanism for the activation of upstream open reading frames and suggest that mRNA structure and proximal near-cognate initiation codons encode a widespread regulatory program for translation initiation that is sensitive to RNA helicase function.
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Project description:Suppressing spurious cryptic transcription by a repressive intragenic chromatin state featuring trimethylated lysine 36 on histone H3 (H3K36me3) and DNA methylation is critical for maintaining self-renewal capacity in mouse embryonic stem cells. In yeast and nematodes, such cryptic transcription is elevated with age, and reducing the levels of age-associated cryptic transcription extends yeast lifespan. Whether cryptic transcription is also increased during mammalian aging is unknown. We show for the first time an age-associated elevation in cryptic transcription in several stem cell populations, including murine hematopoietic stem cells (mHSCs) and neuronal stem cells (NSCs) and human mesenchymal stem cells (hMSCs). Using DECAP-seq, we mapped and quantified age-associated cryptic transcription in hMSCs aged in vitro. Regions with significant age-associated cryptic transcription have a unique chromatin signature: decreased H3K36me3 and increased H3K4me1, H3K4me3, and H3K27ac with age. Furthermore, genomic regions undergoing such age-dependent chromatin changes resemble known promoter sequences and are bound by the promoter-associated protein TBP even in young cells. Hence, the more permissive chromatin state at intragenic cryptic promoters likely underlies the increase of cryptic transcription in aged mammalian stem cells.
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