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EnD-Seq and AppEnD: Sequencing 3' ends to identify non-templated tails and degradation intermediates

ABSTRACT: EnD-Seq and AppEnD: Sequencing 3' ends to identify non-templated tails and degradation intermediates

PROVIDER: PRJNA497386 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8074771_1.fastq.gz	Fastqsanger.gz
	SRR8074771_2.fastq.gz	Fastqsanger.gz
	SRR8074772_1.fastq.gz	Fastqsanger.gz
	SRR8074772_2.fastq.gz	Fastqsanger.gz
	SRR8074773_1.fastq.gz	Fastqsanger.gz

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EnD-Seq and AppEnD: Sequencing 3' ends to identify non-templated tails and degradation intermediates

Project description:EnD-Seq and AppEnD: Sequencing 3' ends to identify non-templated tails and degradation intermediates

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EnD-Seq and AppEnD: Sequencing 3' ends to identify non-templated tails and degradation intermediates

Project description:Existing methods for detecting RNA intermediates resulting from exonuclease degradation are low-throughput and laborious. In addition, mapping the 3’ ends of RNA molecules to the genome after high-throughput sequencing is challenging, particularly if the 3’ ends contain posttranscriptional modifications. To address these problems, we developed EnD-Seq, a high-throughput sequencing protocol that preserves the 3’ end of RNA molecules, and AppEnD, a computational method for analyzing high-throughput sequencing data. Together these allow determination of the 3’ ends of RNA molecules, including nontemplated additions. Applying EnD-Seq and AppEnD to histone mRNAs revealed that a significant fraction of cytoplasmic histone mRNAs end in one or two uridines, which have replaced the 1-2 nts at the 3’ end of mature histone mRNA maintaining the length of the histone transcripts. Histone genes in fly embryos and ovaries show the same pattern, but with different tail nucleotide compositions. We increase the sensitivity of EnD-Seq by using cDNA priming to specifically enrich low-abundance tails of known sequence composition allowing identification of degradation intermediates. In addition, we show the broad applicability of our computational approach by using AppEnD to gain insight into 3’ additions from diverse types of sequencing data, including data from small capped RNA sequencing and some alternative polyadenylation protocols.

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EnD-Seq and AppEnD: Sequencing 3' ends to identify non-templated tails and degradation intermediates

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3' RACE-seq of reporter LINE-1 and control mRNAs' 3' non-templated ends in 293T 'wild-type', XRN1 KO, DCP2 KO, DCP2 plus XRN1 KO cells

Project description:Human LINE-1 retrotransposons can mobilize in the human genome by a copy-and-paste machanism involving RNA intermediates. LINE-1 mRNA 3' ends play a major role in initiation of reverse transcription (a step retrotransposition). To assess the impact of RNA 3’ end dynamics of LINE-1 mRNA on its retrotransposition potential in human cells we analyzed 3' non-templated ends on LINE-1, and control GAPDH and PABPC4 mRNAs by 3' RACE-seq. RNA was retrieved from multiple clonal 293T cells wild-type or knock-out in eitehr XRN1, DCP2, or both (DCP2 plus XRN1). Publication: D Janecki, R Sen, N Szóstak, A Kajdasz, M Kordys, K Plawgo, D Pandakov, A Philips, Z Warkocki (2024): LINE-1 mRNA 3’ end dynamics shape its biology and retrotransposition potential. Nucleic Acids Research. https://doi.org/10.1093/nar/gkad1251

2024-01-05 | GSE230464 | GEO

3' RACE-seq of endogenous LINE-1 mRNA's 3' non-templated ends in PA-1 control kd, XRN1 kd, DCP2 kd, and DIS3L2 kd (kd by RNAi for 48h = set 1, or 126h = set 2), and 293T wild-type and XRN1 KO.

Project description:Human LINE-1 retrotransposons can mobilize in the human genome by a copy-and-paste machanism involving RNA intermediates. LINE-1 mRNA 3' ends play a major role in initiation of reverse transcription (a step retrotransposition). To assess the impact of RNA 3’ end dynamics of LINE-1 mRNA on its retrotransposition potential in human cells we analyzed 3' non-templated ends on LINE-1 mRNA by 3' RACE-seq. RNA was retrieved from multiple clonal 293T and PA-1 cells wild-type or knock-out in either XRN1, DCP2, or DIS3L2. Publication: D Janecki, R Sen, N Szóstak, A Kajdasz, M Kordys, K Plawgo, D Pandakov, A Philips, Z Warkocki (2024): LINE-1 mRNA 3’ end dynamics shape its biology and retrotransposition potential. Nucleic Acids Research. https://doi.org/10.1093/nar/gkad1251

2024-01-05 | GSE248108 | GEO

NMD-degradome sequencing reveals ribosome-bound intermediates with 3'-end nontemplated nucleotides

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EnD-Seq and AppEnD: sequencing 3' ends to identify nontemplated tails and degradation intermediates.

Project description:Existing methods for detecting RNA intermediates resulting from exonuclease degradation are low-throughput and laborious. In addition, mapping the 3' ends of RNA molecules to the genome after high-throughput sequencing is challenging, particularly if the 3' ends contain post-transcriptional modifications. To address these problems, we developed EnD-Seq, a high-throughput sequencing protocol that preserves the 3' end of RNA molecules, and AppEnD, a computational method for analyzing high-throughput sequencing data. Together these allow determination of the 3' ends of RNA molecules, including nontemplated additions. Applying EnD-Seq and AppEnD to histone mRNAs revealed that a significant fraction of cytoplasmic histone mRNAs end in one or two uridines, which have replaced the 1-2 nt at the 3' end of mature histone mRNA maintaining the length of the histone transcripts. Histone mRNAs in fly embryos and ovaries show the same pattern, but with different tail nucleotide compositions. We increase the sensitivity of EnD-Seq by using cDNA priming to specifically enrich low-abundance tails of known sequence composition allowing identification of degradation intermediates. In addition, we show the broad applicability of our computational approach by using AppEnD to gain insight into 3' additions from diverse types of sequencing data, including data from small capped RNA sequencing and some alternative polyadenylation protocols.

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RPA and Rad27 limit templated and inverted insertions at DNA breaks

Project description:Formation of templated insertions at DNA double-strand breaks (DSBs) is very common in cancer cells. The mechanisms and enzymes regulating these events are largely unknown. Here, we investigated templated insertions in yeast at DSBs using amplicon sequencing across a repaired locus. We document very short (most ~5-34 bp), templated inverted duplications at DSBs. They are generated through a foldback mechanism that utilizes microhomologies adjacent to the DSB. Enzymatic requirements suggest a hybrid mechanism wherein one end requires Polδ-mediated synthesis while the other end is captured by nonhomologous end joining (NHEJ) or rarely by alternative end joining (Alt-EJ). This process is exacerbated in mutants with low levels or mutated RPA (rtt105Δ; rfa1-t33) or extensive resection mutant (sgs1Δ exo1Δ). Templated insertions from various distant genomic locations also increase in these mutants as well as in rad27 and originate from fragile regions of the genome. Among complex insertions, common events are insertions of two sequences, originating from the same locus and with inverted orientation. We propose that these inversions are also formed by microhomology-mediated template switching. Taken together, we propose that a shortage of RPA typical in cancer cells is one possible factor stimulating the formation of templated insertions.

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NMD-degradome sequencing reveals ribosome-bound intermediates with 3'-end nontemplated nucleotides

Project description:NMD-degradome sequencing reveals ribosome-bound intermediates with 3'-end nontemplated nucleotides

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Comprehensive analysis of Poly(A) tails in Mouse Testes and Ovaries using Nanopore direct RNA Sequencing. 

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