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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE121461 | GEO | 2018/11/30

REPOSITORIES: GEO

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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.

2015-06-01 | GSE68471 | GEO

Small RNAs in H9 embryonic stem cells

Project description:Non-templated 3'-end oligouridylation of RNA occurs in many species, including humans. Unlike the familiar phenomenon of polyadenylation, non-templated addition of uridines to RNA is poorly characterized in higher eukaryotes. Recent studies have reported non-templated 3'-end oligouridylation of small RNAs and mRNAs. Oligouridylation is involved in many aspects of microRNA biology from biogenesis to turnover of the mature species and it may also mark long mRNAs for degradation by promoting decapping of the protective 5'-cap structure. To determine the prevalence of oligouridylation in higher eukaryotes, we used next-generation sequencing technology to deeply examine the population of small RNAs in human cells. Our data revealed widespread non-templated nucleotide addition to the 3'-ends of many classes of RNA, with short stretches of uridine being the most frequently added nucleotide. Strand-specific RNA-Seq and paired-end sequencing was used to directly analyze the 3'-ends of small RNAs to identify post-transcriptional RNA processing.

2012-03-21 | E-GEOD-31051 | biostudies-arrayexpress

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

| PRJNA497386 | ENA

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

| PRJNA282807 | ENA

Small RNAs in H9 embryonic stem cells

2012-03-21 | GSE31051 | GEO

TUT7 Catalyzes the Uridylation of the 3’ End for Rapid Degradation of Histone mRNA

Project description:Using high-throughput sequencing of histone mRNAs and degradation intermediates, we find that knockdown of TUT7 reduces both the uridylation at the 3’ end as well as uridylation pattern at the 3’ end, and only had a small effect on the uridylation in the stemloop uridylation of the major during histone mRNA degradation. Knockdown of 3’ hEXO also altered the uridylation of histone mRNAs, revealing a dynamic equilibrium between 3’ hEXO digestion and TUT7 uridylation, suggesting that TUT7 and 3’ hExo function together in trimming and uridylating histone mRNAs.

2016-08-29 | GSE85361 | GEO

Deep sequencing shows multiple oligouridylations are required for 3' to 5' degradation of histone mRNAs on polyribosomes

2014-03-20 | GSE54922 | GEO

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

Deep sequencing shows multiple oligouridylations are required for 3' to 5' degradation of histone mRNAs on polyribosomes

Project description:We developed a customized RNA-Seq strategy to identify the 3' termini of degradation intermediates of histone mRNAs. Using this strategy, we identified two types of oligouridylated degradation intermediates: RNAs ending at different sites of the 3' side of the stemloop that resulted from initial degradation by 3'hExo and intermediates near the stop codon and within the coding region. We established a method for identifying degradation intermediates for replication-depdendent histone mRNAs. Histone mRNAs are present in large amounts in S-phase cells. Inhibition of DNA replication results in a rapid degradation of histone mRNA. We developed an approach to selectively amplify the 3' ends of histone mRNA molecules, including degradation intermediates, which we then sequenced. Using a novel bioinformatics platform we identified degradation intermediates including many that had untemplated nts added to the 3' end. We identified a large variety of degradation intermediates that contained oligo(U) tails. We were able to deduce the 3' to 5' pathway of histone mRNA degradation. RNA-seq with a custom 3' linker: The samples analyzed included two biological replicates of RNAs before and 20 min after HU treatment (samples 1, 26, 27) capped RNAs from cells treated with HU for 20 min (samples 2,3,16,17) RNA isolated from polyribosomes 20 min after HU treatment, from untreated cells (samples 4-9) and cells treated with pactamycin for 5 min (samples 10-15); from cells in which the exosome subunit Pml/SC100 had been knocked down (samples 22-23) and cells treated with the control siRNA (samples 20-21). Please note that, for Sample 2 and Sample 3, the sequencer was unable to recover both ends in the mate pairs for these two sequencing runs, but we chose to use these data anyway since the reads that were recovered included the 3' linker that we used to determine the presence of untemplated additions.

2014-03-20 | E-GEOD-54922 | biostudies-arrayexpress

Digital Restriction Enzyme Analysis of Methylation (DREAM) of SW48 colon cancer cell line genomic DNA.

Project description:We analyzed levels of 5-methyl cytosine nnnn CCCGGG target sites by sequential restriction digest by SmaI and XmaI enzymes, ligating Illumina adaptors to the restriction fragments and reading methylation-specific signatures at the ends of restriction fragments by paired ends Illumina high throughput sequencing. Digital restriction enzyme analysis of methylation (DREAM) was performed to determine the methylation profile of SW48 colon cancer cell line genomic DNA. Genomic DNA spiked in with unmethylated, partially methylated and fully methylated standards was sequentially cut at CCCGGG sites with the methylation-sensitive enzyme SmaI (blunt ends) and its methylation-tolerant neoschizomer XmaI (5'CCGG overhangs), creating different end sequences that represented methylation status of the CCCGGG sites. These end sequences were analyzed by Illumina high throughput sequencing. Methylation status at individual CCCGGG sites across the genome was determined by counting the methylated reads with the CCGGG signature and unmethylated reads with the GGG signature at the beginnings of the sequencing reads after alignment to the human genome.

2015-09-08 | E-GEOD-66296 | biostudies-arrayexpress

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