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Homo sapiens

ABSTRACT: Ultra short and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution

PROVIDER: PRJNA145401 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR345514.fastq.gz	Fastqsanger.gz
	SRR345515.fastq.gz	Fastqsanger.gz
	SRR345516.fastq.gz	Fastqsanger.gz
	SRR345517.fastq.gz	Fastqsanger.gz
	SRR345518.fastq.gz	Fastqsanger.gz

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Ultra short and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution

Project description:RNA synthesis and decay rates determine the steady-state levels of cellular RNAs. Metabolic tagging of newly transcribed RNA by 4-thiouridine (4sU) can reveal the relative contributions of RNA synthesis and decay rates. The kinetics of RNA processing, however, so far remained unresolved. Here, we show that ultra-short 4sU-tagging not only provides snap-shot pictures of eukaryotic gene expression but, when combined with progressive 4sU-tagging and RNA-seq, reveals global RNA processing kinetics at nucleotide resolution. Using this method, we identified classes of rapidly and slowly spliced/degraded introns. Interestingly, each class of splicing kinetics was characterized by a distinct association with intron length, gene length and splice site strength. For a large group of introns, we also observed long lasting retention in the primary transcript, but efficient secondary splicing or degradation at later time points. Finally, we show that processing of most, but not all small nucleolar (sno)RNA-containing introns is remarkably inefficient with the majority of introns being spliced and degraded rather than processed into mature snoRNAs. In summary, our study yields unparalleled insights into the kinetics of RNA processing and provides the tools to study molecular mechanisms of RNA processing and their contribution to the regulation of gene expression. 4sU-tagging was performed in human DG75 B-cells by adding 500 uM 4sU to cell culture medium for 5, 10, 15, 20 or 60 min. Following isolation of total cellular RNA, this was separated into nascent and untagged, pre-existing RNA. Nascent RNA as well as total and untagged RNA from 60 min 4sU-tagging were subjected to SOLiD sequencing (SOLiD II) obtaining 35 nt reads.

2012-04-25 | E-GEOD-31653 | biostudies-arrayexpress

Ultra short and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution

2012-04-26 | GSE31653 | GEO

Mapping Human Transient Transcriptomes Using Single Nucleotide Resolution 4sU Sequencing (SNU-Seq)

Project description:Genomes are pervasively transcribed leading to stable and unstable transcripts that define functional regions of genomes and contribute to cellular phenotypes. Defining comprehensive nascent transcriptomes is pivotal to understand gene regulation, disease processes, and the impact of extracellular signals on cells. However, currently employed methods are laborious, technically challenging and costly. We developed single-nucleotide resolution 4sU-sequencing (SNU-Seq), involving pulse labelling, biotinylation and direct isolation of nascent transcripts. Artificial poly-(A)-tailing of the 3’ most nucleotide of nascent transcripts ensures oligo-d(T) primer-based library preparation and sequencing using commercial 3’ RNA-Seq kits. We show that SNU-Seq is a cost-effective new method generating even read profiles across transcription units. We used SNU-Seq to identify transcription elongation parameters, to map usage of polyadenylation (PAS) sites and novel enhancers. Remarkably, 4sU labelled nascent RNA accumulates short ~100nt transcripts that map to the 5’ end of genes. We show that isolation of these short nascent RNA and sequencing the 5’ and 3’ ends using size-selected SNU-Seq (ssSNU-Seq) provides highly sensitive annotations of mapped and novel TSSs, promoter-proximal pause/termination sites. Thus, SNU-seq and ssSNU-seq combined yield comprehensive transcriptomics data at low cost with high spatial and temporal resolution.

2022-04-01 | GSE179306 | GEO

Addition-elimination mechanism activated nucleotide transition sequencing for RNA dynamics profiling

Project description:The temporal information of intracellular transcripts is essential to understand their functional roles, yet routine RNA-seq methods only measure RNA species at a steady state. Here we develop addition-elimination mechanism activated nucleotide transition sequencing (AENT-seq) for transcriptome-wide profiling of RNA dynamics. In AENT-seq, intracellular transcripts are metabolically labeled with 4-thiouridine (4sU), and then isolated total RNA are subjected to 4sU-to-cytosine (C) transition reactions based on a mild addition-elimination chemistry. N2H4•H2O is demonstrated to convert 4sU to 4-hydrazino cytosine (C*) in RNA-friendly aqueous conditions. The C* is recorded as natural C during extension reactions with a high efficiency of ~95%. This 4sU-to-C transition marks 4sU-containing nascent transcripts, and it enables subsequent sequencing analysis of RNA dynamics with single-nucleotide resolution. We applies our AENT-seq to simply investigate transcript dynamic information of several genes involved in cancer progression and metastasis. This method avoids harsh chemical conditions and harmful reagents involved in previous approaches, allowing rapid dissemination and extensive applications of the technique.

2021-03-09 | GSE168485 | GEO

Effect of 4sU labeling concentrations on quantification bias in nucleotide conversion RNA-seq data

Project description:To investigate the effect of increasing 4sU labeling concentrations on quantification bias in nucleotide conversion RNA-seq, we labeled 3 different cell lines with increasing concentrations of 4sU. We then measured 4sU dropout in 4sU samples compared to 4sU naive samples.

2024-02-06 | GSE229504 | GEO

Effect of 4sU labeling durations on quantification bias in nucleotide conversion RNA-seq data

Project description:To investigate the effect of labeling durations on quantification bias in nucleotide conversion RNA-seq, we labeled NIH cells with 4sU. We then measured 4sU dropout in 4sU samples compared to 4sU naive samples.

2024-02-06 | GSE229506 | GEO

Gene expression and 4sUDRB-seq for NF90/NF110 of human scramble and KD HeLa cells.

Project description:We have optimized the metabolic tagging of newly transcribed RNAs by 4-thiouridine (4sU) to identify newly-transcribed (nascent) circRNAs from human HeLa cells.

2017-06-23 | GSE90874 | GEO

A high resolution sequencing and modeling identifies distinct dynamic RNA regulatory strategies

Project description:To monitor the relative regulatory contributions of RNA production, processing and degradation, we sampled RNA from mouse DCs every 15 minutes, for the first 3 hours of their response to LPS (13 samples in total), following a short (10 minute) metabolic labeling pulse with 4sU preceding the sampled time point. We isolated RNA from each of these samples in two ways. First, to comprehensively measure total RNA regardless of its transcription time, we isolated RNA depleted of rRNA. Second, to measure only newly made RNA, we isolated 4sU-labeled RNA, which could only have been transcribed during the 10 minute labeling pulse and is thus enriched for short-lived transcripts, including mRNA precursors and processing intermediates. We deeply sequenced each isolated RNA population to provide the necessary depth to study exons, introns, and splicing junctions across the transcriptome. We showed our approach general utility by also applying it to lincRNAs and early zebrafish development data Time-course mRNA profiles of mouse DCs that were stimulated with LPS, and labeled with a short (10 minute) 4sU pulse (0-3 hours, 15 min. intervals, 13 time points).

2014-12-16 | E-GEOD-56977 | biostudies-arrayexpress

Mapping Human Transient Transcriptomes Using Single Nucleotide Resolution 4sU Sequencing (SNU-Seq)

Project description:Mapping Human Transient Transcriptomes Using Single Nucleotide Resolution 4sU Sequencing (SNU-Seq)

| PRJNA746479 | ENA

Effect of TUC-seq protocol versus SLAM-seq on quantification bias in nucleotide conversion RNA-seq data

Project description:To investigate the effect of TUC-seq versus SLAM-seq on quantification bias in nucleotide conversion RNA-seq, we performed SLAM-seq and TUC-seq on NIH cells. We then measured 4sU dropout in 4sU samples compared to 4sU naive samples.

2024-02-06 | GSE253169 | GEO

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