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Revealing nascent RNA processing dynamics with nano-COP

ABSTRACT: Revealing nascent RNA processing dynamics with nano-COP

PROVIDER: PRJNA645078 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12183700_1.fastq.gz	Fastqsanger.gz
	SRR12183701_1.fastq.gz	Fastqsanger.gz
	SRR12183702_1.fastq.gz	Fastqsanger.gz
	SRR12183703_1.fastq.gz	Fastqsanger.gz
	SRR12183704_1.fastq.gz	Fastqsanger.gz

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Revealing nascent RNA processing dynamics with nano-COP

Project description:During maturation, eukaryotic precursor RNAs undergo processing events including intron splicing, 3’-end cleavage, and polyadenylation. Here, we describe nanopore analysis of CO-transcriptional Processing (nano-COP), a method for probing the timing and patterns of RNA processing. An extension of native elongating transcript sequencing (NET-seq), which quantifies transcription genome-wide through short-read sequencing of nascent RNA 3’ ends, nano-COP uses long-read nascent RNA sequencing to observe global patterns of RNA processing. First, nascent RNA is stringently purified through a combination of 4-thiouridine metabolic labeling and cellular fractionation. In contrast to cDNA or short-read–based approaches relying on reverse transcription or amplification, the sample is sequenced directly through nanopores to reveal the native context of nascent RNA. nano-COP identifies both active transcription sites and splice isoforms of single RNA molecules during synthesis, providing insight into patterns of intron removal and the physical coupling between transcription and splicing. The nano-COP protocol yields data within 3 days.

2021-01-06 | GSE154079 | GEO

Revealing nascent RNA processing dynamics with nano-COP.

Project description:During maturation, eukaryotic precursor RNAs undergo processing events including intron splicing, 3'-end cleavage, and polyadenylation. Here we describe nanopore analysis of co-transcriptional processing (nano-COP), a method for probing the timing and patterns of RNA processing. An extension of native elongating transcript sequencing, which quantifies transcription genome-wide through short-read sequencing of nascent RNA 3' ends, nano-COP uses long-read nascent RNA sequencing to observe global patterns of RNA processing. First, nascent RNA is stringently purified through a combination of 4-thiouridine metabolic labeling and cellular fractionation. In contrast to cDNA or short-read-based approaches relying on reverse transcription or amplification, the sample is sequenced directly through nanopores to reveal the native context of nascent RNA. nano-COP identifies both active transcription sites and splice isoforms of single RNA molecules during synthesis, providing insight into patterns of intron removal and the physical coupling between transcription and splicing. The nano-COP protocol yields data within 3 d.

| S-EPMC8713461 | biostudies-literature

Human co-transcriptional splicing kinetics and coordination revealed by direct nascent RNA sequencing

Project description:Human genes have numerous exons that are differentially spliced within pre-mRNA. Understanding how multiple splicing events are coordinated across nascent transcripts requires quantitative analyses of transient RNA processing events in living cells. We developed nanopore analysis of CO-transcriptional Processing (nano-COP), in which nascent RNAs are directly sequenced through nanopores, exposing the dynamics and patterns of RNA splicing without biases introduced by amplification. nano-COP showed that in both human and Drosophila cells, co-transcriptional splicing occurs after RNA polymerase II transcribes several kilobases of pre-mRNA, suggesting that metazoan splicing transpires distally from the transcription machinery. Inhibition of the branch site recognition complex SF3B globally abolished co-transcriptional splicing in both species. Our findings revealed that splicing order does not strictly follow the order of transcription and is influenced by cis-regulatory elements. In human cells, introns with delayed splicing frequently neighbor alternative exons and are associated with RNA-binding factors. Moreover, neighboring introns in human cells tend to be spliced concurrently, implying that splicing occurs cooperatively. Thus, nano-COP unveils the organizational complexity of metazoan RNA processing.

2019-12-12 | GSE123191 | GEO

POINT technology illuminates the processing of polymerase-associated intact nascent transcripts

Project description:Mammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly due to technical limitations in purifying authentic nascent transcripts. We present a new approach to purify and profile nascent RNA, called Polymerase Intact Nascent Transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5’ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano) and profiles whole transcription units (POINT-seq). In particular, we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5’P-RNA at polyadenylation sites. Furthermore, POINT-nano reveals that splicing occurs either immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing.

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Panicum virgatum strain:COP | cultivar:COP

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Project description:First we developed a new, simple, and robust assay called CoP (Column Purified chromatin) for profiling of accessible regions by directly purifying fragmentized crosslinked chromatin with DNA purification column. The CoP chromatin regions by CoP-seq are consistent with the accessible regions detected by ATAC-seq and FARIE-seq. Then we integrated CoP-seq and Hi-C technique (Hi-CoP) for interactions of accessible chromatin regions which represent active cis-regulatory elements in cells. Our data showed that Hi-CoP assay can robustly detect interactions of regulatory regions.

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Project description:We report that slow transcription by mutant RNA pol II caused accumulation of polyadenylated histone mRNAs that extend past the stem loop processing site. UV irradiation, which decelerates pol II elongation, also induced long poly(A)+ histone transcripts. Inhibition of 3’ processing by slow pol II correlates with failure to recruit SLBP to histone genes. Chemical probing of nascent RNA structure showed that the stem loop fails to fold in transcripts made by slow pol II, thereby explaining the absence of SLBP and failure to process 3’ ends. These results show that regulation of transcription speed can modulate pre-mRNA processing by changing nascent RNA structure, and suggest a mechanism by which alternative processing could be controlled

2018-02-23 | GSE109652 | GEO

Alfa2-COP is involved in early secretory traffic in Arabidopsis and it is required for plant growth

Project description:The loss-of-function mutants of alfa1-COP (alfa1-COP-1) and alfa2-COP (alfa2-COP1), the two alfa-COP isoforms of Arabidopsis. Unlike in alfa1-COP mutant, the alfa2-COP mutant displays defects in plant growth, including small rosettes, stems and roots and mislocalization of p24a5, a protein containing a C-terminal dylisine motif involved in COPI binding. In addition, it exhibited abnormal morphology of the Golgi apparatus. Global expression analyses of the alfa2-COP mutant versus wild-type Arabidopsis plants reveal altered expression of plant cell wall-associated genes. A high expression of the COPII sec31A isoform was observed in the alfa2-COP mutant that also occurs in a mutant of an upstream gene of COPI assembly, the ARF-GEF GNL1

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Single-cell nascent transcription is sparse and heterogeneous, revealing cellular plasticity [single_cell]

Project description:Single-cell nascent transcription is sparse and heterogeneous, revealing cellular plasticity [single_cell]

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Project description:Single-cell nascent transcription is sparse and heterogeneous, revealing cellular plasticity [EU_seq]

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