Revealing nascent RNA processing dynamics with nano-COP
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA645078 | ENA |
REPOSITORIES: ENA
Dataset's files
| 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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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.
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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.
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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.
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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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Project description:Single-cell nascent RNA sequencing is essential for understanding how a genome drives cell diversity. We developed scFLUENT-seq, a single-cell method that captures genome-wide transcription with brief metabolic labeling. Our analysis shows that only 3~6% of the genome is transcribed per cell in a 10-minute window, compared to over 80% in bulk, revealing significant variability in how individual cells interpret the genome. Notably, substantial transcription occurs in intergenic regions, particularly in heterochromatin, with high stochasticity. Moreover, promoter-associated antisense and genic sense transcription rarely co-occur in the same cell. Distal intergenic transcription correlates poorly with gene activity but links to increased genome-wide transcriptional diversity, which marks cellular plasticity and may precede cell-state shifts. Furthermore, mRNA synthesis and decay are uncoupled at the single-cell level, unlike intergenic ncRNA, suggesting specialized mechanisms counteracting stochastic noncoding production. In summary, scFLUENT-seq captures the full transcriptional spectrum, revealing the heterogeneity and regulatory complexity underlying cellular plasticity.
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