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

Sousa-Luís, Rui; Dujardin, Geneviève; Zukher, Inna; Kimurâ, Hiroshi; Weldon, Carika; Carmo‐Fonseca, Maria; Proudfoot, Nick J.; Nojima, Takayuki

doi:10.1016/j.molcel.2021.02.034

Cited by 70 publications

(68 citation statements)

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“…5a ). Similar results were also observed in human Xrn2 depletion cell line detected by short-read-based method POINT-5 [ 25 ]. In addition, accumulation of the 3′ cleavage products is not influenced by splicing, as genes with or without splicing have 3′ cleavage products enriched in atxrn3 and aligned accurately at the poly(A) site (Fig.…”

Section: Resultssupporting

confidence: 84%

“…GRO-seq [ 21 ] and PRO-seq [ 22 ] performed a run-on with isolated nuclei, which might compromise the termination machinery. NET-seq [ 23 ], mNET-seq [ 24 ], and POINT-seq [ 25 ] require immunoprecipitation of Pol II, which might miss terminating RNAs that are no longer associated with Pol II, such as the cleaved and polyadenylated pre-mRNAs after Pol II passing through the poly(A) site, and cannot distinguish between individual peaks caused by Pol II accumulation and co-transcriptional cleavage [ 20 – 24 ]. TT-seq [ 17 ] was indeed able to define transcription termination sites, but due to the limited read length of Illumina sequencing and the RNA fragmentation step during library construction, it also cannot distinguish whether the readthrough transcripts are cleaved or not.…”

Section: Introductionmentioning

confidence: 99%

“…In particular, the Oxford Nanopore direct RNA sequencing (ONT DRS) can recognize and distinguish various forms of RNA modifications that cause unique changes of current when RNA molecule is passing through the nanopore [ 26 ]. The applications of long-read sequencing (LRS) in characterizing full-length nascent RNAs have revealed co-transcriptional splicing kinetics in a variety of species by simultaneously tracking splicing status and position of Pol II elongation on the same RNA molecule, such as nano-COP in human and drosophila [ 33 , 34 ], LRS of nascent RNA in yeast and mouse [ 35 , 36 ], POINT-nano in human [ 25 ], and recent work from our group in Arabidopsis [ 37 ]. Here, we demonstrate the application of single-molecule nascent RNA sequencing in studying transcription termination by simultaneously recording Pol II readthrough distance, cleavage status, and polyadenylation on the same RNA molecule, and revealed the global landscape of transcription termination in Arabidopsis.…”

Section: Introductionmentioning

confidence: 99%

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Landscape of transcription termination in Arabidopsis revealed by single-molecule nascent RNA sequencing

Liu

Hong

et al. 2021

Genome Biol

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Background The dynamic process of transcription termination produces transient RNA intermediates that are difficult to distinguish from each other via short-read sequencing methods. Results Here, we use single-molecule nascent RNA sequencing to characterize the various forms of transient RNAs during termination at genome-wide scale in wildtype Arabidopsis and in atxrn3, fpa, and met1 mutants. Our data reveal a wide range of termination windows among genes, ranging from ~ 50 nt to over 1000 nt. We also observe efficient termination before downstream tRNA genes, suggesting that chromatin structure around the promoter region of tRNA genes may block pol II elongation. 5′ Cleaved readthrough transcription in atxrn3 with delayed termination can run into downstream genes to produce normally spliced and polyadenylated mRNAs in the absence of their own transcription initiation. Consistent with previous reports, we also observe long chimeric transcripts with cryptic splicing in fpa mutant; but loss of CG DNA methylation has no obvious impact on termination in the met1 mutant. Conclusions Our method is applicable to establish a comprehensive termination landscape in a broad range of species.

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Section: Resultssupporting

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Landscape of transcription termination in Arabidopsis revealed by single-molecule nascent RNA sequencing

Liu

Hong

et al. 2021

Genome Biol

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“…Taken together, our results show that splicing in Drosophila embryos can be completed soon after transcription of the 3′ splice site, as previously reported in other cellular models ( Martin et al 2013 ; Carrillo Oesterreich et al 2016 ; Reimer et al 2021 ; Sousa-Luís et al 2021 ). However, a question that remains open is whether splicing influences the kinetics of Pol II elongation.…”

Section: Discussionsupporting

confidence: 90%

“…The lowest of these estimates, such as the few-second estimate reported in Martin et al (2013) , are consistent with splicing being completed right after the 3′ splice site is transcribed. The most recent breakthroughs in the field followed the development of long-read nascent RNA sequencing technologies ( Drexler et al 2020 ; Reimer et al 2021 ; Sousa-Luís et al 2021 ). Collectively, these studies performed in human and Drosophila cultured cell lines show that although many introns are immediately excised as soon as the downstream exon emerges from Pol II, a subset remains unspliced and progressively undergoes delayed splicing while Pol II transcribes further ( Drexler et al 2020 ; Reimer et al 2021 ; Sousa-Luís et al 2021 ).…”

Section: Introductionmentioning

confidence: 99%

Transcription and splicing dynamics during early Drosophila development

Prudêncio

Savisaar

Rebelo

et al. 2021

RNA

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Widespread co-transcriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either Saccharomyces cerevisiae or metazoan cultured cell lines. Here, we adapted native elongating transcript sequencing technology (NET-seq) to measure co-transcriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. Our results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. We found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nucleotides of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. We studied the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. We further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, our data unravels novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.

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Integrating transcription and splicing into cell fate: Transcription factors on the block

2022

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Transcription factors (TFs) are present in all life forms and conserved across great evolutionary distances in eukaryotes. From yeast to complex multicellular organisms, they are pivotal players of cell fate decision by orchestrating gene expression at diverse molecular layers. Notably, TFs fine-tune gene expression by coordinating RNA fate at both the expression and splicing levels.They regulate alternative splicing, an essential mechanism for cell plasticity, allowing the production of many mRNA and protein isoforms in precise cell and tissue contexts. Despite this apparent role in splicing, how TFs integrate transcription and splicing to ultimately orchestrate diverse cell functions and cell fate decisions remains puzzling. We depict substantial studies in various model organisms underlining the key role of TFs in alternative splicing for promoting tissue-specific functions and cell fate. Furthermore, we emphasize recent advances describing the molecular link between the transcriptional and splicing activities of TFs. As TFs can bind both DNA and/or RNA to regulate transcription and splicing, we further discuss their flexibility and compatibility for DNA and RNA substrates. Finally, we propose several models integrating transcription and splicing activities of TFs in the coordination and diversification of cell and tissue identities.

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

Abstract: Highlights d POINT methodology dissects intact nascent RNA processing d Specificity of Xrn2 exonuclease in co-transcriptional RNA degradation d Splicing suppresses Xrn2-dependent premature termination d Different kinetic classes of co-transcriptional splicing in human genes

Cited by 70 publications

References 68 publications

Landscape of transcription termination in Arabidopsis revealed by single-molecule nascent RNA sequencing

Landscape of transcription termination in Arabidopsis revealed by single-molecule nascent RNA sequencing

Transcription and splicing dynamics during early Drosophila development

Integrating transcription and splicing into cell fate: Transcription factors on the block

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