FLEP-seq: simultaneous detection of RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale by single-molecule nascent RNA sequencing

Yanping Long, Jinbu Jia, Weipeng Mo, Xianhao Jin, Jixian Zhai

Published: 2021-07-29 DOI: 10.1038/s41596-021-00581-7

FLEP-seq

nascent RNA sequencing

cotranscriptional splicing

polyadenylation

kinetics

AI 解读

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Elongation, splicing and polyadenylation are fundamental steps of transcription, and studying their coordination requires simultaneous monitoring of these dynamic processes on one transcript. We recently developed a full-length nascent RNA sequencing meth
Introduction
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Anticipated results
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ACKNOWLEDGMENTS

Herzel, L., Ottoz, D. S. M., Alpert, T. & Neugebauer, K. M. Splicing and transcription touch base: co-transcriptional spliceosome assembly and function. Nat. Rev. Mol. Cell Biol. 18, 637–650 (2017).
Naftelberg, S., Schor, I. E., Ast, G. & Kornblihtt, A. R. Regulation of alternative splicing through coupling with transcription and chromatin structure. Annu. Rev. Biochem. 84, 165–198 (2015).
Bentley, D. L. Coupling mRNA processing with transcription in time and space. Nat. Rev. Genet. 15, 163–175 (2014).
Carrillo Oesterreich, F. et al. Splicing of nascent RNA coincides with intron exit from RNA Polymerase II. Cell 165, 372–381 (2016).
Herzel, L., Straube, K. & Neugebauer, K. M. Long-read sequencing of nascent RNA reveals coupling among RNA processing events. Genome Res. 28, 1008–1019 (2018).
Reimer, K. A., Mimoso, C. A., Adelman, K. & Neugebauer, K. M. Co-transcriptional splicing regulates 3’ end cleavage during mammalian erythropoiesis. Mol. Cell http://doi.org/10.1016/j.molcel.2020.12.018 (2021).
Drexler, H. L., Choquet, K. & Churchman, L. S. Splicing kinetics and coordination revealed by direct nascent RNA sequencing through nanopores. Mol. Cell 77, 985–998.e988 (2020).
Drexler, H. L. et al. Revealing nascent RNA processing dynamics with nano-COP. Nat. Protoc. http://doi.org/10.1038/s41596-020-00469-y (2021).
Sousa-Luis, R. et al. POINT technology illuminates the processing of polymerase-associated intact nascent transcripts. Mol. Cell 81, 1935–1950.e6 (2021).
Wissink, E. M., Vihervaara, A., Tippens, N. D. & Lis, J. T. Nascent RNA analyses: tracking transcription and its regulation. Nat. Rev. Genet. 20, 705–723 (2019).
Mayer, A. et al. Native elongating transcript sequencing reveals human transcriptional activity at nucleotide resolution. Cell 161, 541–554 (2015).
Bhatt, DevM. et al. Transcript dynamics of proinflammatory genes revealed by sequence analysis of subcellular RNA fractions. Cell 150, 279–290 (2012).
Nechaev, S. et al. Global analysis of short RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila. Science 327, 335 (2010).
Churchman, L. S. & Weissman, J. S. Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature 469, 368–373 (2011).
Nojima, T. et al. Mammalian NET-Seq reveals genome-wide nascent transcription coupled to RNA processing. Cell 161, 526–540 (2015).
Core, L. J., Waterfall, J. J. & Lis, J. T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845 (2008).
Kwak, H., Fuda, N. J., Core, L. J. & Lis, J. T. Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339, 950–953 (2013).
Judd, J. et al. A rapid, sensitive, scalable method for Precision Run-On sequencing (PRO-seq). Preprint at bioRxiv http://doi.org/10.1101/2020.05.18.102277 (2020).
Rabani, M. et al. Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells. Nat. Biotechnol. 29, 436–442 (2011).
Schwalb, B. et al. TT-seq maps the human transient transcriptome. Science 352, 1225 (2016).
Herzog, V. A. et al. Thiol-linked alkylation of RNA to assess expression dynamics. Nat. Methods 14, 1198–1204 (2017).
Schofield, J. A., Duffy, E. E., Kiefer, L., Sullivan, M. C. & Simon, M. D. TimeLapse-seq: adding a temporal dimension to RNA sequencing through nucleoside recoding. Nat. Methods 15, 221–225 (2018).
Shepard, P. J. et al. Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq. RNA 17, 761–772 (2011).
Harrison, P. F. et al. PAT-seq: a method to study the integration of 3’-UTR dynamics with gene expression in the eukaryotic transcriptome. RNA 21, 1502–1510 (2015).
Ozsolak, F. et al. Direct RNA sequencing. Nature 461, 814–818 (2009).
Chang, H., Lim, J., Ha, M. & Kim, V. N. TAIL-seq: genome-wide determination of poly(A) tail length and 3′ end modifications. Mol. Cell 53 (2014).
Lim, J., Lee, M., Son, A., Chang, H. & Kim, V. N. mTAIL-seq reveals dynamic poly(A) tail regulation in oocyte-to-embryo development. Genes Dev. 30, 1671–1682 (2016).
Subtelny, A. O., Eichhorn, S. W., Chen, G. R., Sive, H. & Bartel, D. P. Poly(A)-tail profiling reveals an embryonic switch in translational control. Nature 508, 66–71 (2014).
Legnini, I., Alles, J., Karaiskos, N., Ayoub, S. & Rajewsky, N. FLAM-seq: full-length mRNA sequencing reveals principles of poly(A) tail length control. Nat. Methods 16, 879–886 (2019).
Liu, Y., Nie, H., Liu, H. & Lu, F. Poly(A) inclusive RNA isoform sequencing (PAIso−seq) reveals wide-spread non-adenosine residues within RNA poly(A) tails. Nat. Commun. 10, 5292 (2019).
Parker, M. T. et al. Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m6A modification. eLife 9, e49658 (2020).
Workman, R. E. et al. Nanopore native RNA sequencing of a human poly(A) transcriptome. Nat. Methods 16, 1297–1305 (2019).
Roach, N. P. et al. The full-length transcriptome of C. elegans using direct RNA sequencing. Genome Res 30, 299–312 (2020).
Kim, D. et al. The architecture of SARS-CoV-2 transcriptome. Cell 181, 914–921.e910 (2020).
Jia, J. et al. Post-transcriptional splicing of nascent RNA contributes to widespread intron retention in plants. Nat. Plants 6, 780–788 (2020).
Boutz, P. L., Bhutkar, A. & Sharp, P. A. Detained introns are a novel, widespread class of post-transcriptionally spliced introns. Genes Dev. 29, 63–80 (2015).
Mauger, O., Lemoine, F. & Scheiffele, P. Targeted intron retention and excision for rapid gene regulation in response to neuronal activity. Neuron 92, 1266–1278 (2016).
Naro, C. et al. An orchestrated intron retention program in meiosis controls timely usage of transcripts during germ cell differentiation. Dev. Cell 41, 82–93.e84 (2017).
Gahura, O. et al. Prp45 affects Prp22 partition in spliceosomal complexes and splicing efficiency of non-consensus substrates. J. Cell. Biochem. 106, 139–151 (2009).
Siatecka, M., Reyes, J. L. & Konarska, M. M. Functional interactions of Prp8 with both splice sites at the spliceosomal catalytic center. Genes Dev. 13, 1983–1993 (1999).
Zhu, D. et al. The features and regulation of co-transcriptional splicing in Arabidopsis. Mol. Plant 13, 278–294 (2020).
Krause, M. et al. tailfindr: alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing. RNA 25, 1229–1241 (2019).
Li, H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34, 3094–3100 (2018).
Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Camacho, C. et al. BLAST+: architecture and applications. BMC Bioinformatics 10, 421 (2009).
Cheng, C.-Y. et al. Araport11: a complete reannotation of the Arabidopsis thaliana reference genome. Plant J. 89, 789–804 (2017).

FLEP-seq: simultaneous detection of RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale by single-molecule nascent RNA sequencing

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