RNA Structure Probing to Characterize RNA-protein Interactions on a Low Abundance Pre-mRNA in Living Cells

Overview

Journal RNA

Specialty Molecular Biology

Date 2020 Dec 14

PMID 33310817

Citations 5

Authors

Jodi L Bubenik

Melissa Hale

Ona McConnell

Eric Wang

Maurice S Swanson

Robert Spitale

J Andrew Berglund

Affiliations

Soon will be listed here.

Abstract

In vivo RNA structure analysis has become a powerful tool in molecular biology, largely due to the coupling of an increasingly diverse set of chemical approaches with high-throughput sequencing. This has resulted in a transition from single target to transcriptome-wide approaches. However, these methods require sequencing depths that preclude studying low abundance targets, which are not sufficiently captured in transcriptome-wide approaches. Here we present a ligation-free method to enrich for low abundance RNA sequences, which improves the diversity of molecules analyzed and results in improved analysis. In addition, this method is compatible with any choice of chemical adduct or read-out approach. We utilized this approach to study an autoregulated event in the pre-mRNA of the splicing factor, muscleblind-like splicing regulator 1 (MBNL1).

Citing Articles

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References

Smola M, Rice G, Busan S, Siegfried N, Weeks K . Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis. Nat Protoc. 2015; 10(11):1643-69. PMC: 4900152. DOI: 10.1038/nprot.2015.103. View

Jacob A, Smith C . Intron retention as a component of regulated gene expression programs. Hum Genet. 2017; 136(9):1043-1057. PMC: 5602073. DOI: 10.1007/s00439-017-1791-x. View

Spitale R, Flynn R, Zhang Q, Crisalli P, Lee B, Jung J . Structural imprints in vivo decode RNA regulatory mechanisms. Nature. 2015; 519(7544):486-90. PMC: 4376618. DOI: 10.1038/nature14263. View

Flynn R, Zhang Q, Spitale R, Lee B, Mumbach M, Chang H . Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE. Nat Protoc. 2016; 11(2):273-90. PMC: 4896316. DOI: 10.1038/nprot.2016.011. View

Spitale R, Crisalli P, Flynn R, Torre E, Kool E, Chang H . RNA SHAPE analysis in living cells. Nat Chem Biol. 2012; 9(1):18-20. PMC: 3706714. DOI: 10.1038/nchembio.1131. View

Schroder A, Baumstark T, Riesner D . Chemical mapping of co-existing RNA structures. Nucleic Acids Res. 1998; 26(14):3449-50. PMC: 147709. DOI: 10.1093/nar/26.14.3449. View

Montes M, Sanford B, Comiskey D, Chandler D . RNA Splicing and Disease: Animal Models to Therapies. Trends Genet. 2018; 35(1):68-87. PMC: 6339821. DOI: 10.1016/j.tig.2018.10.002. View

Kedde M, Van Kouwenhove M, Zwart W, Vrielink J, Elkon R, Agami R . A Pumilio-induced RNA structure switch in p27-3' UTR controls miR-221 and miR-222 accessibility. Nat Cell Biol. 2010; 12(10):1014-20. DOI: 10.1038/ncb2105. View

Guo L, Adams R, Wan H, Huston N, Potapova O, Olson S . Sequencing and Structure Probing of Long RNAs Using MarathonRT: A Next-Generation Reverse Transcriptase. J Mol Biol. 2020; 432(10):3338-3352. PMC: 7556701. DOI: 10.1016/j.jmb.2020.03.022. View

10.

Mockenhaupt S, Makeyev E . Non-coding functions of alternative pre-mRNA splicing in development. Semin Cell Dev Biol. 2015; 47-48:32-9. PMC: 4683092. DOI: 10.1016/j.semcdb.2015.10.018. View

11.

Ni J, Grate L, Donohue J, Preston C, Nobida N, OBrien G . Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay. Genes Dev. 2007; 21(6):708-18. PMC: 1820944. DOI: 10.1101/gad.1525507. View

12.

Pan Q, Shai O, Lee L, Frey B, Blencowe B . Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet. 2008; 40(12):1413-5. DOI: 10.1038/ng.259. View

13.

Baralle F, Giudice J . Alternative splicing as a regulator of development and tissue identity. Nat Rev Mol Cell Biol. 2017; 18(7):437-451. PMC: 6839889. DOI: 10.1038/nrm.2017.27. View

14.

Kelemen O, Convertini P, Zhang Z, Wen Y, Shen M, Falaleeva M . Function of alternative splicing. Gene. 2012; 514(1):1-30. PMC: 5632952. DOI: 10.1016/j.gene.2012.07.083. View

15.

Ule J, Blencowe B . Alternative Splicing Regulatory Networks: Functions, Mechanisms, and Evolution. Mol Cell. 2019; 76(2):329-345. DOI: 10.1016/j.molcel.2019.09.017. View

16.

Smola M, Christy T, Inoue K, Nicholson C, Friedersdorf M, Keene J . SHAPE reveals transcript-wide interactions, complex structural domains, and protein interactions across the Xist lncRNA in living cells. Proc Natl Acad Sci U S A. 2016; 113(37):10322-7. PMC: 5027438. DOI: 10.1073/pnas.1600008113. View

17.

Mitchell 3rd D, Renda A, Douds C, Babitzke P, Assmann S, Bevilacqua P . In vivo RNA structural probing of uracil and guanine base-pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). RNA. 2018; 25(1):147-157. PMC: 6298566. DOI: 10.1261/rna.067868.118. View

18.

Larman B, Dethoff E, Weeks K . Packaged and Free Satellite Tobacco Mosaic Virus (STMV) RNA Genomes Adopt Distinct Conformational States. Biochemistry. 2017; 56(16):2175-2183. PMC: 5837821. DOI: 10.1021/acs.biochem.6b01166. View

19.

Frank F, Kavousi N, Bountali A, Dammer E, Mourtada-Maarabouni M, Ortlund E . The lncRNA Growth Arrest Specific 5 Regulates Cell Survival via Distinct Structural Modules with Independent Functions. Cell Rep. 2020; 32(3):107933. DOI: 10.1016/j.celrep.2020.107933. View

20.

Sztuba-Solinska J, Rausch J, Smith R, Miller J, Whitby D, Le Grice S . Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA: a structural scaffold for nuclear, cytoplasmic and viral proteins. Nucleic Acids Res. 2017; 45(11):6805-6821. PMC: 5499733. DOI: 10.1093/nar/gkx241. View