A Synthetic Small Molecule Stalls Pre-mRNA Splicing by Promoting an Early-stage U2AF2-RNA Complex

Overview

Journal Cell Chem Biol

Publisher Cell Press

Specialty Biochemistry

Date 2021 Mar 10

PMID 33689684

Citations 14

Authors

Rakesh Chatrikhi

Callen F Feeney

Mary J Pulvino

Georgios Alachouzos

Andrew J MacRae

Zackary Falls

Sumit Rai

William W Brennessel

Jermaine L Jenkins

Matthew J Walter

Timothy A Graubert

Ram Samudrala

Melissa S Jurica

Alison J Frontier

Clara L Kielkopf

Affiliations

Soon will be listed here.

Abstract

Dysregulated pre-mRNA splicing is an emerging Achilles heel of cancers and myelodysplasias. To expand the currently limited portfolio of small-molecule drug leads, we screened for chemical modulators of the U2AF complex, which nucleates spliceosome assembly and is mutated in myelodysplasias. A hit compound specifically enhances RNA binding by a U2AF2 subunit. Remarkably, the compound inhibits splicing of representative substrates and stalls spliceosome assembly at the stage of U2AF function. Computational docking, together with structure-guided mutagenesis, indicates that the compound bridges the tandem U2AF2 RNA recognition motifs via hydrophobic and electrostatic moieties. Cells expressing a cancer-associated U2AF1 mutant are preferentially killed by treatment with the compound. Altogether, our results highlight the potential of trapping early spliceosome assembly as an effective pharmacological means to manipulate pre-mRNA splicing. By extension, we suggest that stabilizing assembly intermediates may offer a useful approach for small-molecule inhibition of macromolecular machines.

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References

Zhang , Chung , OLDENBURG . A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen. 2000; 4(2):67-73. DOI: 10.1177/108705719900400206. View

Maji D, Glasser E, Henderson S, Galardi J, Pulvino M, Jenkins J . Representative cancer-associated U2AF2 mutations alter RNA interactions and splicing. J Biol Chem. 2020; 295(50):17148-17157. PMC: 7863893. DOI: 10.1074/jbc.RA120.015339. View

Sivaramakrishnan M, McCarthy K, Campagne S, Huber S, Meier S, Augustin A . Binding to SMN2 pre-mRNA-protein complex elicits specificity for small molecule splicing modifiers. Nat Commun. 2017; 8(1):1476. PMC: 5684323. DOI: 10.1038/s41467-017-01559-4. View

Jurica M, Licklider L, Gygi S, Grigorieff N, Moore M . Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis. RNA. 2002; 8(4):426-39. PMC: 1370266. DOI: 10.1017/s1355838202021088. View

Chun-Wei Lee S, Dvinge H, Kim E, Cho H, Micol J, Chung Y . Modulation of splicing catalysis for therapeutic targeting of leukemia with mutations in genes encoding spliceosomal proteins. Nat Med. 2016; 22(6):672-8. PMC: 4899191. DOI: 10.1038/nm.4097. View

Zarnack K, Konig J, Tajnik M, Martincorena I, Eustermann S, Stevant I . Direct competition between hnRNP C and U2AF65 protects the transcriptome from the exonization of Alu elements. Cell. 2013; 152(3):453-66. PMC: 3629564. DOI: 10.1016/j.cell.2012.12.023. View

Graubert T, Shen D, Ding L, Okeyo-Owuor T, Lunn C, Shao J . Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes. Nat Genet. 2011; 44(1):53-7. PMC: 3247063. DOI: 10.1038/ng.1031. View

Paolella B, Gibson W, Urbanski L, Alberta J, Zack T, Bandopadhayay P . Copy-number and gene dependency analysis reveals partial copy loss of wild-type SF3B1 as a novel cancer vulnerability. Elife. 2017; 6. PMC: 5357138. DOI: 10.7554/eLife.23268. View

Kralovicova J, Borovska I, Kubickova M, Lukavsky P, Vorechovsky I . Cancer-Associated Substitutions in RNA Recognition Motifs of PUF60 and U2AF65 Reveal Residues Required for Correct Folding and 3' Splice-Site Selection. Cancers (Basel). 2020; 12(7). PMC: 7408900. DOI: 10.3390/cancers12071865. View

10.

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

11.

Agrawal A, Salsi E, Chatrikhi R, Henderson S, Jenkins J, Green M . An extended U2AF(65)-RNA-binding domain recognizes the 3' splice site signal. Nat Commun. 2016; 7:10950. PMC: 4786784. DOI: 10.1038/ncomms10950. View

12.

Obeng E, Chappell R, Seiler M, Chen M, Campagna D, Schmidt P . Physiologic Expression of Sf3b1(K700E) Causes Impaired Erythropoiesis, Aberrant Splicing, and Sensitivity to Therapeutic Spliceosome Modulation. Cancer Cell. 2016; 30(3):404-417. PMC: 5023069. DOI: 10.1016/j.ccell.2016.08.006. View

13.

Sickmier E, Frato K, Shen H, Paranawithana S, Green M, Kielkopf C . Structural basis for polypyrimidine tract recognition by the essential pre-mRNA splicing factor U2AF65. Mol Cell. 2006; 23(1):49-59. PMC: 2043114. DOI: 10.1016/j.molcel.2006.05.025. View

14.

Smith M, Choudhary G, Pellagatti A, Choi K, Bolanos L, Bhagat T . U2AF1 mutations induce oncogenic IRAK4 isoforms and activate innate immune pathways in myeloid malignancies. Nat Cell Biol. 2019; 21(5):640-650. PMC: 6679973. DOI: 10.1038/s41556-019-0314-5. View

15.

Wang W, Maucuer A, Gupta A, Manceau V, Thickman K, Bauer W . Structure of phosphorylated SF1 bound to U2AF⁶⁵ in an essential splicing factor complex. Structure. 2013; 21(2):197-208. PMC: 3570649. DOI: 10.1016/j.str.2012.10.020. View

16.

Dignam J, Lebovitz R, Roeder R . Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983; 11(5):1475-89. PMC: 325809. DOI: 10.1093/nar/11.5.1475. View

17.

Effenberger K, Perriman R, Bray W, Lokey R, Ares Jr M, Jurica M . A high-throughput splicing assay identifies new classes of inhibitors of human and yeast spliceosomes. J Biomol Screen. 2013; 18(9):1110-20. PMC: 3902173. DOI: 10.1177/1087057113493117. View

18.

Singh R, Valcarcel J, Green M . Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins. Science. 1995; 268(5214):1173-6. DOI: 10.1126/science.7761834. View

19.

Cho S, Moon H, Loh T, Jang H, Liu Y, Zhou J . Splicing inhibition of U2AF65 leads to alternative exon skipping. Proc Natl Acad Sci U S A. 2015; 112(32):9926-31. PMC: 4538632. DOI: 10.1073/pnas.1500639112. View

20.

Selenko P, Gregorovic G, Sprangers R, Stier G, Rhani Z, Kramer A . Structural basis for the molecular recognition between human splicing factors U2AF65 and SF1/mBBP. Mol Cell. 2003; 11(4):965-76. DOI: 10.1016/s1097-2765(03)00115-1. View