Identification of phenothiazine derivatives as UHM-binding inhibitors of early spliceosome assembly

Nat Commun. 2020 Nov 6;11(1):5621. doi: 10.1038/s41467-020-19514-1.


Interactions between U2AF homology motifs (UHMs) and U2AF ligand motifs (ULMs) play a crucial role in early spliceosome assembly in eukaryotic gene regulation. UHM-ULM interactions mediate heterodimerization of the constitutive splicing factors U2AF65 and U2AF35 and between other splicing factors that regulate spliceosome assembly at the 3' splice site, where UHM domains of alternative splicing factors, such as SPF45 and PUF60, contribute to alternative splicing regulation. Here, we performed high-throughput screening using fluorescence polarization assays with hit validation by NMR and identified phenothiazines as general inhibitors of UHM-ULM interactions. NMR studies show that these compounds occupy the tryptophan binding pocket of UHM domains. Co-crystal structures of the inhibitors with the PUF60 UHM domain and medicinal chemistry provide structure-activity-relationships and reveal functional groups important for binding. These inhibitors inhibit early spliceosome assembly on pre-mRNA substrates in vitro. Our data show that spliceosome assembly can be inhibited by targeting UHM-ULM interactions by small molecules, thus extending the toolkit of splicing modulators for structural and biochemical studies of the spliceosome and splicing regulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing
  • Humans
  • Phenothiazines / chemistry*
  • Phenothiazines / pharmacology*
  • Protein Binding / drug effects
  • Protein Domains
  • RNA Precursors / genetics
  • RNA Precursors / metabolism
  • RNA Splicing Factors / chemistry
  • RNA Splicing Factors / genetics
  • RNA Splicing Factors / metabolism
  • Repressor Proteins / chemistry
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism
  • Spliceosomes / drug effects*
  • Spliceosomes / genetics
  • Spliceosomes / metabolism*
  • Splicing Factor U2AF / chemistry
  • Splicing Factor U2AF / genetics
  • Splicing Factor U2AF / metabolism


  • Phenothiazines
  • RBM17 protein, human
  • RNA Precursors
  • RNA Splicing Factors
  • Repressor Proteins
  • Splicing Factor U2AF
  • U2AF1 protein, human
  • U2AF2 protein, human
  • poly-U binding splicing factor 60KDa
  • phenothiazine