Splicing modulators act at the branch point adenosine binding pocket defined by the PHF5A-SF3b complex

Nat Commun. 2017 May 25:8:15522. doi: 10.1038/ncomms15522.

Abstract

Pladienolide, herboxidiene and spliceostatin have been identified as splicing modulators that target SF3B1 in the SF3b subcomplex. Here we report that PHF5A, another component of this subcomplex, is also targeted by these compounds. Mutations in PHF5A-Y36, SF3B1-K1071, SF3B1-R1074 and SF3B1-V1078 confer resistance to these modulators, suggesting a common interaction site. RNA-seq analysis reveals that PHF5A-Y36C has minimal effect on basal splicing but inhibits the global action of splicing modulators. Moreover, PHF5A-Y36C alters splicing modulator-induced intron-retention/exon-skipping profile, which correlates with the differential GC content between adjacent introns and exons. We determine the crystal structure of human PHF5A demonstrating that Y36 is located on a highly conserved surface. Analysis of the cryo-EM spliceosome Bact complex shows that the resistance mutations cluster in a pocket surrounding the branch point adenosine, suggesting a competitive mode of action. Collectively, we propose that PHF5A-SF3B1 forms a central node for binding to these splicing modulators.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine / chemistry*
  • Alternative Splicing*
  • Carrier Proteins / chemistry*
  • Cell Proliferation
  • Cell Survival
  • Cryoelectron Microscopy
  • Crystallography, X-Ray
  • Epoxy Compounds / chemistry
  • Exons
  • Fatty Alcohols / chemistry
  • HCT116 Cells
  • Humans
  • Introns
  • Macrolides / chemistry
  • Mass Spectrometry
  • Mutagenesis, Site-Directed
  • Mutation
  • Myeloid Cell Leukemia Sequence 1 Protein / chemistry
  • Phosphoproteins / chemistry*
  • Phosphoproteins / metabolism
  • Protein Binding
  • Protein Conformation
  • Pyrans / chemistry
  • RNA Interference
  • RNA Splicing Factors / chemistry*
  • RNA Splicing Factors / metabolism
  • RNA-Binding Proteins
  • Recombinant Proteins / chemistry
  • Sequence Analysis, RNA
  • Spiro Compounds / chemistry
  • Spliceosomes / metabolism
  • Trans-Activators

Substances

  • Carrier Proteins
  • Epoxy Compounds
  • Fatty Alcohols
  • MCL1 protein, human
  • Macrolides
  • Myeloid Cell Leukemia Sequence 1 Protein
  • PHF5A protein, human
  • Phosphoproteins
  • Pyrans
  • RNA Splicing Factors
  • RNA-Binding Proteins
  • Recombinant Proteins
  • SF3B1 protein, human
  • Spiro Compounds
  • Trans-Activators
  • pladienolide B
  • spliceostatin A
  • herboxidiene
  • Adenosine