U2AF65 assemblies drive sequence-specific splice site recognition

EMBO Rep. 2019 Aug;20(8):e47604. doi: 10.15252/embr.201847604. Epub 2019 Jul 4.

Abstract

The essential splicing factor U2AF65 is known to help anchoring U2 snRNP at the branch site. Its C-terminal UHM domain interacts with ULM motifs of SF3b155, an U2 snRNP protein. Here, we report a cooperative binding of U2AF65 and the related protein CAPERα to the multi-ULM domain of SF3b155. In addition, we show that the RS domain of U2AF65 drives a liquid-liquid phase separation that is amplified by intronic RNA with repeated pyrimidine tracts. In cells, knockdown of either U2AF65 or CAPERα improves the inclusion of cassette exons that are preceded by such repeated pyrimidine-rich motifs. These results support a model in which liquid-like assemblies of U2AF65 and CAPERα on repetitive pyrimidine-rich RNA sequences are driven by their RS domains, and facilitate the recruitment of the multi-ULM domain of SF3b155. We anticipate that posttranslational modifications and proteins recruited in dynamical U2AF65 and CAPERα condensates may further contribute to the complex mechanisms leading to specific splice site choice that occurs in cells.

Keywords: RBM39; SF3b1; U2AF2; liquid-liquid phase separation; splicing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing*
  • Cloning, Molecular
  • Computational Biology
  • DNA, Complementary / genetics
  • DNA, Complementary / metabolism
  • Gene Expression
  • Gene Expression Profiling
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • HeLa Cells
  • Humans
  • Nucleotide Motifs
  • Phosphoproteins / genetics*
  • Phosphoproteins / metabolism
  • RNA Splicing Factors / genetics*
  • RNA Splicing Factors / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • RNA-Binding Proteins / antagonists & inhibitors
  • RNA-Binding Proteins / genetics*
  • RNA-Binding Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism
  • Ribonucleoprotein, U2 Small Nuclear / genetics
  • Ribonucleoprotein, U2 Small Nuclear / metabolism
  • Spliceosomes / genetics*
  • Spliceosomes / metabolism
  • Splicing Factor U2AF / antagonists & inhibitors
  • Splicing Factor U2AF / genetics*
  • Splicing Factor U2AF / metabolism

Substances

  • DNA, Complementary
  • HCC1 autoantigen
  • Phosphoproteins
  • RBM17 protein, human
  • RNA Splicing Factors
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • Recombinant Proteins
  • Repressor Proteins
  • Ribonucleoprotein, U2 Small Nuclear
  • SF3B1 protein, human
  • Splicing Factor U2AF
  • U2AF2 protein, human
  • poly-U binding splicing factor 60KDa