A systematic, label-free method for identifying RNA-associated proteins in vivo provides insights into vertebrate ciliary beating machinery

Dev Biol. 2020 Nov 1;467(1-2):108-117. doi: 10.1016/j.ydbio.2020.08.008. Epub 2020 Sep 6.


Cell-type specific RNA-associated proteins are essential for development and homeostasis in animals. Despite a massive recent effort to systematically identify RNA-associated proteins, we currently have few comprehensive rosters of cell-type specific RNA-associated proteins in vertebrate tissues. Here, we demonstrate the feasibility of determining the RNA-associated proteome of a defined vertebrate embryonic tissue using DIF-FRAC, a systematic and universal (i.e., label-free) method. Application of DIF-FRAC to cultured tissue explants of Xenopus mucociliary epithelium identified dozens of known RNA-associated proteins as expected, but also several novel RNA-associated proteins, including proteins related to assembly of the mitotic spindle and regulation of ciliary beating. In particular, we show that the inner dynein arm tether Cfap44 is an RNA-associated protein that localizes not only to axonemes, but also to liquid-like organelles in the cytoplasm called DynAPs. This result led us to discover that DynAPs are generally enriched for RNA. Together, these data provide a useful resource for a deeper understanding of mucociliary epithelia and demonstrate that DIF-FRAC will be broadly applicable for systematic identification of RNA-associated proteins from embryonic tissues.

Keywords: Cilia; DIF-FRAC; Proteomics; RNA; Xenopus.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cilia / metabolism*
  • Embryo, Nonmammalian / metabolism*
  • Epithelium / embryology
  • RNA-Binding Proteins / metabolism*
  • Tissue Culture Techniques
  • Xenopus
  • Xenopus Proteins / metabolism*


  • RNA-Binding Proteins
  • Xenopus Proteins