Are Cajal Bodies Droplet Organelles?

Abstract

Cajal Bodies(CBs) are subnuclear organelles responsible for spliceosomal snRNP maturation in higher eukaryotes. Vertebrates must maintain high snRNP levels during development, and a loss of CBs results in embryonic arrest due to defects in splicing. Like nucleoli and other cellular bodies, CBs are not delimited by lipid bilayers and are RNA-rich. Current models for the formation of such “droplet organelles” invoke phase transitions promoted by intrinsically disordered regions (IDRs) of key components. CBs are scaffolded by the protein coilin, which is assumed to be largely disordered; however, coilin's biochemical and biophysical properties have not been fully examined. It is currently unclear which coilin regions are structured and whether coilin itself mediates a phase transition. For example, a protein or RNA interaction partner may do so. We have purified coilin and subjected it to circular dichroism spectroscopy, finding that at least portions of coilin can adopt an ordered state. In vivo staining with amyloid sensitive fluorescent dyes indicates that this structure is not cross-beta-sheet. Protein folds predicted with RaptorX include: 1) a ubiquitin-like fold at the N-terminus followed by 2) an IDR and 3) a C-terminal tudor-like domain. Prior immunoprecipitation results have shown that the N-terminal domain mediates direct or indirect coilin “self-interaction”, and UV crosslinking revealed RNA binding properties. We have identified single amino acid mutations that disrupt CBs, suggesting specific functional sites within coilin are currently employing a protein-protein crosslinker (DPS) to implicate interactors. The major species appears to consist of coilin-coilin dimers. Thus far, our findings are consistent with a model wherein the IDR promotes a phase transition yet two structured regions impose specificity: an N-terminal coilin-coilin interaction domain and a C-terminal domain that binds to SMN, snRNP proteins and possibly snRNA.