The 30th International Conference on Arabidopsis Research (ICAR 2019)

413 / 2019-01-22 00:46:37

The Arabidopsis coiled coil protein FLL2 promotes formation of liquid droplets to enhance polyadenylation

Arabidopsis,FCA,phase separation,coiled coil

Plenary track > Epigenetics

Liquid-liquid phase separation (LLPS) is an important mechanism in the formation of dynamic non-membranous cellular bodies. However, the requirements for LLPS in vivo are still poorly understood. Here, we show that the Arabidopsis RNA-binding protein FCA is contained in liquid droplets in the nucleus and phase separates in vitro. FCA reduces transcriptional read-through at many sites in the Arabidopsis genome, including the FLC antisense transcript COOLAIR, by promoting proximal polyadenylation. To identify regulators of LLPS in vivo we undertook a genetic suppressor screen for loss of FCA function. This identified a functional requirement for a coiled coil protein, FLL2. A Glu to Lys mutation in FLL2, predicted to disrupt a salt bridge, compromised formation of FCA nuclear droplets as well as proximal polyadenylation of FCA targets, but not binding of FCA to target RNA. Ectopic expression of FLL2 increased the size and number of FCA nuclear droplets. Crosslinking with formaldehyde captured in vivo interactions between FLL2, FCA and the polymerase and nuclease modules of the RNA 3’ end processing machinery. The 3’ RNA processing components were found to colocalize with FCA in vivo. We conclude that FLL2 promotes formation of liquid droplets that enhance polyadenylation at specific poly A sites. Our findings reveal the functional importance of LLPS in facilitating polyadenylation and expand our understanding of the principles governing the formation of liquid-like organelles.