An important component of cellular biochemistry is the concentration of proteins and nucleic acids in non-membranous compartments1,2. These biomolecular condensates are formed from processes including liquid-liquid phase separation (LLPS). The multivalent interactions necessary for LLPS have been studied extensively in vitro1,3. However, what regulates LLPS in vivo is still poorly understood. Here, we identify an in vivo regulator of LLPS through a genetic suppressor screen for loss of function of the Arabidopsis RNA-binding protein FCA. FCA contains prion-like domains that phase-separate in vitro, and exhibits behavior in vivo consistent with phase separation. The mutant screen identified a functional requirement for a coiled coil protein, FLL2, in FCA nuclear body formation. FCA reduces transcriptional read-through by promoting proximal polyadenylation at many sites in the Arabidopsis genome3,4. FLL2 was required to promote this proximal polyadenylation, but not binding of FCA to target RNA. Ectopic expression of FLL2 increased the size and number of FCA nuclear bodies. Crosslinking with formaldehyde captured in vivo interactions between FLL2, FCA and the polymerase and nuclease modules of the RNA 3’ end processing machinery. These 3’ RNA processing components were found to colocalize with FCA in the nuclear bodies in vivo. We conclude that FLL2 promotes liquid-liquid phase separation, important for dynamics of polyadenylation complexes at specific poly A sites. Our findings show that coiled coil proteins can promote LLPS, expanding our understanding of the principles governing the in vivo dynamics of liquid-like bodies.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that play important regulatory roles in gene expression in plants and animals. The biogenesis of miRNAs involves the transcription of primary miRNAs (pri-miRNAs) by RNA polymerase II (RNAPII) and subsequent processing by Dicer or Dicer-like (DCL) proteins. Here we show that the Elongator complex is involved in miRNA biogenesis in Arabidopsis. Disruption of Elongator reduces RNAPII occupancy at miRNA loci and pri-miRNA transcription. We also show that Elongator interacts with the DCL1-containing Dicing complex and lack of Elongator impairs DCL1 localization in the nuclear Dicing body. Finally, we show that pri-miRNA transcripts as well as DCL1 associate with the chromatin of miRNA genes and the chromatin association of DCL1 is compromised in the absence of Elongator. Our results suggest that Elongator functions in both transcription and processing of pri-miRNAs and probably couples these two processes.

Graphical Abstract Highlights d Tocopherols positively regulate miRNA biogenesis d Tocopherols are required for PAP accumulation d PAP positively regulates miRNA biogenesis d Retrograde regulation of miR398 confers heat tolerance In Brief As key regulators of gene expression, miRNAs themselves are subjected to sophisticated regulation. Fang et al. report that chloroplast retrograde signaling involving tocopherols (vitamin E) and PAP, a retrograde inhibitor of the nuclear XRN exonucleases, regulates miRNA biogenesis in Arabidopsis and confers heat tolerance.

RNA polymerases IV and V (Pol IV and Pol V) are required for the generation of noncoding RNAs in RNA-directed DNA methylation (RdDM). Their subunit compositions resemble that of Pol II. The mechanism and accessory factors involved in their assembly remain largely unknown. In this study, we identified mutant alleles of (), (), and () that cause defects in RdDM in We found that Pol IV-dependent small interfering RNAs and Pol V-dependent transcripts were greatly reduced in the mutants. NRPE1, the largest subunit of Pol V, failed to associate with other Pol V subunits in the and mutants, suggesting the involvement of IYO and QQT2 in Pol V assembly. In addition, we found that IYO and QQT2 were mutually dependent for their association with the NRPE3 subassembly prior to the assembly of Pol V holoenzyme. Finally, we show that IYO and QQT2 are similarly required for the assembly of Pol II and Pol IV. Our findings reveal IYO and QQT2 as cofactors for the assembly of Pol II, Pol IV, and Pol V and provide mechanistic insights into how RNA polymerases are assembled in plants.