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Arabidopsis FLL2 promotes liquid–liquid phase separation of polyadenylation complexes

Nature volume 569, pages 265–269 (2019)Cite this article

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Abstract

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 that include liquid–liquid phase separation. The multivalent interactions necessary for liquid–liquid phase separation have been extensively studied in vitro1,3. However, the regulation of this process in vivo is poorly understood. Here we identify an in vivo regulator of liquid–liquid phase separation through a genetic screen targeting factors required for Arabidopsis RNA-binding protein FCA function. FCA contains prion-like domains that phase-separate in vitro, and exhibits behaviour in vivo that is consistent with phase separation. The mutant screen identified a functional requirement for FLL2, a coiled-coil protein, in the formation of FCA nuclear bodies. 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 the 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 colocalized with FCA in the nuclear bodies in vivo, which indicates that FCA nuclear bodies compartmentalize 3′-end processing factors to enhance polyadenylation at specific sites. Our findings show that coiled-coil proteins can promote liquid–liquid phase separation, which expands our understanding of the principles that govern the in vivo dynamics of liquid-like bodies.

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Fig. 1: FCA phase separates in vitro, and exhibits behaviour in vivo that is consistent with phase separation.
Fig. 2: The coiled-coil domain protein FLL2 is required for the function of FCA.
Fig. 3: FLL2 promotes the phase separation of FCA to form nuclear bodies.
Fig. 4: FCA and FLL2 associate with 3′-end processing factors and are important for polyadenylation at specific sites.

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Data availability

Full lists of mass spectrometry are provided as Supplementary Tables 1–4. All the other raw data that support the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank M. M. Babu (MRC, Laboratory of Molecular Biology) and M. Howard (John Innes Centre) for discussions. This work was supported by the European Research Council grant ‘MEXTIM’, Wellcome Trust grant 210654/Z/18/Z, the BBSRC Institute Strategic Programme GEN (BB/P013511/1), the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant 800318, the JSPS overseas research fellowship to R.I., and the Medical Research Council grant U105192713.

Reviewer information

Nature thanks Julia Chekanova, Jessica Hennacy, Martin Jonikas and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. Ryo Ishikawa

    Present address: Graduate School of Agricultural Science, Kobe University, Kobe, Japan

  2. Fuquan Liu

    Present address: Institute of Global Food Security, School of Biological Sciences, Queen’s University Belfast, Belfast, UK

  3. These authors contributed equally: Xiaofeng Fang, Liang Wang, Ryo Ishikawa

Authors and Affiliations

  1. John Innes Centre, Norwich, UK

    Xiaofeng Fang, Ryo Ishikawa, Yaoxi Li, Fuquan Liu, Grant Calder & Caroline Dean

  2. Beijing Advanced Innovation Center for Structural Biology, Tsinghua University-Peking University Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China

    Liang Wang & Pilong Li

  3. MRC Laboratory of Molecular Biology, Cambridge, UK

    Marc Fiedler

  4. Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany

    Beth Rowan & Detlef Weigel

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Contributions

X.F. and C.D. conceived the study. X.F., Y.L. and G.C. performed all in vivo imaging experiments and analyses. X.F. performed the mass spectrometry experiments. R.I., B.R., D.W., M. F. and F.L. did the genetic identification and analysis of the sof78 mutant. L.W. and P.L. performed and analysed in vitro phase-separation assays. X.F., P.L. and C.D. wrote the manuscript, and all authors contributed ideas and reviewed the manuscript.

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Correspondence to Pilong Li or Caroline Dean.

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Extended data figures and tables

Extended Data Fig. 1 Transgenic FCA–eGFP is functionally equivalent to endogenous FCA.

a, Top, genomic FCA locus indicating upstream and downstream genes (grey), and the position of the fca-1 mutation. Bottom, illustration of transgenic FCA-eGFP construct. Thick black boxes indicate exons, thin black boxes indicate UTRs and black lines indicate introns. b, Flowering time of indicated plants grown in a long-day photoperiod. Data are presented as mean ± s.d. (n = 20). Asterisk indicates a significant difference (P = 0.0001, two-tailed t-test). c, Expression of spliced FLC relative to UBC in the indicated plants. Data are presented as mean ± s.d. (n = 3). Asterisk indicates a significant difference (P = 0.0004, two-tailed t-test). d, Expression of spliced FCAγ relative to UBC in the indicated plants. Data are presented as mean ± s.d. (n = 3). Asterisk indicates a significant difference (P = 0.0003, two-tailed t-test). e, The protein levels of FCA and FCA–eGFP in the indicated plants as determined by western blot. Asterisks indicate non-specific signals. Data are representative of two independent experiments. For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Fig. 2 FCA undergoes phase separation in vitro.

a, Coomassie staining of indicated protein samples before and after TEV cleavage of the MBP tag. Arrowheads indicate the proteins labelled on top of the gel. Data are representative of three independent experiments. b, Phase separation of GFP–FCA in the presence of Arabidopsis total RNA was tested using 3.13 μM GFP–FCA and RNA ranging from 0.09 to 1.3 μg ml−1. Scale bars, 10 μm. Data are representative of three independent experiments. c, In vitro phase separation assay of GFP–FCA–PrLD at various protein concentrations. Scale bar, 50 μm. Data are representative of three independent experiments. d, FRAP of GFP–FCA puncta. Time 0 indicates the time of the photobleaching pulse. Scale bar, 1 μm. Data are representative of eight independent experiments. e, Plot showing the time course of the recovery after photobleaching GFP–FCA puncta. Data are presented as mean ± s.d. (n = 8). f, GFP–FCA puncta do not grow in size or coalesce with each other. Time points are indicated above (in minutes). Scale bar, 10 μm. Data are representative of three independent experiments. g, FRAP of GFP–FCA puncta in the presence of 10% (w/v) PEG. Time 0 indicates the time of the photobleaching pulse. Scale bar, 2 μm. Data are representative of nine independent experiments. h, Plot showing the time course of the recovery after photobleaching GFP–FCA puncta in the presence of 10% (w/v) PEG. Data are presented as mean ± s.d. (n = 9). i, Fusion of GFP–FCA puncta in the presence of 10% (w/v) PEG. Time points are indicated above (in minutes). Scale bar, 2 μm. Data are representative of three independent experiments. j, FRAP of GFP–FCA puncta in the presence of Arabidopsis total RNA. Time 0 indicates the time of the photobleaching pulse. Scale bar, 1 μm. Data are representative of eight independent experiments. k, Plot showing the time course of the recovery after photobleaching GFP–FCA puncta in the presence of Arabidopsis total RNA. Data are presented as mean ± s.d. (n = 8).

Source data

Extended Data Fig. 3 Characterization of the sof78 mutation.

a, The seed development (top) and the petal number (bottom) of sof78 mutant and Ler wild type. Photos are representative of at least five independent experiments. b, FLC–LUC bioluminescence signal of indicated plants taken by charge-coupled device camera. Data are representative of three independent experiments. c, Expression of spliced FLC relative to UBC in the indicated genotypes. Data are presented as mean ± s.d. (n = 4). Asterisk indicates a significant difference (P = 0.0014, two-tailed t-test). d, Flowering time of indicated plants grown in long-day photoperiod. Data are presented as mean ± s.d. (n = 20). Asterisk indicates a significant difference (P < 0.0001, two-tailed t-test). e, PCR with reverse transcription (RT–PCR) detection of FLC and UBC transcripts, or PCR amplification of indicated fragments from genomic DNA. Data are representative of three independent experiments. f, Flowering time of indicated plants grown in long-day photoperiod. Data are presented as mean ± s.d. (n = 20). Asterisk indicates a significant difference (P < 0.0001, two-tailed t-test). g, Flowering time of indicated plants grown in long-day photoperiod. Data are presented as mean ± s.d. (n = 8). Asterisks indicate significant differences (P ≤ 0.0077, two-tailed t-test). h, Top, genomic FLL2 locus, indicating upstream and downstream genes and positions of sof78 mutation and fll2-2 transfer DNA insertion. Bottom, illustration of transgenic FLL2-eYFP construct. Thick black boxes indicate exons, thin black boxes indicate UTRs and black lines indicate introns. i, RT–PCR detection of FLL2 and UBC transcripts in Col-0 and fll2-2 plants. Data are representative of three independent experiments. j, Expression of spliced FLC relative to UBC in the indicated genotypes. Data are presented as mean ± s.d. (n = 4). k, Flowering time of indicated plants grown in long-day photoperiod. Data are presented as mean ± s.d. (n = 12). l, Phylogenetic tree of proteins of the FLX family. The tree was drawn using the PHYLIP program. Bootstrap values from 1,000 trials are shown. m, FLC–LUC bioluminescence signal of indicated plants taken using a charge-coupled device camera. Data are representative of three independent experiments. n, Flowering time of indicated plants grown in long-day photoperiod. Data are presented as mean ± s.d. (n = 10). Asterisk indicates a significant difference (P < 0.0001, two-tailed t-test).

Source data

Extended Data Fig. 4 FLL2 encodes a coiled-coil domain protein.

a, A fragment (55–243 amino acids) of FLL2 protein was blasted against the ‘PDB_mmCIF70_5_Oct’ database using HHpred of the MPI Bioinformatics Toolkit (https://toolkit.tuebingen.mpg.de/#/). The top 10 hits are shown. When the probability is larger than 95%, the homology is nearly certain. b, The alignment between coiled-coil domains of FLL2 and human ROCK1. Black arrowhead indicates the Glu that is mutated in sof78. c, A salt bridge was formed between Glu and Arg (indicated by red arrowheads in b) on two molecules of ROCK1. Data were obtained from the Protein Contacts Atlas (http://www.mrc-lmb.cam.ac.uk/pca/). d, Plot showing the sequence conservation of FLL2. Analysis was done using the HmmerWeb server (https://www.ebi.ac.uk/Tools/hmmer/) by searching with A. thaliana FLL2 within the taxonomy of plants ‘Ensembl genomes plants’, which yielded 520 homologues within the Streptophyta. The HMM logo shows the conservation for each amino acid for the 520 homologues. Black arrowheads indicate the two amino acids that are predicted to form a salt bridge.

Extended Data Fig. 5 Characterization of FLL1 and FLL3.

a, Colocalization of FLL1–YFP and FLL3–YFP with FCA–CFP in tobacco leaf nuclei. Scale bars, 5 μm. Data are representative of three independent experiments. b, c, Top, protein domain structures of FLL1 and FLL3. Bottom, predictions of PrLDs and disordered regions by PLAAC and D2P2 algorithms, respectively. d, Interactions of FCA with FLL proteins in yeast two-hybrid assay. Combinations of constructs were transformed into yeast AH109 strain, and assayed on stringent medium. Three independent colonies were tested. AD, activating domain; BD, binding domain; E.V., empty vector. Data are representative of three independent experiments.

Extended Data Fig. 6 FLL2 promotes the formation of FCA nuclear bodies.

a, An example image showing the FCA–eGFP nuclear bodies in sof78-mutant background. A seven-day-old Arabidopsis root tip was observed under the confocal microscope. The region indicated in the left panel is shown magnified in the right panel. Scale bars, 5 μm. Image is representative of eight independent experiments. b, A tobacco nucleus overexpressing FCA–YFP and FLL2. Scale bar, 5 μm. Data are representative of six independent experiments. c, Half-bleach of the FCA–YFP body indicated in b. Time 0 indicates the time of the photobleaching pulse. Scale bar, 1 μm. Data are representative of six independent experiments. d, Plot showing the time course of the recovery after photobleaching the FCA body. Data are presented as mean ± s.d. (n = 6). e, The effect of FLL2 overexpression on the pattern of FCA–CFP nuclear bodies, assayed in tobacco leaf nuclei. Scale bars, 5 μm. Data are representative of three independent experiments. f, The protein level of FCA–CFP in indicated samples as determined by western blot. Asterisks indicate non-specific signal. Data are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Fig. 7 Analysis of FCA, FPA, FY and CPSF30.

a, In vivo formaldehyde crosslinking gives heterogenous FCA complexes that are much larger than those obtained without crosslinking. Nuclear extracts were prepared from crosslinked or non-crosslinked plants; half of the extracts were mixed with NuPAGE LDS sample buffer and boiled at 70 °C and, for the remaining half, extracts were reverse-crosslinked by heating at 95 °C. Samples were analysed by western blot using FCA antibody. Data are representative of two independent experiments. For gel source data, see Supplementary Fig. 1. b, c, Top, the annotated functional domains of FPA (b) and FY (c). Bottom, predictions of PrLDs and disordered regions by PLAAC and D2P2 algorithms, respectively. d, The colocalization of CPSF30–YFP with FCA–CFP. CPSF30–YFP alone (top) or together with FCA–CFP (middle and bottom) are expressed in tobacco leaves. Images are representative of three independent experiments. Scale bars, 20 μm (top and middle), 5 μm (bottom).

Extended Data Fig. 8 Effect of sof78 mutation on the binding of FCA to the nascent transcripts of COOLAIR and unannotated segments, and proximal polyadenylation of unannotated segments.

a–e, RNA immunoprecipitation and qPCR analysis of FCA enrichment on the transcripts of COOLAIR (a), AT1G28140 and unannotated segment 2 (UA2) (b), AT1G62820 and unannotated segment 5 (UA5) (c), AT4G24660 and unannotated segment 16 (UA16) (d), and AT3G23100 and distal XRCC4 (XRCC4d) (e). Gene structures are shown above the plots. Data are presented as mean ± s.d. (n = 3). Asterisks indicate significant differences (P ≤ 0.0381, two-tailed t-test). Short black lines indicate positions of the primers that were used for qPCR amplification. f–i, The expression levels of distally polyadenylated isoforms of UA2 (f), UA5 (g), UA16 (h) and XRCC4d (i) in the indicated plants relative to wild type. Data are presented as mean ± s.d. (n = 3). Asterisks indicate significant differences (P ≤ 0.0099, two-tailed t-test).

Extended Data Fig. 9 A working model for the role of the coiled-coil protein FLL2 to promote nuclear bodies that are important for polyadenylation at specific sites.

At efficient polyadenylation sites, the cleavage and polyadenylation specificity factor complex specifically recognizes the cis-acting motif upstream of the cleavage site, catalyses pre-mRNA cleavage and recruits polyadenylation polymerase to initiate polyadenylation. At other sites, phase-separated FCA bodies compartmentalize 3′-end processing factors to enhance polyadenylation.

Source data

Extended Data Table 1 List of proteins identified by FCA and FLL2–HA affinity purification, with or without formaldehyde crosslinking
Full size table

Supplementary information

Supplementary Figure 1

This file contains the uncropped scans with size marker indications.

Reporting Summary

Supplementary Table 1

A list of proteins identified by CLNIP–MS of FCA. The nuclear extract was prepared from crosslinked Col-0 plants and immunoprecipitated with anti-FCA antibody. The immunoprecipitates were then analysed by mass spectrometry. Three independent biological replicates were shown. IP, immunoprecipitation.

Supplementary Table 2

List of proteins identified in IP–MS of FCA without crosslinking. The nuclear extract was prepared from non-crosslinked Col-0 plants and immunoprecipitated with anti-FCA antibody. The immunoprecipitates were then analysed by mass spectrometry. Two independent biological replicates were shown. IP, immunoprecipitation.

Supplementary Table 3

List of proteins identified by CLNIP–MS of FLL2–HA. Nuclear extract was prepared from crosslinked pFLL2::FLL2-HA/sof78 transgenic plants and immunoprecipitated with anti-HA magnetic beads. The immunoprecipitates were then analysed by mass spectrometry. Three independent biological replicates were shown. IP, immunoprecipitation.

Supplementary Table 4

List of proteins identified by CLNIP–MS of FLD-Flag. Nuclear extract was prepared from crosslinked pFLD::FLD-FLAG/fld-4 transgenic plants and immunoprecipitated with anti-Flag magnetic beads. The immunoprecipitates were then analysed by mass spectrometry. IP, immunoprecipitation.

Supplementary Table 5

List of primers used in this study.

Video 1

FRAP of FCA–GFP nuclear body formed in vivo. An FCA–GFP nuclear body was bleached. Recovery was recorded for every second (sec) after bleaching. Scale bar, 5 μm. Data are representative of seven independent experiments.

Video 2

Fusion of two FCA–GFP nuclear bodies formed in vivo. Time-lapse imaging of two FCA–GFP nuclear bodies every 15 seconds. White arrowheads indicate the two bodies that undergo fusion. Scale bar, 2 μm. Data are representative of three independent experiments.

Video 3

FRAP of GFP–FCA–PrLD droplet formed in vitro. A region inside the GFP–FCA–PrLD droplet was bleached. Recovery was recorded for the indicated time. Scale bar, 2 μm. Data are representative of twelve independent experiments.

Video 4

Fusion of GFP–FCA–PrLD droplets formed in vitro. Time-lapse imaging of GFP–FCA–PrLD droplets for the indicated time. Scale bar, 10 μm. Data are representative of three independent experiments.

Video 5

Half-bleach assay of FCA–YFP nuclear body formed in tobacco leaves. About half of an FCA–YFP nuclear body was bleached. Recovery was recorded for every 233 milliseconds (msec) after bleaching. Scale bar, 1 μm. Data are representative of six independent experiments.

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Fang, X., Wang, L., Ishikawa, R. et al. Arabidopsis FLL2 promotes liquid–liquid phase separation of polyadenylation complexes. Nature 569, 265–269 (2019). https://doi.org/10.1038/s41586-019-1165-8

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