CoChat
Sign Up
Cited by 13Circadian rhythm and melatoninProtein Structure and DynamicsLight effects on plants

The DOI matches its Crossref record; 3 of the 3 references it cites were found in a registry or an index.

Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock

Open access
Tariq, Daniyal, Maurici, Nicole, Bartholomai, Bradley M, Chandrasekaran, Siddarth, Dunlap, Jay C, Bah, Alaji, Crane, Brian R·2024·eLife
DOI 10.7554/elife.90259PMC PMC10963029

Published version of a preprint

Open this paper in CoChat — free accountOpen PDF

Abstract

Circadian clocks are composed of transcription-translation negative feedback loops that pace rhythms of gene expression to the diurnal cycle. In the filamentous fungus Neurospora crassa, the proteins F requency ( F RQ), the F RQ-interacting RNA helicase (FRH), and C asein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the white-collar complex (WCC). FRQ orchestrates key molecular interactions of the clock despite containing little predicted tertiary structure. Spin labeling and pulse-dipolar electron spin resonance spectroscopy provide domain-specific structural insights into the 989-residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state, but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid–liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic clocks.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.7554/elife.90259

Cites

3 of 3 references verified0 citation errors · 0 not checkable

Statistics

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Flagged first, then by citations

Research works printed but not linked: 92

  1. Babinchak, 2019, The role of liquid-liquid phase separation in aggregation of the TDP-43 low-complexity domain, The Journal of Biological Chemistry, 294, 6306, 10.1074/jbc.RA118.007222 · DOI
  2. Baker, 2009, Quantitative proteomics reveals a dynamic interactome and phase-specific phosphorylation in the Neurospora circadian clock, Molecular Cell, 34, 354, 10.1016/j.molcel.2009.04.023 · DOI
  3. Bartholomai, 2022, Cell biological dissection of the neurospora circadian oscillator: tools, approaches and fundamental observations, biochemistry and genetics
  4. Bartholomai, 2022, PRD-2 mediates clock-regulated perinuclear localization of clock gene RNAs within the circadian cycle of neurospora, PNAS, 119, 10.1073/pnas.2203078119 · DOI
  5. Bateman, 2021, UniProt: the universal protein knowledgebase in 2021, Nucleic Acids Research, 49, D480, 10.1093/nar/gkaa1100 · DOI · Paper page
  6. Bellay, 2011, Bringing order to protein disorder through comparative genomics and genetic interactions, Genome Biology, 12, 10.1186/gb-2011-12-2-r14 · DOI
  7. Berliner, 1982, A novel reversible thiol-specific spin label: papain active site labeling and inhibition, Analytical Biochemistry, 119, 450, 10.1016/0003-2697(82)90612-1 · DOI
  8. Bolognesi, 2016, A concentration-dependent liquid phase separation can cause toxicity upon increased protein expression, Cell Reports, 16, 222, 10.1016/j.celrep.2016.05.076 · DOI
  9. Bonucci, 2020, A combined NMR and EPR investigation on the effect of the disordered RGG regions in the structure and the activity of the RRM domain of FUS, Scientific Reports, 10, 10.1038/s41598-020-77899-x · DOI
  10. Brady, 2017, Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation, PNAS, 114, E8194, 10.1073/pnas.1706197114 · DOI
  11. Caragliano, 2022, Human cytomegalovirus forms phase-separated compartments at viral genomes to facilitate viral replication, Cell Reports, 38, 10.1016/j.celrep.2022.110469 · DOI
  12. Cha, 2015, Mechanism of the neurospora circadian clock, a FREQUENCY-centric view, Biochemistry, 54, 150, 10.1021/bi5005624 · DOI
  13. Chandrasekaran, 2021, Tuning flavin environment to detect and control light-induced conformational switching in Drosophila cryptochrome, Communications Biology, 4, 10.1038/s42003-021-01766-2 · DOI
  14. Cheng, 2001, Coiled-coil domain-mediated FRQ-FRQ interaction is essential for its circadian clock function in neurospora, The EMBO Journal, 20, 101, 10.1093/emboj/20.1.101 · DOI
  15. Cheng, 2005, Regulation of the neurospora circadian clock by an rna helicase, Genes & Development, 19, 234, 10.1101/gad.1266805 · DOI
  16. Cohen, 2014, Dynamic localization of the cyanobacterial circadian clock proteins, Current Biology, 24, 1836, 10.1016/j.cub.2014.07.036 · DOI
  17. Colot, 2006, A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors, PNAS, 103, 10352, 10.1073/pnas.0601456103 · DOI
  18. Conrad, 2016, Structure of the frequency-interacting RNA helicase: a protein interaction hub for the circadian clock, The EMBO Journal, 35, 1707, 10.15252/embj.201694327 · DOI
  19. Crane, 2014, Interactive features of proteins composing eukaryotic circadian clocks, Annual Review of Biochemistry, 83, 191, 10.1146/annurev-biochem-060713-035644 · DOI
  20. Diernfellner, 2007, Long and short isoforms of neurospora clock protein FRQ support temperature-compensated circadian rhythms, FEBS Letters, 581, 5759, 10.1016/j.febslet.2007.11.043 · DOI
  21. Dignon, 2020, Biomolecular phase separation: from molecular driving forces to macroscopic properties, Annual Review of Physical Chemistry, 71, 53, 10.1146/annurev-physchem-071819-113553 · DOI
  22. Dunlap, 2017, Making time: conservation of biological clocks from fungi to animals, Microbiology Spectrum, 5, 10.1128/microbiolspec.FUNK-0039-2016 · DOI
  23. Dunlap, 2018, Just-so stories and origin myths: phosphorylation and structural disorder in circadian clock proteins, Molecular Cell, 69, 165, 10.1016/j.molcel.2017.11.028 · DOI
  24. Dunleavy, 2023, Enzymatic spin-labeling of protein n- and c-termini for electron paramagnetic resonance spectroscopy, Bioconjugate Chemistry, 1, 10.1021/acs.bioconjchem.3c00029 · DOI
  25. Durand, 2010, NADPH oxidase activator p67(phox) behaves in solution as a multidomain protein with semi-flexible linkers, Journal of Structural Biology, 169, 45, 10.1016/j.jsb.2009.08.009 · DOI
  26. Emmanouilidis, 2021, NMR and EPR reveal a compaction of the RNA-binding protein FUS upon droplet formation, Nature Chemical Biology, 17, 608, 10.1038/s41589-021-00752-3 · DOI
  27. Franck, 2015, Focus: two-dimensional electron-electron double resonance and molecular motions: the challenge of higher frequencies, The Journal of Chemical Physics, 142, 10.1063/1.4917322 · DOI
  28. Freed, 2005, ESR and molecular Dynamics, 239, 10.1007/0-306-48533-8_9 · DOI
  29. Froehlich, 2002, White collar-1, a circadian blue light photoreceptor, binding to the frequency promoter, Science, 297, 815, 10.1126/science.1073681 · DOI · Paper page
  30. Gasset-Rosa, 2019, Cytoplasmic tdp-43 de-mixing independent of stress granules drives inhibition of nuclear import, loss of nuclear tdp-43, and cell death, Neuron, 102, 339, 10.1016/j.neuron.2019.02.038 · DOI
  31. Gonzalez, 2021, Mechanism of karyopherin-β2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X), Scientific Reports, 11, 10.1038/s41598-021-83196-y · DOI
  32. Guo, 2010, Functional significance of FRH in regulating the phosphorylation and stability of neurospora circadian clock protein FRQ, The Journal of Biological Chemistry, 285, 11508, 10.1074/jbc.M109.071688 · DOI
  33. Holehouse, 2017, CIDER: resources to analyze sequence-ensemble relationships of intrinsically disordered proteins, Biophysical Journal, 112, 16, 10.1016/j.bpj.2016.11.3200 · DOI
  34. Hopkins, 2017, BioXTAS RAW: improvements to a free open-source program for small-angle X-ray scattering data reduction and analysis, Journal of Applied Crystallography, 50, 1545, 10.1107/S1600576717011438 · DOI
  35. Hu, 2021, FRQ-CK1 interaction underlies temperature compensation of the neurospora circadian clock, mBio, 12, 10.1128/mBio.01425-21 · DOI
  36. Hubbell, 2000, Identifying conformational changes with site-directed spin labeling, Nature Structural Biology, 7, 735, 10.1038/78956 · DOI
  37. Hurley, 2013, Conserved RNA helicase FRH acts nonenzymatically to support the intrinsically disordered neurospora clock protein FRQ, Molecular Cell, 52, 832, 10.1016/j.molcel.2013.11.005 · DOI
  38. Hurley, 2016, Circadian oscillators: around the transcription-translation feedback loop and on to output, Trends in Biochemical Sciences, 41, 834, 10.1016/j.tibs.2016.07.009 · DOI
  39. Jankowski, 2022, The formation of a fuzzy complex in the negative arm regulates the robustness of the circadian clock, 10.1101/2022.01.04.474980 · DOI · Paper page
  40. Jung, 2020, A prion-like domain in ELF3 functions as a thermosensor in Arabidopsis, Nature, 585, 256, 10.1038/s41586-020-2644-7 · DOI
  41. Kataoka, 1997, Structural characterization of the molten globule of alpha-lactalbumin by solution X-ray scattering, Protein Science, 6, 422, 10.1002/pro.5560060219 · DOI
  42. Kikhney, 2015, A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins, FEBS Letters, 589, 2570, 10.1016/j.febslet.2015.08.027 · DOI
  43. Kyte, 1982, A simple method for displaying the hydropathic character of a protein, Journal of Molecular Biology, 157, 105, 10.1016/0022-2836(82)90515-0 · DOI
  44. Larrondo, 2015, Circadian rhythms decoupling circadian clock protein turnover from circadian period determination, Science, 347, 10.1126/science.1257277 · DOI
  45. Larson, 2017, Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin, Nature, 547, 236, 10.1038/nature22822 · DOI
  46. Lauinger, 2014, The RNA helicase FRH is an ATP-dependent regulator of CK1a in the circadian clock of Neurospora crassa, Nature Communications, 5, 10.1038/ncomms4598 · DOI
  47. Lin, 2019, Narrow equilibrium window for complex coacervation of tau and RNA under cellular conditions, eLife, 8, 10.7554/eLife.42571 · DOI
  48. Lin, 2021, Liquid-liquid phase separation of tau driven by hydrophobic interaction facilitates fibrillization of tau, Journal of Molecular Biology, 433, 10.1016/j.jmb.2020.166731 · DOI
  49. Liu, 1997, Thermally regulated translational control of FRQ mediates aspects of temperature responses in the neurospora circadian clock, Cell, 89, 477, 10.1016/s0092-8674(00)80228-7 · DOI
  50. Liu, 2017, Ionic strength sensing in living cells acs chemical biology, ACS Chem. Biol, 12, 2510, 10.1021/acschembio.7b00348 · DOI
  51. Liu, 2019, FRQ-CK1 interaction determines the period of circadian rhythms in neurospora, Nature Communications, 10, 10.1038/s41467-019-12239-w · DOI
  52. Martin, 2020, Intrinsically disordered protein regions and phase separation: sequence determinants of assembly or lack thereof, Emerging Topics in Life Sciences, 4, 307, 10.1042/ETLS20190164 · DOI
  53. Martin, 2021, Small-angle X-ray scattering experiments of monodisperse intrinsically disordered protein samples close to the solubility limit, Methods in Enzymology, 646, 185, 10.1016/bs.mie.2020.07.002 · DOI
  54. Marzoll, 2022, Casein kinase 1 and disordered clock proteins form functionally equivalent, phospho-based circadian modules in fungi and mammals, PNAS, 119, 10.1073/pnas.2118286119 · DOI
  55. Mehra, 2009, A role for casein kinase 2 in the mechanism underlying circadian temperature compensation, Cell, 137, 749, 10.1016/j.cell.2009.03.019 · DOI
  56. Mészáros, 2018, IUPred2A: context-dependent prediction of protein disorder as a function of redox state and protein binding, Nucleic Acids Research, 46, W329, 10.1093/nar/gky384 · DOI
  57. Meyer, 2006, PER-TIM interactions in living Drosophila cells: an interval timer for the circadian clock, Science, 311, 226, 10.1126/science.1118126 · DOI
  58. Muok, 2018, Nucleotide spin labeling for esr spectroscopy of atp-binding proteins, Applied Magnetic Resonance, 49, 1385, 10.1007/s00723-018-1070-6 · DOI
  59. Nguyen, 2015, Site-specific n-terminal labeling utelase 1 and thiodepsipeptide angewandte chemie-international edition, Angew Chem Int Ed Engl, 54, 15694, 10.1002/anie.201506810 · DOI
  60. Nott, 2015, Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles, Molecular Cell, 57, 936, 10.1016/j.molcel.2015.01.013 · DOI · Paper page
  61. Pattanayak, 2020, Daily cycles of reversible protein condensation in cyanobacteria, Cell Reports, 32, 10.1016/j.celrep.2020.108032 · DOI
  62. Pedersen, 2020, Production of intrinsically disordered proteins for biophysical studies, Tips and Tricks Methods in Molecular Biology, 2141, 195, 10.1007/978-1-0716-0524-0 · DOI
  63. Pelham, 2020, Intrinsic disorder is an essential characteristic of components in the conserved circadian circuit, Cell Communication and Signaling, 18, 10.1186/s12964-020-00658-y · DOI
  64. Pelham, 2021, Conformational Changes in the Negative Arm of the Circadian Clock Correlate with Dynamic Interactomes Involved in Post-Transcriptionally Regulated Processes, 10.1101/2021.11.20.469315 · DOI
  65. Posey, 2018, Phase separation of intrinsically disordered proteins, Methods in Enzymology, 611, 1, 10.1016/bs.mie.2018.09.035 · DOI
  66. Querfurth, 2007, Posttranslational regulation of neurospora circadian clock by ck1a-dependent phosphorylation cold spring harbor symposia on quantitative biology, Cold Spring Harb Symp Quant Biol, 72, 177, 10.1101/sqb.2007.72.025 · DOI
  67. Querfurth, 2011, Circadian conformational change of the neurospora clock protein FREQUENCY triggered by clustered hyperphosphorylation of a basic domain, Molecular Cell, 43, 713, 10.1016/j.molcel.2011.06.033 · DOI
  68. Rambo, 2011, Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law, Biopolymers, 95, 559, 10.1002/bip.21638 · DOI · Paper page
  69. Rust, 2007, Ordered phosphorylation governs oscillation of a three-protein circadian clock, Science, 318, 809, 10.1126/science.1148596 · DOI
  70. Sagar, 2020, Structural analyses of intrinsically disordered proteins by small-angle X-ray scattering, 249, 10.1007/978-1-0716-0524-0 · DOI
  71. Schindelin, 2012, Fiji: an open-source platform for biological-image analysis, Nature Methods, 9, 676, 10.1038/nmeth.2019 · DOI · Paper page
  72. Seal, 2021, Evolution of cpeb4 dynamics across its liquid-liquid phase separation transition, The Journal of Physical Chemistry. B, 125, 12947, 10.1021/acs.jpcb.1c06696 · DOI
  73. Selmke, 2018, Open and closed form of maltose binding protein in its native and molten globule state as studied by electron paramagnetic resonance spectroscopy, Biochemistry, 57, 5507, 10.1021/acs.biochem.8b00322 · DOI
  74. Shapiro, 2021, Protein phase separation arising from intrinsic disorder: first-principles to bespoke applications, The Journal of Physical Chemistry. B, 125, 6740, 10.1021/acs.jpcb.1c01146 · DOI
  75. Somjee, 2020, Exploring relationships between the density of charged tracts within disordered regions and phase separation, Pacific Symposium on Biocomputing, 25, 207
  76. Srivastava, 2017, A new wavelet denoising method for experimental time-domain signals: pulsed dipolar electron spin resonance, The Journal of Physical Chemistry. A, 121, 2452, 10.1021/acs.jpca.7b00183 · DOI
  77. Srivastava, 2017, Singular value decomposition method to determine distance distributions in pulsed dipolar electron spin resonance, The Journal of Physical Chemistry Letters, 8, 5648, 10.1021/acs.jpclett.7b02379 · DOI
  78. Srivastava, 2019, Singular value decomposition method to determine distance distributions in pulsed dipolar electron spin resonance: Ii. Estimating uncertainty, The Journal of Physical Chemistry. A, 123, 359, 10.1021/acs.jpca.8b07673 · DOI
  79. Steinegger, 2018, Clustering huge protein sequence sets in linear time, Nature Communications, 9, 10.1038/s41467-018-04964-5 · DOI · Paper page
  80. Szabó, 2022, Charged sequence motifs increase the propensity towards liquid-liquid phase separation, FEBS Letters, 596, 1013, 10.1002/1873-3468.14294 · DOI
  81. Tang, 2009, Setting the pace of the neurospora circadian clock by multiple independent FRQ phosphorylation events, PNAS, 106, 10722, 10.1073/pnas.0904898106 · DOI
  82. Tatavosian, 2019, Nuclear condensates of the polycomb protein chromobox 2 (CBX2) assemble through phase separation, The Journal of Biological Chemistry, 294, 1451, 10.1074/jbc.RA118.006620 · DOI
  83. Trewhella, 2017, 2017 publication guidelines for structural modelling of small-angle scattering data from biomolecules in solution: an update, Acta Crystallographica. Section D, Structural Biology, 73, 710, 10.1107/S2059798317011597 · DOI
  84. van Son, 2015, Double electron-electron spin resonance tracks flavodoxin folding, The Journal of Physical Chemistry. B, 119, 13507, 10.1021/acs.jpcb.5b00856 · DOI
  85. Vernon, 2018, Pi-Pi contacts are an overlooked protein feature relevant to phase separation, eLife, 7, 10.7554/eLife.31486 · DOI
  86. Vogel, 1956, A convenient growth medium for neurospora (Medium N), Microb. Genet. Bull, 13, 42
  87. Wang, 2019, The phospho-code determining circadian feedback loop closure and output in neurospora, Molecular Cell, 74, 771, 10.1016/j.molcel.2019.03.003 · DOI
  88. Wang, 2023, Optimized fluorescent proteins for 4-color and photoconvertible live-cell imaging in Neurospora crassa, Fungal Genetics and Biology, 164, 103763, 10.1016/j.fgb.2022.103763 · DOI
  89. Wright, 2015, Intrinsically disordered proteins in cellular signalling and regulation, Nature Reviews. Molecular Cell Biology, 16, 18, 10.1038/nrm3920 · DOI
  90. Xiao, 2021, Clock proteins regulate spatiotemporal organization of clock genes to control circadian rhythms, PNAS, 118, 10.1073/pnas.2019756118 · DOI
  91. Xie, 2023, Mammalian Circadian Clock Proteins Form Dynamic Interacting Microbodies Distinct from Phase Separation, 10.1101/2023.10.19.563153 · DOI
  92. Zhou, 2013, Non-optimal codon usage affects expression, structure and function of clock protein FRQ, Nature, 495, 111, 10.1038/nature11833 · DOI

Metadata

Authors
Tariq, D., Maurici, N., Bartholomai, B. M., Chandrasekaran, S., Dunlap, J. C., Bah, A., & Crane, B. R.
Affiliations
Dartmouth College (US)
Corresponding author
Bah, Alaji
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2024-03-25
Volume
12
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.90259
PMC
PMC10963029
OpenAlex
W4388971907
Cited by
13
References
3
Citations by year
2026: 5 · 2025: 3 · 2024: 3
FWCI
1.94
Type
journal article
Language
en
Open access
Licence
Locations
  • eLife (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Institute of General Medical Sciences (R35GM122535, R35GM118021, R35GM138097, 1F31GM143890, T32008704, R24GM146107, P30GM103485)
  • Pew Charitable Trusts
Topics
  • Life Sciences › Neuroscience › Endocrine and Autonomic Systems › Circadian rhythm and melatonin
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Light effects on plants
Keywords
circadian clock, Neurospora crassa, intrinsically disordered proteins, phosphorylation, liquid-liquid phase separation, casein kinase 1, white collar complex, protein flexibility, oligomeric state, spin labeling, live imaging, temperature dependence
MeSH terms
Phase Separation, Circadian Rhythm, Fungal Proteins, Neurospora crassa, Phosphorylation, Circadian Clocks

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review