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

Tariq, Daniyal1 ; Crane, Brian1 ; Bah, Alaji2

Published Feb 27, 2024 on Dryad. https://doi.org/10.5061/dryad.pk0p2ngwh

Data files

Feb 27, 2024 version files 97.30 MB

Raw_Data.zip

97.27 MB
README.md

8.08 KB
scripts.zip

15.84 KB

Abstract

Circadian clocks are composed of molecular oscillators that pace rhythms of gene expression to the diurnal cycle. Therein, transcriptional-translational negative feedback loops (TTFLs) generate oscillating levels of transcriptional repressor proteins that regulate their own gene expression. In the filamentous fungus Neurospora crassa, the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH) and Casein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the White-Collar complex (WCC). A key question concerns how FRQ orchestrates molecular interactions at the core of the clock despite containing little predicted tertiary structure. We present the reconstitution and biophysical characterization of FRQ and the FFC in unphosphorylated and highly phosphorylated states. Site-specific spin labeling and pulse-dipolar ESR spectroscopy provides domain-specific structural details on the full-length, 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 circadian clocks.

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

https://doi.org/10.5061/dryad.pk0p2ngwh

Description of the data and file structure

These files represent the raw data from the ESR, SAXS, UV-Vis, MALS, and microscopy experiments. The main folder titled "Raw Data" contains various sub-folders that are labeled by experiment type.

The "Microscopy Images" sub-folder contains all the microscopy images showing FRQ droplets +/- binding partners under different conditions. Within this folder, there are 3 sub-folders.
1. "Co-LLPS" contains co-phase-separation microscopy images with FRQ and either FRH or CK1. This folder also has the images from the CheY control.
2. "np-FRQ and p-FRQ Concentration Titration +_- hexanediol" contains microscopy images of FRQ at different concentrations +/- hexanediol, which is used to dissolve droplets and show that they are indeed liquid-like.
3. "np-FRQ Cysless Temperature" contains microscopy images of FRQ undergoing phase separation at different temperatures and correlates to the radio ATP based assay shown in the paper Figure 7.
The "Raw ESR Data" folder contains the raw data from all the CW-ESR and DEER experiments shown in the paper.
1. The "CW ESR" sub-folder contains the raw files for the Cw ESR experiments done with the various single cysteine mutants shown in Figure 3 of the paper as well as the N and C terminus of FRQ. Each filename contains the site of the spin-label attachment.
  1. Each file is a “.DTA” format that is the raw output from the spectrometer, containing the field strength and intensity (at those field strengths) values. These files are processed by the easyspin software package in MATLAB.
2. The "DEER" sub-folder contains the raw data files from all of the DEER experiments shown in the paper. Each filename contains the site of the spin-label attachment.
  1. Each file is a “.DTA” format that is the raw output from the spectrometer. These files contain the time points and echo intensity values.These files are processed by the easyspin software package in MATLAB.
The "Raw SAXS Data" folder contains the files used to generate the dimensionless kratky plots and p(r) distributions shown in the paper. The filenames reflect which type of plots they were used to generate.
1. “PhosFRQ_NonPhosFRQ_Dimensionless_Kratky.csv” contains the data used to generate the dimensionless Kratky plot showin in Figure 2.
  1. Column 1 has the qRG values for np-FRQ
  2. Column 2 has the qRGI^2(Q)/I0 values for np-FRQ
  3. Column 3 has the qRG values for p-FRQ
  4. Column 4 has the qRGI^2(Q)/I0 values for p-FRQ
2. “PhosFFC_NonPhosFFC_Dimensionless_Kratky.csv”
  1. Column 1 has the qRG values for np-FFC
  2. Column 2 has the qRGI^2(Q)/I0 values for np-FFC
  3. Column 3 has the qRG values for p-FFC
  4. Column 4 has the qRGI^2(Q)/I0 values for p-FFC
The "Raw_SEC_MALS_Data" folder contains the csv files used to plot the analytical SEC trace for the FFC complex in Figure 4 and the MALS trace for FRQ shown in Figure 2.
1. “FFC_AnalyticalSEC.csv” contains the data used to plot the FFC analytical SEC trace.
  1. Column 1 contains the elution volume(in mLs)
  2. Column 2 contains the corresponding UV absorbance at 280 nm for the volume in Column 1.
2. “PhosFRQ_NonPhosFRQ_SECMALS.csv” contains the data used to plot the SEC-MALS trace shown in Figure 2.
  1. Column 1 has the elution volumes for the concentrated np-FRQ sample
  2. Column 2 has the UV absorption values for the concentrated np-FRQ sample
  3. Column 3 has the elution volumes for the concentrated np-FRQ sample that were chosen for molecular weight determination
  4. Column 4 has the molecular weight estimates across the chosen peak region for the np-FRQ sample
  5. Column 5 has the elution volumes for the np-FRQ 4X diluted sample
  6. Column 6 has the UV absorption values for the concentrated np-FRQ 4X diluted sample
  7. Column 7 has the elution volumes for the concentrated np-FRQ 4X sample that were chosen for molecular weight determination
  8. Column 8 has the molecular weight estimates across the chosen peak region for the np-FRQ 4X diluted sample
  9. Column 9 has the elution volumes for the p-FRQ sample
  10. Column 10 has the UV absorption values for the concentrated p-FRQ sample
  11. Column 11 has the elution volumes for the concentrated p-FRQ sample that were chosen for molecular weight determination
  12. Column 12 has the molecular weight estimates across the chosen peak region for the p-FRQ sample

The "Temperature Concentration Phase Diagram UV-Vis Data" folder contains the csv files with the fit used to generate the temperature and concentration dependent phase diagram in Figure 6 as well as the results of the concentration titration turbidity assays done for both phosphorylated and non-phosphorylated FRQ.

The file “np-FRQ Concentration Titration (5-15 uM)” contains the following data:
1. Column 1: The temperature at which absorbance, or scattering, at 300 nm was recorded.
2. Columns 2 and 3: Two replicates of np-FRQ at 5 uM and their absorbance values
3. Columns 4 and 5: Two replicates of np-FRQ at 10 uM and their absorbance values
4. Columns 6 and 7: Two replicates of np-FRQ at 15 uM and their absorbance values
5. Columns 8 and 9: Two replicates of Buffer without protein and their absorbance values used as a control and baseline

2. The file “p-FRQ Concentration Titration (5-15 uM)” contains the following data:

Column 1: The temperature at which absorbance, or scattering, at 300 nm was recorded.
Columns 2 and 3: Two replicates of p-FRQ at 5 uM and their absorbance values
Columns 4: No data as there is no third replicate for p-FRQ at 5 uM
Columns 5 and 6: Two replicates of p-FRQ at 10 uM and their absorbance values

5. Columns 7: No data as there is no third replicate for p-FRQ at 10 uM

Columns 8, 9, and 10: Three replicates of p-FRQ at 15 uM and their absorbance values
Columns 11, 12, and 13: Three replicates for Buffer without protein and their absorbance values used as a control and baseline
The file “Nonlin fit of Temperature-Concentration Phase Diagram” contains information calculated by GraphPad using the data from the files “np-FRQ Concentration Titration (5-15 uM)” and “p-FRQ Concentration Titration (5-15 uM)”.
The "Proteome LLPS Propensity and Disorder Scores" folder contains two files:
1. The first file contains the IUPRED disorder scores for FRQ, hPER, dPER as well the proteomes for each of these organisms (Neurospora crassa, Drosophila melanogaster and humans)
  1. Column 1: The disorder scores for FRQ and its homologs
  2. Column 2: The disorder scores for the Neurospora crassa proteome
  3. Column 3: The disorder scores for dPER and its homologs
  4. Column 4: The disorder scores for the Drosophila melanogaster proteome
  5. Column 5: The disorder scores for hPER and its homologs
  6. Column 6: The disorder scores for the human proteome
2. The second file contains the LLPS propensity scores for FRQ, hPER, dPER as well the proteomes for each of these organisms (Neurospora crassa, *Drosophila melanogaster *and humans) based on the method described in the following paper: https://elifesciences.org/articles/31486
  1. Column 1: The LLPS propensity scores for FRQ and its homologs
  2. Column 2: The LLPS propensity for the Neurospora crassa proteome
  3. Column 3: The LLPS propensity for dPER and its homologs
  4. Column 4: The LLPS propensity for the Drosophila melanogaster proteome
  5. Column 5: The LLPS propensity for hPER and its homologs
  6. Column 6: The LLPS propensity for the human proteome