(A) Yeast 2-hybrid analyses of interaction between the FBF-2 PUM domain fused to a GAL4 activation domain (A.D.) and LST-1 fragments fused to the LexA DNA-binding domain (D.B.D.). A negative control …
Source data for Figure 1A-Yeast two-hybrid of WT FBF-2 (A.D.) and LST-1 truncations (D.B.D.).
Source data for Figure 1B-Yeast two-hybrid of LST-1 point mutants (A.D.) and WT FBF-2 (D.B.D.).
To quantify binding activity, fusion proteins are introduced into yeast with a ‘bait’ protein (purple) fused to the LexA DNA-binding domain. The LexA protein provides a tether to the promoter region …
Yeast-two hybrid assays were conducted with LST-1 residues 55–105 fused to the Lex-A DNA-binding domain (D.B.D.) and the GAL4 activation domain (A.D.) was fused to the PUM domain of the indicated …
Source data for Figure 1—figure supplement 2-Yeast two-hybrid of PUF protein homologs (A.D.) and WT LST-1 (D.B.D.).
(A) Crystal structure of an FBF-2/LST-1/RNA ternary complex. FBF-2 is shown as a ribbon diagram with cylindrical helices. PUM repeats are colored alternately red and blue. RNA recognition side …
Source data for Figure 2D-Yeast two-hybrid of LST-1 point mutants (D.B.D.) and WT FBF-2 (A.D.).
Source data for Figure 2E-Yeast two-hybrid of FBF-2 point mutants (A.D.) and WT LST-1 (D.B.D.).
(A) The essential residue LST-1 L83 interacts with FBF-2 at the base of the FBF-2 R7-R8 loop. FBF-2 L444 and Y479 at the R7-R8 loop are shown with space-filling atoms. (B) Yeast 2-hybrid analyses …
Source data for Figure 3B-Yeast two-hybrid of FBF-2 point mutants (A.D.) and WT LST-1 (D.B.D.).
Source data for Figure 3C-Yeast two-hybrid of FBF-2 point mutants (A.D.) and WT LST-1 (D.B.D.).
(A) FBF-2 recognizes the central nucleotides in a compact RNA using repeats 4 and 5. The crystal structure of the FBF-2/LST-1/RNA ternary complex is shown with FBF-2 displayed as a ribbon diagram …
(A) Diagram of the SEQRS procedure. (B) Motif from SEQRS analysis of the FBF-2/LST-1 complex. (C) Motif from SEQRS analysis of FBF-2. Inset, superposition of the upstream C pocket in structures of …
Source data for Figure 5B,C-Sequences for MEMEs.
Source data for Figure 5E-mRNA targets for GO term enrichment.
Triangles above the gels indicate increasing concentrations of FBF-2 from 0.49 to 4000 nM. The left lanes in each gel contained no protein. Kd values for triplicate experiments are presented in Table…
Source data for Figure 5—figure supplement 1 and Table 2-Kd values for triplicate measurements.
Resolution range | 39.7–2.1 (2.174–2.1) |
---|---|
Space group | P 1 |
Unit cell dimensions a, b, c (Å) α, β, γ (°) | 42.75, 74.38, 81.55 107.17, 104.40, 101.76 |
Total reflections* | 180,242 (13587) |
Unique reflections | 26,619 (4934) |
Multiplicity | 6.8 (7.0) |
Completeness (%) | 96.6 (95.3) |
Mean I/sigma(I) | 11.8 (2.5) |
Wilson B-factor | 41.2 |
R-merge | 0.101 (0.795) |
R-meas | 0.109 (0.858) |
R-pim | 0.041 (0.322) |
CC1/2 | 0.995 (0.885) |
Refinement | |
Reflections used in refinement | 50,102 (4931) |
Reflections used for R-free | 2000 (197) |
R-work | 0.198 (0.296) |
R-free | 0.240 (0.343) |
Number of atoms | |
protein | 6565 |
RNA | 266 |
Solvent | 189 |
RMSD bonds (Å) | 0.003 |
RMSD angles (°) | 0.82 |
Ramachandran favored (%) | 98.38 |
Ramachandran allowed (%) | 1.62 |
Ramachandran outliers (%) | 0.00 |
Average B-factors (Å2) | |
protein | 53.6 |
RNA | 76.7 |
solvent | 52.2 |
*Statistics for the highest-resolution shell are shown in parentheses.
RNA | 87654 321 rpt C-UGUGA-AUG (8) C-UGUGCCAUA (9) 12345 pos2 | FBF-2, Kd (nM) | Krel2 | FBF-2/LST-1, Kd (nM) | Krel3 |
---|---|---|---|---|---|
gld-1 FBEa | CAUGUGCCAUA | 12.4 ± 2.0 | 1 | 46.4 ± 5.0 | 1 |
gld-1 –2U | UAUGUGCCAUA | 32.2 ± 4.7 | 2.6 | 101.3 ± 13.2 | 2.2 |
gld-1 G4A | CAUGUACCAUA | 12.0 ± 1.4 | 1 | 34.4 ± 5.6 | 0.7 |
gld-1 C5A | CAUGUGACAUA | 27.1 ± 5.4 | 2.2 | 79.2 ± 8.8 | 1.7 |
cFBE-7 | C-UGUGA-AU | 22.0 ± 2.7 | 2.1 | 111.7 ± 7.7 | 2.9 |
cFBE | C-UGUGA-AUG | 10.3 ± 2.9 | 1 | 38.7 ± 5.0 | 1 |
cFBE −1U | U-UGUGA-AUG | 46.5 ± 4.3 | 4.5 | 175.9 ± 37.8 | 4.5 |
PBE | C-UGUAU-AUA | 56.8 ± 13.7 | 5.5 | 814.0 ± 180 | 21 |
cFBE G4A | C-UGUAA-AUG | 18.8 ± 3.0 | 1.8 | 82.7 ± 16.0 | 2.1 |
cFBE A5C | C-UGUGC-AUG | 19.5 ± 2.5 | 1.9 | 82.3 ± 18.8 | 2.1 |
cFBE A5U | C-UGUGU-AUG | 25.5 ± 5.5 | 2.5 | 133.2 ± 23.8 | 3.4 |
cFBE G8A | C-UGUGA-AUA | 21.1 ± 2.5 | 1.9 | 84.4 ± 20.2 | 2.2 |
1Representative EMSA gels and binding curves are shown in Figure 5—figure supplement 1. Source data for the three technical replicate EMSAs are included in Figure 5—figure supplement 1—source data 1.
2RNA sequences of the cFBE compact element and gld-1 FBEa motif are shown with the FBF-2 repeat (rpt) that binds to the respective nucleotide above and the RNA motif position below. Nucleotides in boldface differ from the sequences of the gld-1 FBEa motif (top four lines) or the cFBE.
3Relative Kd values (Krel) are calculated with respect to the Kd for binding to the gld-1 FBEa motif (top four lines) or the cFBE.
Protein | RNA sequence | Base +4 | Terminal AU position | Count | Ratio compact/ |
---|---|---|---|---|---|
extended | |||||
87654 321 repeatCUGUGA AUG (8mer)CUGUGCCAUA (9mer) | |||||
FBF-2 | CTGTA..AT | A | +8U | 119374 | 0.21 |
FBF-2 | CTGTA. AT | A | +7U | 24819 | |
FBF-2 | CTGTG..AT | G | +8U | 1970 | 2.8 |
FBF-2 | CTGTG. AT | G | +7U | 5506 | |
Complex | CTGTA..AT | A | +8U | 170 | 0.7 |
Complex | CTGTA. AT | A | +7U | 118 | |
Complex | CTGTG..AT | G | +8U | 113 | 1.1 |
Complex | CTGTG. AT | G | +7U | 126 | |
CLIP | CTGTA..AT | A | +8U | 266 | 0.44 |
CLIP | CTGTA. AT | A | +7U | 117 | |
CLIP | CTGTG..AT | G | +8U | 92 | 1.1 |
CLIP | CTGTG. AT | G | +7U | 102 |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Caenorhabditis elegans) | LST-1 | UniprotKB: P91820_(CAEEL) | ||
Gene (Caenorhabditis elegans) | FBF-2 | UniprotKB: Q09312_(CAEEL) | ||
Strain, strain background (Saccharomyces cerevisiae)) | L40 | ATCC | Cat. #: MYA-3332 | Yeast 2-hybrid strain |
Strain, strain background (Escherichia coli) | DH5-alpha | Thermo Fisher | Cat. #: 18265017 | Chemically competent cells |
Strain, strain background (Escherichia coli) | BL21-CodonPlus (DE3)-RIL | Agilent | Cat. #: 230245 | Competent cells |
Recombinant DNA reagent | pACT2 (plasmid) | PMID: 21372189 | GenBank Accession #: U29899 | Yeast two-hybrid expression vector with Gal4 activation domain fusion |
Recombinant DNA reagent | pBTM116 (plasmid) | Clonetech | Vojtek et al., 1993 | Yeast two hybrid vector with LexA DNA binding ORF |
Recombinant DNA reagent | pSMT3 (plasmid) | provided by Dr. Christopher Lima | Mossessova and Lima (2000) | Encodes an N-terminal His6-SUMO fusion tag followed by a TEV protease cleavage site |
Recombinant DNA reagent | pGEX4T-3 (plasmid) | GE Healthcare | Cat. #: 27-4583-01 | Bacterial vector for expressing fusion proteins with a thrombin site |
Recombinant DNA reagent | pMAL-C2T (plasmid) | New England Biolabs | Accession #: JF795283 | Bacterial vector for cytoplasmic expression of maltose-binding protein fusion |
Sequence-based reagent | Yeast tRNA | Thermo Fisher | Cat. #: 15401011 | Carrier for nucleic acid precipitation |
Peptide, recombinant protein | TURBO DNase | Thermo Fisher | Cat. #: AM2238 | |
Peptide, recombinant protein | ImProm-II reverse transcription reaction | Promega | Cat. #: A3803 | |
Peptide, recombinant protein | GoTaq reaction | Promega | Cat. #: M7123 | |
Peptide, recombinant protein | T4 polynucleotide kinase | New England Biolabs | Cat. #: M0201S | |
Peptide, recombinant protein | lysozyme | Thermo Fisher | Cat. #: 89833 | |
Commercial assay or kit | β-Glo reagent | Promega | Cat. #: E4720 | |
Commercial assay or kit | Phusion High- Fidelity PCR Kit | Thermo Fisher | Cat. #: F553S | |
Commercial assay or kit | AmpliScribe T7-Flash Transcription Kit | Lucigen | Cat. #: ASF3507 | |
Chemical compound, drug | EDTA-free Protease Inhibitor | Roche | Cat. #: 11836170001 | |
Chemical compound, drug | Amylose resin | New England Biolabs | Cat. #: E8021S | |
Chemical compound, drug | Glutathione agarose resin | Gold Biotechnology | Cat. #: G-250 | |
Chemical compound, drug | Ni-NTA resin | Qiagen | Cat. #: 30210 | |
Chemical compound, drug | reduced glutathione | Sigma-Aldrich | Cat. #: G4251 | |
Chemical compound, drug | Glutathione magnetic beads | Thermo Fisher | Cat. #: 78602 | |
Software, algorithm | HKL2000 | http://www.hkl-xray.com/ | Otwinowski and Minor, 1997 | |
Software, algorithm | Phaser | http://www.ccp4.ac.uk/html/phaser.html | McCoy et al., 2007 | |
Software, algorithm | Phenix | https://www.phenix-online.org | Adams et al., 2010 | |
Software, algorithm | Coot | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot | Emsley and Cowtan, 2004 | |
Software, algorithm | MEME | http://meme-suite.org/ | Bailey et al., 2006 | |
Software, algorithm | Enrichr | https://amp.pharm.mssm.edu/Enrichr/ | Kuleshov et al., 2016 | |
Software, algorithm | ImageQuant Version 5.1 | GE Healthcare | ||
Software, algorithm | GraphPad Prism 7 | GraphPad | ||
Software, algorithm | Matlab R2008a | MathWorks |