A crystal structure of a collaborative RNA regulatory complex reveals mechanisms to refine target specificity

  1. Chen Qiu
  2. Vandita D Bhat
  3. Sanjana Rajeev
  4. Chi Zhang
  5. Alexa E Lasley
  6. Robert N Wine
  7. Zachary T Campbell  Is a corresponding author
  8. Traci M Tanaka Hall  Is a corresponding author
  1. National Institute of Environmental Health Sciences, National Institutes of Health, United States
  2. University of Texas at Dallas, United States
5 figures, 4 tables and 1 additional file

Figures

Figure 1 with 2 supplements
Identification of a minimal fragment of LST-1 that interacts with FBF-2.

(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 …

https://doi.org/10.7554/eLife.48968.002
Figure 1—source data 1

Source data for Figure 1A-Yeast two-hybrid of WT FBF-2 (A.D.) and LST-1 truncations (D.B.D.).

https://doi.org/10.7554/eLife.48968.005
Figure 1—source data 2

Source data for Figure 1B-Yeast two-hybrid of LST-1 point mutants (A.D.) and WT FBF-2 (D.B.D.).

https://doi.org/10.7554/eLife.48968.006
Figure 1—figure supplement 1
A schematic of the yeast two-hybrid assay.

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 …

https://doi.org/10.7554/eLife.48968.003
Figure 1—figure supplement 2
LST-1 interacts with FBF but not homologous PUF proteins.

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 …

https://doi.org/10.7554/eLife.48968.004
Figure 1—figure supplement 2—source data 1

Source data for Figure 1—figure supplement 2-Yeast two-hybrid of PUF protein homologs (A.D.) and WT LST-1 (D.B.D.).

https://doi.org/10.7554/eLife.48968.007
Figure 2 with 1 supplement
Crystal structure of an FBF-2/LST-1/RNA ternary complex reveals hotspots for protein-protein interaction.

(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 …

https://doi.org/10.7554/eLife.48968.009
Figure 2—source data 1

Source data for Figure 2D-Yeast two-hybrid of LST-1 point mutants (D.B.D.) and WT FBF-2 (A.D.).

https://doi.org/10.7554/eLife.48968.011
Figure 2—source data 2

Source data for Figure 2E-Yeast two-hybrid of FBF-2 point mutants (A.D.) and WT LST-1 (D.B.D.).

https://doi.org/10.7554/eLife.48968.012
Figure 2—figure supplement 1
Fo-Fc simulated annealing omit map for the LST-1 peptide, contoured at 3 σ.
https://doi.org/10.7554/eLife.48968.010
The FBF-2 R7-R8 loop is essential for interaction with LST-1.

(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 …

https://doi.org/10.7554/eLife.48968.013
Figure 3—source data 1

Source data for Figure 3B-Yeast two-hybrid of FBF-2 point mutants (A.D.) and WT LST-1 (D.B.D.).

https://doi.org/10.7554/eLife.48968.014
Figure 3—source data 2

Source data for Figure 3C-Yeast two-hybrid of FBF-2 point mutants (A.D.) and WT LST-1 (D.B.D.).

https://doi.org/10.7554/eLife.48968.015
Figure 4 with 1 supplement
FBF-2 in the ternary complex binds to RNA using a 1:1 recognition mode and its curvature is more pronounced.

(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 …

https://doi.org/10.7554/eLife.48968.016
Figure 4—figure supplement 1
Fo-Fc simulated annealing omit map for the cFBE RNA nucleotides 4–6, contoured at 3 σ.
https://doi.org/10.7554/eLife.48968.017
Figure 5 with 1 supplement
SEQRS analysis of FBF-2/LST-1 and FBF-2 reveals distinct specificities.

(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 …

https://doi.org/10.7554/eLife.48968.018
Figure 5—source data 1

Source data for Figure 5B,C-Sequences for MEMEs.

https://doi.org/10.7554/eLife.48968.020
Figure 5—source data 2

Source data for Figure 5E-mRNA targets for GO term enrichment.

https://doi.org/10.7554/eLife.48968.021
Figure 5—figure supplement 1
Representative EMSA gels and corresponding binding curves are shown for binding to gld-1 (A) and compact FBE (cFBE, (B) RNAs.

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…

https://doi.org/10.7554/eLife.48968.019
Figure 5—figure supplement 1—source data 1

Source data for Figure 5—figure supplement 1 and Table 2-Kd values for triplicate measurements.

https://doi.org/10.7554/eLife.48968.022

Tables

Table 1
X-ray data collection and refinement statistics.
https://doi.org/10.7554/eLife.48968.008
Resolution range39.7–2.1 (2.174–2.1)
Space groupP 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 reflections26,619 (4934)
Multiplicity6.8 (7.0)
Completeness (%)96.6 (95.3)
Mean I/sigma(I)11.8 (2.5)
Wilson B-factor41.2
R-merge0.101 (0.795)
R-meas0.109 (0.858)
R-pim0.041 (0.322)
CC1/20.995 (0.885)
Refinement
Reflections used in refinement50,102 (4931)
Reflections used for R-free2000 (197)
R-work0.198 (0.296)
R-free0.240 (0.343)
Number of atoms
protein6565
RNA266
Solvent189
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)
protein53.6
RNA76.7
solvent52.2
  1. *Statistics for the highest-resolution shell are shown in parentheses.

Table 2
RNA-binding analyses of FBF-2 and FBF-2/LST-11.
https://doi.org/10.7554/eLife.48968.023
RNA87654 321 rpt
C-UGUGA-AUG (8)
C-UGUGCCAUA (9)
12345 pos2
FBF-2,
Kd (nM)
Krel2FBF-2/LST-1,
Kd (nM)
Krel3
gld-1 FBEaCAUGUGCCAUA12.4 ± 2.0146.4 ± 5.01
gld-1 –2UUAUGUGCCAUA32.2 ± 4.72.6101.3 ± 13.22.2
gld-1 G4ACAUGUACCAUA12.0 ± 1.4134.4 ± 5.60.7
gld-1 C5ACAUGUGACAUA27.1 ± 5.42.279.2 ± 8.81.7
cFBE-7C-UGUGA-AU22.0 ± 2.72.1111.7 ± 7.72.9
cFBEC-UGUGA-AUG10.3 ± 2.9138.7 ± 5.01
cFBE −1UU-UGUGA-AUG46.5 ± 4.34.5175.9 ± 37.84.5
PBEC-UGUAU-AUA56.8 ± 13.75.5814.0 ± 18021
cFBE G4AC-UGUAA-AUG18.8 ± 3.01.882.7 ± 16.02.1
cFBE A5CC-UGUGC-AUG19.5 ± 2.51.982.3 ± 18.82.1
cFBE A5UC-UGUGU-AUG25.5 ± 5.52.5133.2 ± 23.83.4
cFBE G8AC-UGUGA-AUA21.1 ± 2.51.984.4 ± 20.22.2
  1. 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.

  2. 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.

Table 3
SEQRS enrichment for specific sequence elements.
https://doi.org/10.7554/eLife.48968.024
ProteinRNA sequenceBase +4Terminal AU positionCountRatio compact/
extended
87654 321 repeatCUGUGA AUG (8mer)CUGUGCCAUA (9mer)
FBF-2CTGTA..ATA+8U1193740.21
FBF-2CTGTA. ATA+7U24819
FBF-2CTGTG..ATG+8U19702.8
FBF-2CTGTG. ATG+7U5506
ComplexCTGTA..ATA+8U1700.7
ComplexCTGTA. ATA+7U118
ComplexCTGTG..ATG+8U1131.1
ComplexCTGTG. ATG+7U126
CLIPCTGTA..ATA+8U2660.44
CLIPCTGTA. ATA+7U117
CLIPCTGTG..ATG+8U921.1
CLIPCTGTG. ATG+7U102
Key resources table
Reagent type (species)
or resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene (Caenorhabditis elegans)LST-1UniprotKB: P91820_(CAEEL)
Gene (Caenorhabditis elegans)FBF-2UniprotKB: Q09312_(CAEEL)
Strain, strain background (Saccharomyces cerevisiae))L40ATCCCat. #: MYA-3332Yeast 2-hybrid strain
Strain, strain background (Escherichia coli)DH5-alphaThermo FisherCat. #: 18265017Chemically competent cells
Strain, strain background (Escherichia coli)BL21-CodonPlus (DE3)-RILAgilentCat. #: 230245Competent cells
Recombinant DNA reagentpACT2
(plasmid)
PMID: 21372189GenBank Accession #: U29899Yeast two-hybrid expression vector with Gal4 activation domain fusion
Recombinant DNA reagentpBTM116
(plasmid)
ClonetechVojtek et al., 1993Yeast two hybrid vector with LexA DNA binding ORF
Recombinant DNA reagentpSMT3
(plasmid)
provided by Dr. Christopher LimaMossessova and Lima (2000)Encodes an N-terminal His6-SUMO fusion tag followed by a TEV protease cleavage site
Recombinant DNA reagentpGEX4T-3
(plasmid)
GE HealthcareCat. #: 27-4583-01Bacterial vector for expressing fusion proteins with a thrombin site
Recombinant DNA reagentpMAL-C2T
(plasmid)
New England BiolabsAccession #: JF795283Bacterial vector for cytoplasmic expression of maltose-binding protein fusion
Sequence-based reagentYeast tRNAThermo FisherCat. #: 15401011
Carrier for nucleic acid precipitation
Peptide, recombinant proteinTURBO DNaseThermo FisherCat. #: AM2238
Peptide, recombinant proteinImProm-II reverse transcription reactionPromegaCat. #: A3803
Peptide, recombinant proteinGoTaq reactionPromegaCat. #: M7123
Peptide, recombinant proteinT4 polynucleotide kinaseNew England BiolabsCat. #: M0201S
Peptide, recombinant proteinlysozymeThermo FisherCat. #: 89833
Commercial assay or kitβ-Glo reagentPromegaCat. #: E4720
Commercial assay or kitPhusion High- Fidelity PCR KitThermo FisherCat. #: F553S
Commercial assay or kitAmpliScribe T7-Flash Transcription KitLucigenCat. #: ASF3507
Chemical compound, drugEDTA-free Protease InhibitorRocheCat. #: 11836170001
Chemical compound, drugAmylose resinNew England BiolabsCat. #: E8021S
Chemical compound, drugGlutathione agarose resinGold BiotechnologyCat. #: G-250
Chemical compound, drugNi-NTA resinQiagenCat. #: 30210
Chemical compound, drugreduced glutathioneSigma-Aldrich
Cat. #: G4251
Chemical compound, drugGlutathione magnetic beadsThermo FisherCat. #: 78602
Software, algorithmHKL2000http://www.hkl-xray.com/Otwinowski and Minor, 1997
Software, algorithmPhaserhttp://www.ccp4.ac.uk/html/phaser.htmlMcCoy et al., 2007
Software, algorithmPhenixhttps://www.phenix-online.orgAdams et al., 2010
Software, algorithmCoothttps://www2.mrc-lmb.cam.ac.uk/personal/pemsley/cootEmsley and Cowtan, 2004
Software, algorithmMEMEhttp://meme-suite.org/Bailey et al., 2006
Software, algorithmEnrichrhttps://amp.pharm.mssm.edu/Enrichr/
Kuleshov et al., 2016
Software, algorithmImageQuant Version 5.1GE Healthcare
Software, algorithmGraphPad Prism 7GraphPad
Software, algorithmMatlab R2008aMathWorks

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