Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio
- University of Michigan, United States
- National Institutes of Health, United States
- University of Texas at Dallas, United States
- University of Minnesota, United States
Figures
Figure 1 with 1 supplement
The Zn finger and C-terminal regions of Nanos collaborate with Pumilio to repress target protein and mRNA expression.
(A) Diagram of Renilla Luciferase (RnLuc) reporters including the hb 3´UTR. WT PRE1 and PRE2 sequences, located within NRE1 and NRE2, respectively, and mutant PRE sequences (mt1, mt2, and mt1,2) are …
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Figure 1—source data 1
Values and statistical analysis of luciferase reporter assays.
The average value of the relative response ratio of Renilla to Firefly luciferase activities (Rn/FF), average percent repression values (%Repress), and Standard Errors of the Mean (SEM) are listed for each condition in the experiments (from four technical replicates, n = 4). The p-values (p-val) resulting from two-tailed t-tests between each measurement and the indicated control are represented in bold (significant) or italics (not significant). Experimental values derived from the same experiment are outlined in boxes.
- https://doi.org/10.7554/eLife.17096.004
Figure 1—figure supplement 1
Nos reduces hb 3´UTR reporter mRNA level in a PRE-dependent manner.
Quantitation of Northern blot detection of the indicated RnLuc hb 3´UTR reporter mRNAs and the 7SL RNA, as a loading control, from total RNA purified from D.mel−2 cells expressing negative control …
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Figure 1—figure supplement 1—source data 1
Values and statistical analysis of Northern blot of luciferase reporter mRNAs.
Quantitation of the Northern blot in Figure 1—figure supplement 1 was performed and the average ratio of Renilla luciferase mRNA to 7SL RNA internal control (Rn/7SL) was calculated to normalize expression values (Normed, n = 3). Standard Error of the Mean (SEM) is reported for each measurment. The p-values (p-val) resulting from two-tailed t-tests between each measurement and the indicated control are represented in bold (significant) or italics (not significant).
- https://doi.org/10.7554/eLife.17096.006
Figure 2 with 3 supplements
Nanos embraces Pumilio and hb RNA to stabilize the ternary complex.
(A) Crystal structure of a Drosophila Nos-Pum-hb NRE2 RNA ternary complex. Pum is shown as a ribbon diagram (yellow), Nos is shown as a surface representation (blue) and hb NRE2 RNA is shown as a …
Figure 2—figure supplement 1
Representative electron density map of the Nos-Pum-hb NRE2 complex.
A 2Fo-Fc composite omit map contoured at 1.2 σ is shown superimposed with the Nos-Pum-hb NRE2 RNA structure at the binding pocket for the −1U. Carbon atoms are blue for Nos and grey for RNA.
Figure 2—figure supplement 2
Nanos induces localized structural changes in Pum upon formation of the Nos-Pum-hb NRE2 RNA ternary complex.
A loop between repeats R7-R8 of Pum (R7-R8 loop) undergoes conformational changes to promote Nos-Pum interactions. The terminal helix of Repeat 8′ (R8′) also changes conformation to interact with …
Figure 2—figure supplement 3
Crystal structure of Nos – Pum – hb NRE2 RNA ternary complex highlights key Pum-RNA and Nos-Pum contacts.
The C-terminal helix of Pum unfolds to promote ternary complex formation, forming additional contacts with the RNA. Important Nos and Pum residues are indicated, although electron density for most …
Figure 3 with 1 supplement
Nanos increases the binding affinity of Pumilio for hunchback RNA.
(A) Diagram of recombinant proteins and RNA ligand used for EMSAs. The amino acid residue boundaries of the Pum RNA-binding domain (PUM-HD, yellow) are represented relative to full-length Pum. For …
Figure 3—figure supplement 1
Recombinant purified Pum and Nos test proteins.
Coomassie blue-stained SDS-polyacrylamide gel loaded with equivalent amounts of purified recombinant Pum and Nos WT and mutant test proteins used for the EMSAs. Molecular weights of protein markers …
Figure 4
Interactions between Nanos and Pumilio are necessary for repression.
(A) Diagrams of Nos and Pum proteins highlighting residues involved in protein-protein interaction. The amino acid sequence of the C-terminal region of Nos is shown. Residues 376–382 that are …
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Figure 4—source data 1
Values and statistical analysis of luciferase reporter assays.
The average value of the relative response ratio of Renilla to Firefly luciferase activities (Rn/FF), average percent repression values (%Repress), and Standard Errors of the Mean (SEM) are listed for each condition in the experiments (from four technical replicates, n = 4). The p-values (p-val) resulting from two-tailed t-tests between each measurement and the indicated control are represented in bold (significant) or italics (not significant). Experimental values derived from the same experiment are outlined in boxes.
- https://doi.org/10.7554/eLife.17096.015
Figure 5
Nanos Zn finger interaction with RNA extends the RNA-binding site and is critical for repression.
(A) Interaction of Nos ZFs with the -1U of hb NRE2. In addition to the base contacts noted in the text, the OH group of Y369Nos and the NH2 group of K368Nos interact with the phosphate group of −1U. …
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Figure 5—source data 1
Values and statistical analysis of luciferase reporter assays.
The average value of the relative response ratio of Renilla to Firefly luciferase activities (Rn/FF), average percent repression values (%Repress), and Standard Errors of the Mean (SEM) are listed for each condition in the experiments (from four technical replicates, n = 4). The p-values (p-val) resulting from two-tailed t-tests between each measurement and the indicated control are represented in bold (significant) or italics (not significant). Experimental values derived from the same experiment are outlined in boxes.
- https://doi.org/10.7554/eLife.17096.017
Figure 6 with 5 supplements
Nanos alters Pumilio RNA-binding specificity.
(A) A representative EMSA with increasing concentrations of Pum in the presence or absence of Nos, performed with radiolabeled CycB NRE RNA shown at the top. The PRE and the NBS are highlighted in …
Figure 6—figure supplement 1
Nos promotes ternary complex formation with Pum and the hb NRE1 RNA.
Representative EMSA with increasing concentrations of Pum in the presence or absence of Nos. Radiolabeled RNA sequence used is shown at the top. The PRE and NBS sequences are highlighted in yellow …
Figure 6—figure supplement 2
Nos promotes ternary complex formation with Pum and the bcd NRE RNA.
Representative EMSA with increasing concentrations of Pum in the presence or absence of Nos. Radiolabeled RNA sequence used is shown at the top. The PRE and NBS sequences are highlighted in yellow …
Figure 6—figure supplement 3
Nos promotes ternary complex formation with Pum and the hb NRE2 +3G RNA.
Representative EMSA with increasing concentrations of Pum in the presence or absence of Nos. Radiolabeled RNA sequence used is shown at the top. The PRE and NBS sequences are highlighted in yellow …
Figure 6—figure supplement 4
Nos and Pum do not bind the mutant hb NRE2 RNA.
Representative EMSAs with increasing concentrations of Pum in the presence or absence of Nos. Radiolabeled RNA sequence used is shown at the top. The PRE and NBS sequences are highlighted in yellow …
Figure 6—figure supplement 5
hb and CycB RNAs form different conformations in complex with Pum and Nos.
Crystal structures of hb and CycB RNAs from ternary complexes with Pum and Nos are shown superimposed with 2Fo-Fc electron density maps contoured at 1.2 σ. The models fit well with their respective …
Figure 7 with 4 supplements
SEQRS analysis of Nos and Pum reveals specificity of RNA-binding activities.
(A) Diagram of the SEQRS procedure. (B) Motif from SEQRS analysis of Pum. (C) Motif from SEQRS analysis of Nos-Pum complex. (D) The Nos-Pum and Pum motifs are preferentially enriched by their …
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Figure 7—source data 1
Related to Figure 7F.
Gene ontology enrichment results for Pum targets from Drosophila adults and embryos that contain SEQRS-derived Pum motif, Nos-Pum motif, or both motifs. (A) Target gene lists used in gene ontology analysis. (B) Gene ontology enrichment analysis of target mRNAs from adults that contain the Nos-Pum motif. (C) Gene ontology enrichment analysis of target mRNAs from adults that contain the Pum motif. (D) Gene ontology enrichment analysis of target mRNAs from adults that contain the Nos-Pum and Pum motif. No categories were significantly enriched. (E) Gene ontology enrichment analysis of target mRNAs from embryos that contain the Nos-Pum motif. (F) Gene ontology enrichment analysis of target mRNAs from embryos that contain the Pum motif. No categories were significantly enriched. (G) Gene ontology enrichment analysis of target mRNAs from embryos that contain both the Nos-Pum and Pum motif. No categories were significantly enriched.
- https://doi.org/10.7554/eLife.17096.025
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Figure 7—source data 2
Related to Figure 7A–E.
SEQRS sequences for Pum and Nos-Pum ternary complex and statistical analysis of motif enrichment in target mRNA 3´UTRs. (A) 20-mer sequences corresponding to the random region of the SEQRS library are reported for two replicates designated as A or B. (B) Top 100 enriched sequences for Pum and Nos-Pum complex. (C) Motif enrichment data and statistics for Pumilio target mRNAs from Drosophila embryos and adults based on Gerber et al, 2006. Sequences were obtained following five rounds of selection.
- https://doi.org/10.7554/eLife.17096.026
Figure 7—figure supplement 1
Comparison of the reproducibility of two replicates of Pum SEQRS.
Scatter plots of all possible 8-mer sequences following five rounds of selection. R2 values are based on linear regression analysis.
Figure 7—figure supplement 2
Comparison of sequences selected in SEQRS for Pum and the Nos-Pum complex.
Scatter plots of all possible 8-mer sequences following five rounds of selection. Orange data points indicate sequences corresponding to (a) hb NRE1, (b) bcd NRE, (c) CycB NRE, and (d) the hb NRE2. …
Figure 7—figure supplement 3
Analysis of upstream nucleotides enriched in SEQRS by Nos in the ternary complex relative to Pum alone.
Relative enrichment values for sequences matching the pattern 5´-NNNNUGUA for the Nos-Pum complex were calculated after subtraction of the sequences bound by Pum alone.
Figure 7—figure supplement 4
Venn diagrams reveal differences in the extent of motif overlap in Pum bound transcripts in embryo (above) versus adult (below) (Gerber et al., 2006).
https://doi.org/10.7554/eLife.17096.030
Figure 8
Nanos expands the Pumilio mRNA target repertoire in cells.
(A) Diagram of the RnLuc 1x NRE reporters with minimal 3´UTRs containing WT or mutant (+3G and ACA) hb NRE2 sequences, the hb NRE1 sequence, or NRE sequences from the Cyclin B (CycB) and bicoid (bcd)…
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Figure 8—source data 1
Values and statistical analysis of luciferase reporter assays.
The average value of the relative response ratio of Renilla to Firefly luciferase activities (Rn/FF), average percent repression values (%Repress), and Standard Errors of the Mean (SEM) are listed for each condition in the experiments (from 4 technical replicates, n = 4). The p-values (p-val) resulting from two-tailed t-tests between each measurement and the indicated control are represented in bold (significant) or italics (not significant). Experimental values derived from the same experiment are outlined in boxes.
- https://doi.org/10.7554/eLife.17096.032
Figure 9
Nanos Zn finger 2 is structurally homologous to HIV nucleocapsid protein Zn knuckles.
(A) Ribbon diagrams of Nos ZFs 1 and 2. The Nos ZFs follow the same structural topology, but ZF1 has longer loops than ZF2 that are N- and C-terminal to the Zn-coordinating histidine residue. (B) …
Tables
Table 1
Data collection and refinement statistics.
| Pum-RNA | Pum-Nos-hb RNA | Pum-Nos-cycB RNA | ||
|---|---|---|---|---|
| PDB ID | 5KLA | 5KL1 | 5KL8 | |
| Data collection | ||||
| Space group | C2 | P6522 | P6522 | |
| Cell dimensions | a, b, c (Å) | 194.9, 29.5, 62.0 | 137.0, 137.0, 221.4 | 135.1, 135.1, 220.4 |
| a, b, g (°) | 90.0, 101.2, 90.0 | 90.0, 90.0, 120.0 | 90.0, 90.0, 120.0 | |
| Resolution (Å) | 50-1.14 (1.16-1.14) | 50-3.70 (3.83-3.70) | 50-4.00 (4.12-4.00) | |
| Rsym | 0.045 (0.387) | 0.128 (0.747) | 0.143 (0.779) | |
| I / σI | 36.9 (2.7) | 19.1 (2.8) | 13.0 (3.6) | |
| Completeness (%) | 99.7 (97.4) | 99.3 (93.2) | 99.3 (100.0) | |
| Redundancy | 4.2 (2.4) | 11.3 (11.0) | 8.9 (8.7) | |
| Refinement | ||||
| Resolution (Å) | 34.46 - 1.14 | 38.3 - 3.70 | 39.0 - 4.00 | |
| No. reflections | 127077 | 13562 | 10715 | |
| Rwork / Rfree (%) | 16.0 / 17.4 | 26.4 / 30.0 | 28.3 / 31.2 | |
| No. atoms | ||||
| Protein | 5532 | 3194 | 3021 | |
| RNA | 253 | 252 | 226 | |
| Water / Solvent | 401 | 0 | 0 | |
| B-factors | ||||
| Protein | 29.0 | 175.5 | 208.6 | |
| RNA | 20.5 | 150.4 | 183.4 | |
| Water / Solvent | 34.9 | - | - | |
| R.m.s deviations | ||||
| Bond lengths (Å) | 0.007 | 0.003 | 0.002 | |
| Bond angles (°) | 0.950 | 0.605 | 0.508 | |
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*Values in parentheses are for highest-resolution shell.
