Abstract
The addition of poly(UG) (‘pUG’) repeats to 3′ termini of mRNAs drives gene silencing and transgenerational epigenetic inheritance in the metazoan Caenorhabditis elegans. pUG tails promote silencing by recruiting an RNA-dependent RNA polymerase (RdRP) that synthesizes small interfering RNAs. Here we show that active pUG tails require a minimum of 11.5 repeats and adopt a quadruplex (G4) structure we term the pUG fold. The pUG fold differs from known G4s in that it has a left-handed backbone similar to Z-RNA, no consecutive guanosines in its sequence, and three G quartets and one U quartet stacked non-sequentially. The compact pUG fold binds six potassium ions and brings the RNA ends into close proximity. The biological importance of the pUG fold is emphasized by our observations that porphyrin molecules bind to the pUG fold and inhibit both gene silencing and binding of RdRP. Moreover, specific 7-deaza substitutions that disrupt the pUG fold neither bind RdRP nor induce RNA silencing. These data define the pUG fold as a previously unrecognized RNA structural motif that drives gene silencing. The pUG fold can also form internally within larger RNA molecules. Approximately 20,000 pUG-fold sequences are found in noncoding regions of human RNAs, suggesting that the fold probably has biological roles beyond gene silencing.
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Data availability
The model for (GU)11.5 bound to NMM has been deposited in the Protein Data Bank under accession code 7MKT. PDB deposition files for PDB 7MKT are provided in Supplementary Data 1. Source data are provided with this paper.
Code availability
The Python script for positional analysis of pUG repeat sequences in the human genome is available for download at https://doi.org/10.5281/zenodo.6964887.
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Acknowledgements
Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under contract no. DE-AC02-06CH11357. Use of Life Sciences Collaborative Access Team was supported by Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant no. 085P1000817). Use of GM/CA@APS was funded by the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006 and P30GM138396). The Collaborative Crystallography Core was supported in part by the Department of Biochemistry, UW Madison endowment. Circular dichroism data were obtained at the University of Wisconsin–Madison Biophysics Instrumentation Facility, which was established with support from the University of Wisconsin–Madison and grants nos. BIR-9512577 (NSF) and S10RR13790 (NIH). This study made use of the National Magnetic Resonance Facility at Madison, which is supported by NIH grant no. P41GM136463. This study was supported by NIH/NIGMS grants no. R01GM050942 to M.W., R01GM088289 to S.G.K. and R35 GM118131 to S.E.B.
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Authors and Affiliations
Contributions
S.R. performed CD experiments, electrophoretic mobility shift assays and RNA crystallization. J.Y. and S.G.K. performed RNA-silencing experiments. E.J.M. and S.R. crystallized (GU)11.5–NMM and (GU)12–NMM complexes. C.A.B. collected diffraction data, solved the crystallographic phase problem, and refined the structure of the (GU)11.5–NMM and (GU)12–NMM complexes. Y.N. created the initial models for the (GU)11.5–NMM and (GU)12–NMM complexes. C.A.E. and R.J.P. made NMR samples and analyzed NMR data along with S.E.B. C.A.E. analyzed genomic data. R.J.P. measured NMM and hemin binding to (GU)11.5. M.T. collected NMR data. E.J.M., R.V. and S.E.B. contributed to interpretation of structural data. M.W., S.G.K. and S.E.B. wrote the manuscript, with input from all authors.
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Nature Structural & Molecular Biology thanks Konstantinos Tzelepis and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Sara Osman, in collaboration with the Nature Structural & Molecular Biology editorial team. Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 Silencing assay with AA substitutions.
a, oma-1(zu405ts) silencing assay with AA substitutions within the pUG tail (GU)12.5. The pUG tail sequence is shown below the plot, with location of AA substitutions indicated at the numbered positions. Data are mean ± s.d. Number of independent experiments (injected animals), n = 9 (no injection), 18 (pUG(12.5)), 8 (1), 10 (2), 8 (3), 9 (4), 6 (5), 9 (6), 21 (7), 10 (8), 10 (9), 10 (10), and 14 (11). **, p-value < 0.005 (p-value = 1.88E-04 (1), 1.58E-04 (2), 6.38E-04 (3), 6.99E-04 (4), 6.17E-04 (5), 7.98E-05 (6), 1.10E-07 (7), 1.60E-04 (8), 8.50E-05 (9), 9.86E-06 (10), and 2.18E-07 (11)) (two-sided Student’s t-test). b, oma-1(zu405ts) silencing assay of (GU)13.5 with AA insertions, sequences indicated as in A. Data are mean ± s.d. Number of independent experiments (injected animals), n = 3 (no injection), 6 (pUG(13.5)), 10 (1), 9 (2), 4 (3), 8 (4), and 9 (5). *, p-value < 0.05 (p-value = 3.35E-02 (1); **, p-value < 0.005 (p-value = 4.92E-03 (4)) (two-sided Student’s t-test). c, CD secondary structure analysis of (GU)13.5 with AA substitution at position 2, compared to (GU)12.
Extended Data Fig. 2 CD monitored thermal denaturation of (GU)11.5 in 150 mM KCl.
CD monitored thermal denaturation of (GU)11.5 in 150 mM KCl. a, Three different wavelengths show a single cooperative melting transition at 51.5 °C. b, Thermal melting data measured from low to high temperature and high to low temperature show minimal hysteresis (< 3 °C).
Extended Data Fig. 3 pUG RNA is unfolded by 7 deaza G substitution.
a, pUG RNA is unfolded by 7 deaza G substitution. Native gel analysis of (GU)12 electrophoretic mobility. Lane1: (AC)12 was used as a marker for single stranded RNA (ssRNA). Lane 2: 7 deaza G substitution of (GU)12 produces ssRNA with the same electrophoretic mobility as (AC)12. Lane 3: (GU)12 RNA runs with anomalously slow electrophoretic mobility. A representative gel is shown from experiments that were performed in triplicate, all of which produced the same results. b, CD analysis of unfolded 7 deaza G substituted (GU)12 compared to (GU)12. c, The pUG fold electrophoretic mobility is concentration independent. Lane 1: double stranded RNA (dsRNA) was enforced by heat annealing (GU)12 to excess (AC)12 complementary ssRNA. Lane 2: ssRNA maker (AC)12. Lanes 3-6: (GU)12 at 10, 5, 1, and 0.5 μM, respectively. A representative gel is shown from experiments that were performed in triplicate, all of which produced the same results.
Extended Data Fig. 4 The pUG fold binds the porphyrins NMM and hemin.
The pUG fold binds the porphyrins NMM and hemin. a, Chemical structure of NMM b, The NMM absorbance of free NMM (2.2 μM, red, λmax=378 nm) displays a hyperchromic shift (black, λmax=397 nM) upon addition of increasing amount of the pUG RNA (GU)11.5. c, Fitting of data in A to an equilibrium binding equation. The results of 3 independent experiments are plotted in black, blue and red. d, Chemical Structure of hemin e, The absorbance of free hemin (7.3 μM red, λmax=370 nm) displays a hyperchromic shift (black, λmax=402 nM) upon addition of increasing amount of the pUG RNA (GU)11.5. f, Fitting of data in B to an equilibrium binding equation. The results of 3 independent experiments are plotted in black, blue and red.
Extended Data Fig. 5 Electron density map for (GU)12-NMM.
a, Electron density map for (GU)12-NMM contoured at 1 r.m.s.d. b, Electron density for NMM. c, Electron density for the G1 quartet. d, Electron density for the G3 quartet. e, Electron density for the G5 quartet. F. Electron density for the U4 quartet.
Extended Data Fig. 6 Residual dipolar coupling analysis of free structure in solution vs. crystal.
Measured residual dipolar couplings (RDCs) vs. predicted RDCs from the (GU)12-NMM crystal structure. NMR RDCs were measured for 13C,15N G-labeled (GU)12 RNA (observed) and plotted against the predicted RDC values from the (GU)12-NMM crystal structure, R2 = 0.95.
Extended Data Fig. 7 End to end distance of A-form vs pUG fold RNA.
End to end distance of A-form vs pUG fold RNA. The sequence of (GU)11.5 is color coded as in Fig. 3, except with end nucleotides highlighted in red. The A-form RNA geometry was modeled using PyMOL software version 2.5.2.
Extended Data Fig. 8 CD spectra of (GU)12 and the (GU)12-NMM complex.
a, CD spectra of (GU)12 and the (GU)12-NMM complex. b, Thermal denaturation of (GU)11.5-NMM complex (1:1) monitored at three different wavelengths. The melting temperature of (GU)11.5-NMM is 59.7 °C.
Extended Data Fig. 9 Number and distribution pUG fold coding sequences with 11.5 or more GT repeats in the human vs C. elegans genomes.
Number and distribution pUG fold coding sequences with 11.5 or more GT repeats in the human vs C. elegans genomes.
Extended Data Fig. 10 Genomic analysis of human intron sequences with dinucleotide repeat tracts of 11.5 or more GU repeats.
Genomic analysis of human intron sequences with dinucleotide repeat tracts of 11.5 or more GU repeats. Hits are plotted with respect to their distance from splice sites.
Supplementary information
Supplementary Information
Supplementary Table 1.
Supplementary Video 1
Video of the RNA structure.
Supplementary Data 1
PDB deposition files for PDB 7MKT, Source Data Figs. 3 and 4 and Extended Data Figs. 5 and 7.
Source data
Source Data Fig. 1
Statistical source data, unprocessed gels.
Source Data Fig. 2
1D and 2D NMR data, unprocessed and processed.
Source Data Fig. 5
Statistical source data, unprocessed gels.
Source Data Fig 6
Unprocessed western blots.
Source Data Extended Data Fig. 1
Statistical source data, unprocessed and processed CD data.
Source Data Extended Data Fig. 2
Unprocessed and processed CD data.
Source Data Extended Data Fig. 3
Unprocessed gels, unprocessed and processed CD data.
Source Data Extended Data Fig. 4
Statistical source data.
Source Data Extended Data Fig 6
Statistical source data.
Source Data Extended Data Fig 8
Unprocessed and processed CD data.
Source Data Extended Data Fig 9
Statistical source data.
Source Data Extended Data Fig 10
Statistical source data.
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Roschdi, S., Yan, J., Nomura, Y. et al. An atypical RNA quadruplex marks RNAs as vectors for gene silencing. Nat Struct Mol Biol 29, 1113–1121 (2022). https://doi.org/10.1038/s41594-022-00854-z
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DOI: https://doi.org/10.1038/s41594-022-00854-z
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