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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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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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.
The authors declare no competing interests.
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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.
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).
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.
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.
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.
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.
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.
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.
Statistical source data, unprocessed gels.
1D and 2D NMR data, unprocessed and processed.
Statistical source data, unprocessed gels.
Unprocessed western blots.
Statistical source data, unprocessed and processed CD data.
Unprocessed and processed CD data.
Unprocessed gels, unprocessed and processed CD data.
Statistical source data.
Statistical source data.
Unprocessed and processed CD data.
Statistical source data.
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