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RdRP-synthesized antisense ribosomal siRNAs silence pre-rRNA via the nuclear RNAi pathway

Nature Structural & Molecular Biology volume 24, pages 258–269 (2017)Cite this article

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Abstract

Expression of rRNA affects cell growth and proliferation, but mechanisms that modulate rRNA levels are poorly understood. We conducted a genetic screen for factors that negatively regulate generation of endogenous short interfering RNA (endo-siRNA) in Caenorhabditis elegans and identified a suppressor of siRNA (susi-1) and antisense ribosomal siRNAs (risiRNAs). risiRNAs show sequence complementary to 18S and 26S rRNAs and require RNA-dependent RNA polymerases (RdRPs) for their production. They act through the nuclear RNA interference (RNAi) pathway to downregulate pre-rRNA. Stress stimuli, including low temperature and UV irradiation, induced the accumulation of risiRNAs. SUSI-1 is a homolog of the human DIS3L2 exonuclease involved in 3′–5′ degradation of oligouridylated RNAs. In susi-1 mutant and in low temperature-treated animals, 3′-tail oligouridylated 26S rRNA accumulated. The injection of oligouridylated rRNA elicited nuclear accumulation of NRDE-3. Our findings identify a new subset of 22G-RNAs that regulate pre-rRNA expression and a mechanism to maintain rRNA homeostasis.

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Figure 1: Accumulation of risiRNA in the suppressor of siRNA susi-1 mutant.
Figure 2: risiRNA belongs to the 22G-RNA class.
Figure 3: Genetic requirements for risiRNA biogenesis.
Figure 4: risiRNAs downregulate expression of pre-rRNA via the Nrde pathway.
Figure 5: Lowering temperature increased expression of risiRNA.
Figure 6: SUSI-1(ceDIS3L2) suppresses risiRNA accumulation.
Figure 7: Oligouridylated 26S rRNA promotes risiRNA generation.
Figure 8: UV irradiation enhances risiRNA accumulation and triggers redistribution of NRDE-3 to the nucleus.
Figure 9: A working model for risiRNA- and NRDE-mediated pre-rRNA regulation.

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Acknowledgements

We are grateful to S. Kenney, X. Fu, B. Buckley, X. Liu, B. Dong, C. Liu and members of S.G.'s lab for their comments. We are grateful to the Caenorhabditis Genetics Center (CGC), the International C. elegans Gene Knockout Consortium and the National Bioresource Project for providing the strains. A. Fire (Stanford University) provided HT115 bacteria expressing the empty vector L4440. This work was supported by grants from the National Natural Science Foundation of China (31371323, 31671346, 91640110 and 81501329), the Fundamental Research Funds for Central Universities (WK2060190018 and WK2070000034) and KJZD-EW-L01-2 to S.G.

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  1. Xufei Zhou and Xuezhu Feng: These authors contributed equally to this work.

Authors and Affiliations

  1. School of Life Sciences, CAS Center for Excellence in Molecular Cell Science, University of Science and Technology of China, Hefei, China

    Xufei Zhou, Xuezhu Feng, Hui Mao, Mu Li, Fei Xu, Kai Hu & Shouhong Guang

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Contributions

X.Z. constructed the transgenes and generated Figures 3,4,5,6,7, Supplementary Figures 3, 4 and 6 and Supplementary Tables 1 and 3. X.F. conducted the genetic screening, identified risiRNA, mapped susi-1 and contributed to Figures 1, 2 and 8, Supplementary Figures 2, 5, 7 and 8 and Supplementary Table 2. H.M. contributed to Figure 8 and Supplementary Figure 8. M.L., F.X. and K.H. contributed to Figure 3b,e. X.Z., X.F. and S.G. designed the project and wrote the manuscript.

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Correspondence to Shouhong Guang.

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Integrated supplementary information

Supplementary Figure 1 Antisense ribosomal siRNAs were enriched in susi-1 mutant.

(A) Deep sequencing of small RNAs in wild-type N2 and susi-1(R457H) mutant animals. The relative abundance of rRNA-related sequences is indicated. (B) Size distribution and 5'-end nucleotide preference of sense rRNA reads in wild-type N2 and susi-1(R457H) animals. (C) Pie charts display the proportion of reads aligning to each genomic feature. The reads corresponding to sense rRNA sequences were excluded from the analysis. (D) Relative abundance of endogenous smalls from wild-type N2 and susi-1(R457H) animals. Source data (for panels B-C) are available on-line.

Source data

Supplementary Figure 2 risiRNA belongs to 22G-RNAs.

(A) Relative abundance of Argonaute-associated risiRNAs from published data sets. (B) risiRNA was pre-treated with or without calf intestinal alkaline phosphatase (CIAP), followed by p32 labeling. Uncropped gel image is shown in Supplementary Data Set 2. (C) risiRNA was pre-treated with guanylyl transferase followed by p32 labeling. Uncropped gel image is shown in Supplementary Data Set 2. (D) risiRNA was pre-labeled with p32 followed by β-elimination reactions. Uncropped gel image is shown in Supplementary Data Set 2. (E) Relative abundance of Argonaute-associated small RNAs from published data sets. The blue dashed lines represent risiRNA. Source data (for panels A, E) are available on-line.

Source data

Supplementary Figure 3 risiRNA silences pre-rRNA through the nuclear RNAi pathway.

(A) Images of C. elegans' embryos expressing risiRNA sensor after feeding exogenous dsRNA targeting gfp and 26S rRNA. (B) Brood size of indicated animals at 20°C. (C) NRDE-3-associated RNAs in the indicated animals were immunoprecipitated and quantified by qRT-PCR. Ratios are presented as +/- exogenous dsRNA. mean ± s.d. n=3 independent animals. Source data (for panels B-C) are available on-line.

Source data

Supplementary Figure 4 Low temperature upregulates risiRNA expression.

(A) Brood size of the indicated animals at different temperatures. (B, C) Total RNA samples were collected from bleached embryos of the indicated genotypes. The abundance of risiRNA was quantified by Taqman qRT-PCR and is shown relative to levels of wild-type animals at 20°C. mean ± s.d. n=3 independent animals. *p<0.05, **p<0.01, ***p<0.001, NS, not significant. two-tailed student t-test. (D) Images of representative seam cells of the indicated animals expressing GFP::NRDE-3. The percentage of nuclear localized NRDE-3 is quantified at the right panel. Source data (for panels A-D) are available on-line.

Source data

Supplementary Figure 5 Lowering temperature triggers risiRNA generation.

(A) Relative abundance of small RNAs in wild-type N2 animals at different temperatures. The blue dashed lines represent risiRNA. (B) Relative abundance of NRDE-3-associated small RNAs at different temperatures. NRDE-3-associated small RNAs at 20°C have been deep sequenced previously. The blue dashed lines represent risiRNA. (C) Relative abundance of NRDE-3-associated small RNAs at different temperatures in eri-1(mg366);gfp::nrde-3 animals. The small RNA deep sequencing data of NRDE-3 immunoprecipitation in eri-1(mg366);dpy-13(e458);dpy-13(RNAi); gfp::nrde-3 animals were re-analyzed here. The blue dashed lines represent risiRNA. Source data (for panels A-C) are available on-line.

Source data

Supplementary Figure 6 SUSI-1 localized to the cytoplasm and was required for fertility.

(A) Images of representative seam cells of indicated animals. susi-1p::mCherry::SUSI-1 rescued the nuclear localization of NRDE-3. (B) Images of representative seam cells of indicated animals expressing GFP::SUSI-1 and its variants. (C) Synergistic fertility defects in eri-1(mg366);susi-1 double mutants. (D) Synergistic embryonic lethality in eri-1(mg366);susi-1 double mutants. The number of counted embryos are shown above each column. Source data (for panels C-D) are available on-line.

Source data

Supplementary Figure 7 TAIL-seq analysis identified nontemplated addition of single nucleotide at the 3' ends of 26S rRNA.

(A, B) Tail-seq of 26S rRNA and the comparison of 3'-end untemplated addition of single nucleotide. Total RNA of indicated animals were isolated from bleached embryos and subjected to Tail-seq assay. The sense 26S rRNA reads were compared to annotated 26S rRNA sequences of WS250 transcriptome assembly. Source data (for panels A-B) are available on-line.

Source data

Supplementary Figure 8 UV irradiation elicits the translocation of NRDE-3 to the nucleus and stimulates risiRNA generation.

(A) Bleached embryos of eri-1(mg366);gfp::nrde-3 animals were exposed to 50 mJ/cm2 UV and the percentage of nuclear localized NRDE-3 was scored at indicated times post irradiation. (B) Bleached embryos of eri-1(mg366);gfp::nrde-3;mCherry::fib-1 animals were exposed to 50 mJ/cm2 UV irradiation and the subcellular localization of GFP::NRDE-3 and mCherry::FIB-1 were visualized. white arrows, nucleoli; red triangle, nucleus. (C) NRDE-3-associated small RNAs were immunoprecipitated and risiRNAs were quantified by Taqman qRT-PCR. mean ± s.d. n=3 independent animals. 18S and 26S, risiRNA sequences; 21UR-1 and 21UR-5045, piRNA sequences; e01g4.5 #1 and #2, endo-siRNA sequences. Source data (for panels C-D) are available on-line. (D) Total RNAs were isolated from bleached embryos after UV irradiation and the abundance of risiRNAs was quantified by qRT-PCR. mean ± s.d. n=3 independent animals.

Source data

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 1–5 (PDF 1879 kb)

Supplementary Data Set 1

NRDE-3 reassociates with siRNAs in susi-1 mutant. (PDF 1120 kb)

Supplementary Data Set 2

NRDE-3 reassociates with siRNAs after 50 mJ/cm2 UV irradiation. (PDF 1980 kb)

Supplementary Data Set 3

Biochemical analysis of risiRNA. (PDF 1135 kb)

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Zhou, X., Feng, X., Mao, H. et al. RdRP-synthesized antisense ribosomal siRNAs silence pre-rRNA via the nuclear RNAi pathway. Nat Struct Mol Biol 24, 258–269 (2017). https://doi.org/10.1038/nsmb.3376

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