Hierarchical roles of mitochondrial Papi and Zucchini in Bombyx germline piRNA biogenesis

Subjects

Abstract

PIWI-interacting RNAs (piRNAs) are small regulatory RNAs that bind to PIWI proteins to control transposons and maintain genome integrity in animal germ lines1,2,3. piRNA 3′ end formation in the silkworm Bombyx mori has been shown to be mediated by the 3′-to-5′ exonuclease Trimmer (Trim; known as PNLDC1 in mammals)4, and piRNA intermediates are bound with PIWI anchored onto mitochondrial Tudor domain protein Papi5. However, it remains unclear whether the Zucchini (Zuc) endonuclease and Nibbler (Nbr) 3′-to-5′ exonuclease, both of which have pivotal roles in piRNA biogenesis in Drosophila6,7,8, are required for piRNA processing in other species. Here we show that the loss of Zuc in Bombyx had no effect on the levels of Trim and Nbr, but resulted in the aberrant accumulation of piRNA intermediates within the Papi complex, and that these were processed to form mature piRNAs by recombinant Zuc. Papi exerted its RNA-binding activity only when bound with PIWI and phosphorylated, suggesting that complex assembly involves a hierarchical process. Both the 5′ and 3′ ends of piRNA intermediates within the Papi complex showed hallmarks of PIWI ‘slicer’ activity, yet no phasing pattern was observed in mature piRNAs. The loss of Zuc did not affect the 5′- and 3′-end formation of the intermediates, strongly supporting the idea that the 5′ end of Bombyx piRNA is formed by PIWI slicer activity, but independently of Zuc, whereas the 3′ end is formed by the Zuc endonuclease. The Bombyx piRNA biogenesis machinery is simpler than that of Drosophila, because Bombyx has no transcriptional silencing machinery that relies on phased piRNAs.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Papi is essential for piRNA biogenesis and piRISC formation in BmN4 cells.
Figure 2: Zuc is essential for piRNA biogenesis and piRISC formation in BmN4 cells.
Figure 3: Papi-associated int-piRNAs are generated by Siwi and Ago3 slicer.
Figure 4: Zuc processes int-piRNAs to mature piRNAs.

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. 1

    Ghildiyal, M. & Zamore, P. D. Small silencing RNAs: an expanding universe. Nat. Rev. Genet. 10, 94–108 (2009)

  2. 2

    Iwasaki, Y. W., Siomi, M. C. & Siomi, H. PIWI-interacting RNA: its biogenesis and functions. Annu. Rev. Biochem. 84, 405–433 (2015)

  3. 3

    Czech, B. & Hannon, G. J. One loop to rule them all: the ping-pong cycle and piRNA-guided silencing. Trends Biochem. Sci. 41, 324–337 (2016)

  4. 4

    Izumi, N. et al. Identification and functional analysis of the pre-piRNA 3′ Trimmer in silkworms. Cell 164, 962–973 (2016)

  5. 5

    Honda, S. et al. Mitochondrial protein BmPAPI modulates the length of mature piRNAs. RNA 19, 1405–1418 (2013)

  6. 6

    Han, B. W., Wang, W., Li, C., Weng, Z. & Zamore, P. D. Noncoding RNA. piRNA-guided transposon cleavage initiates Zucchini-dependent, phased piRNA production. Science 348, 817–821 (2015)

  7. 7

    Mohn, F., Handler, D. & Brennecke, J. Noncoding RNA. piRNA-guided slicing specifies transcripts for Zucchini-dependent, phased piRNA biogenesis. Science 348, 812–817 (2015)

  8. 8

    Hayashi, R. et al. Genetic and mechanistic diversity of piRNA 3′-end formation. Nature 539, 588–592 (2016)

  9. 9

    Brennecke, J. et al. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128, 1089–1103 (2007)

  10. 10

    Gunawardane, L. S. et al. A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila. Science 315, 1587–1590 (2007)

  11. 11

    Homolka, D. et al. PIWI slicing and RNA elements in precursors instruct directional primary piRNA biogenesis. Cell Reports 12, 418–428 (2015)

  12. 12

    Saito, K. et al. A regulatory circuit for piwi by the large Maf gene traffic jam in Drosophila. Nature 461, 1296–1299 (2009)

  13. 13

    Kawaoka, S. et al. The Bombyx ovary-derived cell line endogenously expresses PIWI/PIWI-interacting RNA complexes. RNA 15, 1258–1264 (2009)

  14. 14

    Nishida, K. M. et al. Respective functions of two distinct Siwi complexes assembled during PIWI-interacting RNA biogenesis in Bombyx germ cells. Cell Reports 10, 193–203 (2015)

  15. 15

    Xiol, J. et al. A role for Fkbp6 and the chaperone machinery in piRNA amplification and transposon silencing. Mol. Cell 47, 970–979 (2012)

  16. 16

    Liu, L., Qi, H., Wang, J. & Lin, H. PAPI, a novel TUDOR-domain protein, complexes with AGO3, ME31B and TRAL in the nuage to silence transposition. Development 138, 1863–1873 (2011)

  17. 17

    Ipsaro, J. J., Haase, A. D., Knott, S. R., Joshua-Tor, L. & Hannon, G. J. The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis. Nature 491, 279–283 (2012)

  18. 18

    Nishimasu, H. et al. Structure and function of Zucchini endoribonuclease in piRNA biogenesis. Nature 491, 284–287 (2012)

  19. 19

    Watanabe, T. et al. MITOPLD is a mitochondrial protein essential for nuage formation and piRNA biogenesis in the mouse germline. Dev. Cell 20, 364–375 (2011)

  20. 20

    Feltzin, V. L. et al. The exonuclease Nibbler regulates age-associated traits and modulates piRNA length in Drosophila. Aging Cell 14, 443–452 (2015)

  21. 21

    Horwich, M. D. et al. The Drosophila RNA methyltransferase, DmHen1, modifies germline piRNAs and single-stranded siRNAs in RISC. Curr. Biol. 17, 1265–1272 (2007)

  22. 22

    Saito, K. et al. Pimet, the Drosophila homolog of HEN1, mediates 2′-O-methylation of Piwi- interacting RNAs at their 3′ ends. Genes Dev. 21, 1603–1608 (2007)

  23. 23

    Eddy, E. M. Germ plasm and the differentiation of the germ cell line. Int. Rev. Cytol. 43, 229–280 (1975)

  24. 24

    Nicastro, G., Taylor, I. A. & Ramos, A. KH-RNA interactions: back in the groove. Curr. Opin. Struct. Biol. 30, 63–70 (2015)

  25. 25

    Shoji, K., Suzuki, Y., Sugano, S., Shimada, T. & Katsuma, S. Artificial “ping-pong” cascade of PIWI-interacting RNA in silkworm cells. RNA 23, 86–97 (2017)

  26. 26

    Matsumoto, N. et al. Crystal structure of silkworm PIWI-clade Argonaute Siwi bound to piRNA. Cell 167, 484–497.e9 (2016)

  27. 27

    Saxe, J. P., Chen, M., Zhao, H. & Lin, H. Tdrkh is essential for spermatogenesis and participates in primary piRNA biogenesis in the germline. EMBO J. 32, 1869–1885 (2013)

  28. 28

    Ding, D. et al. PNLDC1 is essential for piRNA 3′ end trimming and transposon silencing during spermatogenesis in mice. Nat. Commun. 8, 819 (2017)

  29. 29

    Handler, D. et al. A systematic analysis of Drosophila TUDOR domain-containing proteins identifies Vreteno and the Tdrd12 family as essential primary piRNA pathway factors. EMBO J. 30, 3977–3993 (2011)

  30. 30

    Sumiyoshi, T. et al. Loss of l(3)mbt leads to acquisition of the ping-pong cycle in Drosophila ovarian somatic cells. Genes Dev. 30, 1617–1622 (2016)

  31. 31

    Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)

  32. 32

    Saldanha, A. J. Java Treeviewextensible visualization of microarray data. Bioinformatics 20, 3246–3248 (2004)

  33. 33

    Fujinoki, M. et al. Identification of 36-kDa flagellar phosphoproteins associated with hamster sperm motility. J. Biochem. 133, 361–369 (2003)

  34. 34

    Fujii, K. et al. Fully automated online multi-dimensional protein profiling system for complex mixtures. J. Chromatogr. A 1057, 107–113 (2004)

  35. 35

    Keller, B. O., Wang, Z. & Li, L. Low-mass proteome analysis based on liquid chromatography fractionation, nanoliter protein concentration/digestion, and microspot matrix-assisted laser desorption ionization mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 782, 317–329 (2002)

  36. 36

    Murota, Y. et al. Yb integrates piRNA intermediates and processing factors into perinuclear bodies to enhance piRISC assembly. Cell Rep. 8, 103–113 (2014)

  37. 37

    Wieckowski, M.R., Giorgi, C., Lebiedzinska, M., Duszynski, J., & Pinton, P. Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells. Nat. Protocols 4, 1582–1590 (2009)

Download references

Acknowledgements

We are grateful to T. Mannen for preparing materials for mass spectrometry, T. Suzuki for comments on our in vitro Zuc processing assays and Y. Ono for support with the bioinformatics. We also thank S. Ohnishi for technical assistance and other members of the Siomi laboratories for discussions and comments on the manuscript. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan to K.M.N., Y.W.I., Y.M., H.S. and M.C.S. R.M. is supported by CREST, the Japan Science and Technology Agency. T.Ka. was supported by grants from the New Energy and Industrial Technology Development Organization, Japan, and Translational Systems Biology and Medicine Initiative from the Ministry of Education, Culture, Sports, Science and Technology of Japan. T.Ko. is a recipient of Molecular Dynamics for Antibody Drug Development, First Program Grant from the Japan Society of Promotion of Science.

Author information

Affiliations

Authors

Contributions

K.M.N. generated monoclonal antibodies and performed biochemical analyses of piRNAs, int-piRNAs and piRNA factors. K.S. carried out in vitro experiments with help from R.M. H.Y. performed protein–protein interaction analyses. Y.M. performed immunofluorescence analyses. Y.W.I. performed bioinformatics analyses. T.Ka. and T.Ko. performed LC–MS/MS analysis. M.C.S. designed the experiments with other authors, supervised and discussed the work, and wrote the manuscript. H.S. discussed and supervised the study. All authors commented on the manuscript.

Corresponding author

Correspondence to Mikiko C. Siomi.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks J. Brennecke, S. Chameyron and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Production of monoclonal antibodies against Papi, Trim and Zuc, and analysis of depletion upon RNA interference treatment.

a, Quantitative PCR with reverse transcription (qRT–PCR) shows that Nbr was efficiently depleted by RNA interference (RNAi) in BmN4 cells. Data are mean ± s.e.m. of three independent experiments. b, Western blotting shows the specificity of anti-Papi, anti-Trim and anti-Zuc monoclonal antibodies raised in this study. HSP60 and tubulin were used as loading controls. The images show the domain structures of Papi, Trim and Zuc. Underlines indicate the antigen regions used for producing the monoclonal antibodies. c, Western blotting shows that Papi and Trim were efficiently depleted by RNAi in BmN4 cells.

Extended Data Figure 2 Siwi/Ago3-associated small RNAs upon Trim or Nbr depletion.

a, Length distribution of transposon-mapped Flag-Siwi- and Flag-Ago3-associated piRNAs. piRNAs appear to be slightly longer when Trim was depleted. b, Sequence logos showing unaffected levels of 1U and 10A under Trim- or Nbr-depleted conditions. c, Strand bias and frequency of piRNAs mapped to each transposon consensus sequence. Depletion of Trim or Nbr has little effect on strand bias or the frequency of piRNAs mapped to each transposon consensus sequence.

Extended Data Figure 3 sDMA modification of Siwi and Ago3.

a, A synthesized short interfering RNA (siRNA) (26 nucleotides) was downshifted by β-elimination, indicating that this siRNA is not 2′-O-methylated. b, The amino acid sequences of the N-terminal regions of wild-type Siwi, the Siwi-9RK mutant, wild-type Ago3 and the Ago3-5RK mutant are shown. Arginine residues shown in red were determined to be sDMAs in BmN4 cells. Arginine residues mutated to lysines are shown in green. c, Representative ETD tandem mass spectra for Siwi and Ago3 peptides, which include arginine modifications. Ac, acetylation; Di, demethylation; Me, monomethylation. Charge, m/z and Mascot score are shown on the top right of each spectrum. All identified Siwi and Ago3 peptides are listed in Supplementary Table 1.

Extended Data Figure 4 Analysis of Siwi and Ago3 mutants.

a, Wild-type Flag–Siwi and Flag–Ago3, but not Flag–Siwi-9RK and Flag-Ago3-5RK mutants, are co-immunoprecipitated with Papi from BmN4 cells. b, Wild-type Flag–Siwi and Flag–Ago3, but not Flag–Siwi-9RK and Flag–Ago3-5RK mutants, are loaded with piRNAs in BmN4 cells. The middle (sDMA) shows that neither the Flag–Siwi-9RK nor Flag–Ago3-5RK mutant reacts with the Y12 antibody, which specifically recognizes sDMA. c, Wild-type Flag–Siwi and Flag–Ago3, but not Flag–Siwi-9RK and Flag–Ago3-5RK mutants, are localized to nuage in BmN4 cells (shown in green). Blue (DAPI staining) indicates the location of the nucleus. Scale bars, 10 μm. d, Papi depletion has little effect on sDMA modification of Flag–Siwi and Flag–Ago3 expressed in BmN4 cells.

Extended Data Figure 5 Papi complex analysis.

a, Top, Flag–Siwi and Flag–Ago3 expressed in BmN4 cells were immunoisolated with anti-Flag antibody and probed with anti-Flag antibody after sequential dilution. Bottom, Flag–Siwi and Flag–Ago3 immunoisolated from BmN4 cells (the same samples as in the top panel) were probed with anti-Siwi and anti-Ago3 antibodies, respectively. Siwi and Ago3 co-immunoprecipitated with Papi were simultaneously probed with anti-Siwi and anti-Ago3 antibodies, respectively. The Papi complex was equally divided into two fractions and each fraction was used for each blot. Examination of the signal intensity revealed that the amount of Siwi within the Papi complex was approximately equal to 1/1.6 volume of Flag–Siwi and that the amount of Ago3 within the Papi complex was approximately equal to 1/16 volume of Flag–Ago3. Comparison of the signal intensity on the top and bottom blots suggests that the ratio of abundance of Siwi and Ago3 in the Papi complex is 10:1. b, Northern blotting shows that the Papi complex contains RT3-1 int-piRNAs. c, Northern blotting shows that Siwi in a form associated with Papi on mitochondria binds RT3-1 int-piRNAs independently of Papi. The Siwi–int-piRNA association is maintained even after Zuc depletion. d, CLIP analysis shows that only the long form, but not the short form, of endogenous Papi in BmN4 cells interacts with RNA in vivo. e, Western blotting using anti-Papi (top) and anti-Flag (second from the top) antibodies shows that wild-type Papi–Flag and the KH mutant are equally expressed in BmN4 cells, in which endogenous Papi has been depleted by RNAi. Western blotting using anti-Myc (third from the top) shows that the levels of Myc–Siwi are approximately equal in the cells. Tubulin was used as a loading control (bottom). Both wild-type Papi–Flag and the KH mutant were mutated to be RNAi resistant. f, Flag–Siwi-9RK and Flag–Ago3-5RK mutants bind with little int-piRNA. g, Flag–Siwi binds with little int-piRNA in Papi-lacking BmN4 cells. h, Northern blotting shows that int-piRNAs are still present in Siwi-depleted BmN4 cells.

Extended Data Figure 6 RNAi efficiency, Siwi–Papi–Zuc interaction and analysis of Papi-associated intermediates and piRNA phasing.

a, Western blotting shows that Zuc is efficiently depleted by RNAi. HSP60 is used as a loading control. b, Western blotting shows that Zuc and Trim are efficiently depleted by RNAi. Zuc and Trim depletion had little effect on the protein levels of Trim and Zuc, respectively. HSP60 is shown as a loading control. c, qRT–PCR shows that the level of Nbr is not affected by Zuc depletion in BmN4 cells. Data are mean ± s.e.m. of three independent experiments. d, Zuc and Papi are detected in the Siwi complex immunoisolated from the mitochondrial fraction of BmN4 cells. e, The size distribution of Papi-associated intermediates mapped to transposons. f, Analyses of phased piRNAs in Papi-associated intermediates. The distance between the 3′ end of the upstream piRNA and the 5′ end of the downstream piRNA on the same genomic strand is analysed.

Extended Data Figure 7 Analyses of Zuc RNA cleavage.

a, Coomassie brilliant blue (CBB)-stained gel showing purified wild-type Zuc and the Zuc(H141A) mutant. b, Wild-type Zuc in a cleaves 1U50 in a dose-dependent manner. c, Detailed analyses of Zuc RNA cleavage. The left gel shows RNA ladders ranging from 14 to 50 nucleotides. The right gel shows RNA ladders ranging from 7 to 15 nucleotides. Relatively ‘strong’ RNA bands are indicated by red arrowheads (s1–s7). ‘Intermediate’ RNA bands are indicated by blue arrowheads (i1–i11). Relatively ‘weak’ RNA bands are indicated by grey arrowheads (w1–w7). 1U29 is an authentic RNA, the sequence of which is identical to that of 1U50 RNA over 1–29 nucleotides from the 5′ end. Classification of strong, intermediate and weak RNA bands is carried out in accordance with the intensity of each band. d, An 80-nucleotide RNA, 1U80, is cleaved by wild-type Zuc. e, Cleavage patterns of 1U50 and 1U80 by wild-type Zuc are compared. f, CBB-stained gel showing purified Flag–Siwi. g, 1U80 pre-loaded onto Flag-Siwi in f is cleaved by wild-type Zuc. h, The Siwi–1U80 RNA complex was first incubated with Papi, which was immunopurified using an anti-Papi antibody, and then treated with wild-type Zuc.

Extended Data Figure 8 A new model for piRNA biogenesis in Bombyx.

a, A model for the ping-pong cycle in Bombyx. Papi is localized on the surface of mitochondria through MLS, whereas KH domains are required for Papi to exhibit RNA-binding activity. The Papi Tudor domain and sDMA modification of Siwi and Ago3 are required for the Siwi–Papi and Ago3–Papi interactions. It remains unclear how Papi is maintained in an RNA-free state before the Papi–PIWI association. Also, it remains unclear how piRISC upon its formation is displaced from Papi, and how piRISC avoids re-association with Papi. Conformational change of piRISC may be involved. b, Drosophila phased piRNA biogenesis involves Zuc–Zuc endonucleolytic cleavage for piRNA 5′ and 3′ end formation. PIWI-slicer-Nbr exonucleolytic trimming and PIWI-slicer-Zuc endonucleolytic cleavage produce piRNAs in the ping-pong cycle. The role of Drosophila Papi remains under discussion. Its functional homologue(s) (shown as X and Y) may function with Zuc and Nbr. Bombyx lacks a gene homologue of Drosophila Piwi, so it does not have to accommodate phased piRNA biogenesis. Because of this, Zuc endonuclease might not be used for piRNA 5′ end formation. However, PIWI-slicer-Zuc endonucleolytic cleavage produces piRNAs in the ping-pong cycle. We infer that the 3′-to-5′ exonuclease cannot trim the 3′ end of the intermediate because Papi impedes this reaction. This model shows that Bombyx piRNAs are produced in a manner that depends on PIWI-slicer and Zuc. There may also be alternative pathways that have less of an effect on the overall levels of piRNA production.

Supplementary information

Life Sciences Reporting Summary (PDF 92 kb)

Supplementary Figures

This file contains uncropped gel data for Supplementary Figure 1 and Extended Data Figures. (PDF 1575 kb)

Supplementary Table 1

This file contains all identified Siwi and Ago3 peptides with arginine modifications. (XLSX 15 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Nishida, K., Sakakibara, K., Iwasaki, Y. et al. Hierarchical roles of mitochondrial Papi and Zucchini in Bombyx germline piRNA biogenesis. Nature 555, 260–264 (2018). https://doi.org/10.1038/nature25788

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.