#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein


Dicer has first been described as the enzyme dedicated to the generation of small RNA fragments from long double stranded RNA. This function of Dicer of playing a central role in the microRNA and short-interfering RNA biogenesis pathways was found to be taking place in the cytoplasm of the cell. However, recent studies reported additional functions of Dicer in the nucleus of human and mouse cell lines, where the protein was proposed to be involved in processing nuclear RNAs as well as in influencing the chromatin state. Consequently, the localization of Dicer within the cell has been highly debated ever since. In this study, we show by biochemical and microscopy techniques that Dicer is only localized in the cytoplasm of embryonic and adult tissues of the mouse. We also exclude the possibility that Dicer only transiently enters the cell nucleus. Our data indicate that nuclear RNA processing is not a conserved feature of mammalian Dicer and highlight the fact that findings from in vitro experiments in cell lines do not always predict the in vivo state within a living organism.


Vyšlo v časopise: Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein. PLoS Genet 12(6): e32767. doi:10.1371/journal.pgen.1006095
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1006095

Souhrn

Dicer has first been described as the enzyme dedicated to the generation of small RNA fragments from long double stranded RNA. This function of Dicer of playing a central role in the microRNA and short-interfering RNA biogenesis pathways was found to be taking place in the cytoplasm of the cell. However, recent studies reported additional functions of Dicer in the nucleus of human and mouse cell lines, where the protein was proposed to be involved in processing nuclear RNAs as well as in influencing the chromatin state. Consequently, the localization of Dicer within the cell has been highly debated ever since. In this study, we show by biochemical and microscopy techniques that Dicer is only localized in the cytoplasm of embryonic and adult tissues of the mouse. We also exclude the possibility that Dicer only transiently enters the cell nucleus. Our data indicate that nuclear RNA processing is not a conserved feature of mammalian Dicer and highlight the fact that findings from in vitro experiments in cell lines do not always predict the in vivo state within a living organism.


Zdroje

1. Cerutti H, Casas-Mollano JA (2006) On the origin and functions of RNA-mediated silencing: from protists to man. Curr Genet 50: 81–99. 16691418

2. O'Carroll D, Schaefer A (2013) General principals of miRNA biogenesis and regulation in the brain. Neuropsychopharmacology 38: 39–54. doi: 10.1038/npp.2012.87 22669168

3. Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, et al. (2002) Prediction of plant microRNA targets. Cell 110: 513–520. 12202040

4. Tang G, Reinhart BJ, Bartel DP, Zamore PD (2003) A biochemical framework for RNA silencing in plants. Genes Dev 17: 49–63. 12514099

5. Guo H, Ingolia NT, Weissman JS, Bartel DP (2010) Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466: 835–840. doi: 10.1038/nature09267 20703300

6. Wu L, Fan J, Belasco JG (2006) MicroRNAs direct rapid deadenylation of mRNA. Proc Natl Acad Sci U S A 103: 4034–4039. 16495412

7. Doench JG, Sharp PA (2004) Specificity of microRNA target selection in translational repression. Genes Dev 18: 504–511. 15014042

8. Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, et al. (2008) Widespread changes in protein synthesis induced by microRNAs. Nature 455: 58–63. doi: 10.1038/nature07228 18668040

9. Baek D, Villen J, Shin C, Camargo FD, Gygi SP, et al. (2008) The impact of microRNAs on protein output. Nature 455: 64–71. doi: 10.1038/nature07242 18668037

10. Giraldez AJ, Mishima Y, Rihel J, Grocock RJ, Van Dongen S, et al. (2006) Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science 312: 75–79. 16484454

11. Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101: 25–33. 10778853

12. Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, et al. (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305: 1437–1441. 15284456

13. Verdel A, Jia S, Gerber S, Sugiyama T, Gygi S, et al. (2004) RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303: 672–676. 14704433

14. Baulcombe D (2004) RNA silencing in plants. Nature 431: 356–363. 15372043

15. Tam OH, Aravin AA, Stein P, Girard A, Murchison EP, et al. (2008) Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 453: 534–538. doi: 10.1038/nature06904 18404147

16. Watanabe T, Totoki Y, Toyoda A, Kaneda M, Kuramochi-Miyagawa S, et al. (2008) Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453: 539–543. doi: 10.1038/nature06908 18404146

17. Li Y, Lu J, Han Y, Fan X, Ding SW (2013) RNA interference functions as an antiviral immunity mechanism in mammals. Science 342: 231–234. doi: 10.1126/science.1241911 24115437

18. Maillard PV, Ciaudo C, Marchais A, Li Y, Jay F, et al. (2013) Antiviral RNA interference in mammalian cells. Science 342: 235–238. doi: 10.1126/science.1241930 24115438

19. Elbashir SM, Lendeckel W, Tuschl T (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15: 188–200. 11157775

20. Kim VN (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol 6: 376–385. 15852042

21. Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409: 363–366. 11201747

22. Kurzynska-Kokorniak A, Koralewska N, Pokornowska M, Urbanowicz A, Tworak A, et al. (2015) The many faces of Dicer: the complexity of the mechanisms regulating Dicer gene expression and enzyme activities. Nucleic Acids Res 43: 4365–4380. doi: 10.1093/nar/gkv328 25883138

23. Rybak-Wolf A, Jens M, Murakawa Y, Herzog M, Landthaler M, et al. (2014) A variety of dicer substrates in human and C. elegans. Cell 159: 1153–1167. doi: 10.1016/j.cell.2014.10.040 25416952

24. Hellwig S, Bass BL (2008) A starvation-induced noncoding RNA modulates expression of Dicer-regulated genes. Proc Natl Acad Sci U S A 105: 12897–12902. doi: 10.1073/pnas.0805118105 18723671

25. Billy E, Brondani V, Zhang H, Muller U, Filipowicz W (2001) Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc Natl Acad Sci U S A 98: 14428–14433. 11724966

26. Provost P, Dishart D, Doucet J, Frendewey D, Samuelsson B, et al. (2002) Ribonuclease activity and RNA binding of recombinant human Dicer. EMBO J 21: 5864–5874. 12411504

27. Gullerova M, Proudfoot NJ (2012) Convergent transcription induces transcriptional gene silencing in fission yeast and mammalian cells. Nat Struct Mol Biol 19: 1193–1201. doi: 10.1038/nsmb.2392 23022730

28. Ando Y, Tomaru Y, Morinaga A, Burroughs AM, Kawaji H, et al. (2011) Nuclear pore complex protein mediated nuclear localization of dicer protein in human cells. PLoS One 6: e23385. doi: 10.1371/journal.pone.0023385 21858095

29. Sinkkonen L, Hugenschmidt T, Filipowicz W, Svoboda P (2010) Dicer is associated with ribosomal DNA chromatin in mammalian cells. PLoS One 5: e12175. doi: 10.1371/journal.pone.0012175 20730047

30. White E, Schlackow M, Kamieniarz-Gdula K, Proudfoot NJ, Gullerova M (2014) Human nuclear Dicer restricts the deleterious accumulation of endogenous double-stranded RNA. Nat Struct Mol Biol 21: 552–559. doi: 10.1038/nsmb.2827 24814348

31. Skourti-Stathaki K, Kamieniarz-Gdula K, Proudfoot NJ (2014) R-loops induce repressive chromatin marks over mammalian gene terminators. Nature 516: 436–439. doi: 10.1038/nature13787 25296254

32. Neve J, Burger K, Li W, Hoque M, Patel R, et al. (2016) Subcellular RNA profiling links splicing and nuclear DICER1 to alternative cleavage and polyadenylation. Genome Res 26: 24–35. doi: 10.1101/gr.193995.115 26546131

33. Doyle M, Badertscher L, Jaskiewicz L, Guttinger S, Jurado S, et al. (2013) The double-stranded RNA binding domain of human Dicer functions as a nuclear localization signal. RNA 19: 1238–1252. doi: 10.1261/rna.039255.113 23882114

34. Ohrt T, Muetze J, Svoboda P, Schwille P (2012) Intracellular localization and routing of miRNA and RNAi pathway components. Curr Top Med Chem 12: 79–88. 22196276

35. Gagnon KT, Li L, Chu Y, Janowski BA, Corey DR (2014) RNAi factors are present and active in human cell nuclei. Cell Rep 6: 211–221. doi: 10.1016/j.celrep.2013.12.013 24388755

36. Matsui M, Li L, Janowski BA, Corey DR (2015) Reduced Expression of Argonaute 1, Argonaute 2, and TRBP Changes Levels and Intracellular Distribution of RNAi Factors. Sci Rep 5: 12855. doi: 10.1038/srep12855 26242502

37. Flemr M, Malik R, Franke V, Nejepinska J, Sedlacek R, et al. (2013) A retrotransposon-driven dicer isoform directs endogenous small interfering RNA production in mouse oocytes. Cell 155: 807–816. doi: 10.1016/j.cell.2013.10.001 24209619

38. Comazzetto S, Di Giacomo M, Rasmussen KD, Much C, Azzi C, et al. (2014) Oligoasthenoteratozoospermia and infertility in mice deficient for miR-34b/c and miR-449 loci. PLoS Genet 10: e1004597. doi: 10.1371/journal.pgen.1004597 25329700

39. Chendrimada TP, Gregory RI, Kumaraswamy E, Norman J, Cooch N, et al. (2005) TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436: 740–744. 15973356

40. Yang J, Bardes ES, Moore JD, Brennan J, Powers MA, et al. (1998) Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1. Genes Dev 12: 2131–2143. 9679058

41. Drake M, Furuta T, Suen KM, Gonzalez G, Liu B, et al. (2014) A requirement for ERK-dependent Dicer phosphorylation in coordinating oocyte-to-embryo transition in C. elegans. Dev Cell 31: 614–628. doi: 10.1016/j.devcel.2014.11.004 25490268

42. Francia S, Michelini F, Saxena A, Tang D, de Hoon M, et al. (2012) Site-specific DICER and DROSHA RNA products control the DNA-damage response. Nature 488: 231–235. doi: 10.1038/nature11179 22722852

43. Wisniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6: 359–362. doi: 10.1038/nmeth.1322 19377485

44. Rappsilber J, Ishihama Y, Mann M (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem 75: 663–670. 12585499

45. Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, et al. (2014) Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics 13: 2513–2526. doi: 10.1074/mcp.M113.031591 24942700

46. Hubner NC, Bird AW, Cox J, Splettstoesser B, Bandilla P, et al. (2010) Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions. J Cell Biol 189: 739–754. doi: 10.1083/jcb.200911091 20479470

47. Pelechano V, Wei W, Steinmetz LM (2016) Genome-wide quantification of 5'-phosphorylated mRNA degradation intermediates for analysis of ribosome dynamics. Nat Protoc 11: 359–376. doi: 10.1038/nprot.2016.026 26820793

Štítky
Genetika Reprodukčná medicína
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#