RNAi is the sequence-specific mRNA degradation guided by siRNAs produced from long dsRNA by RNase Dicer. Proteins executing RNAi are present in mammalian cells but rather sustain the microRNA pathway. Aiming for a systematic analysis of mammalian RNAi, we report here that the main bottleneck for RNAi efficiency is the production of functional siRNAs, which integrates Dicer activity, dsRNA structure, and siRNA targeting efficiency. Unexpectedly, increased expression of Dicer cofactors TARBP2 or PACT reduces RNAi but not microRNA function. Elimination of protein kinase R, a key dsRNA sensor in the interferon response, had minimal positive effects on RNAi activity in fibroblasts. Without high Dicer activity, RNAi can still occur when the initial Dicer cleavage of the substrate yields an efficient siRNA. Efficient mammalian RNAi may use substrates with some features of microRNA precursors, merging both pathways even more than previously suggested. Although optimized endogenous Dicer substrates mimicking miRNA features could evolve for endogenous regulations, the same principles would make antiviral RNAi inefficient as viruses would adapt to avoid efficacy.

Bioinformatics Group Division of Molecular Biology Department of Biology Faculty of Science University of Zagreb Zagreb Croatia

Institute of Molecular Genetics Academy of Sciences of the Czech Republic Prague Czech Republic

Zobrazit více v PubMed

Aken BL, Achuthan P, Akanni W, Amode MR, Bernsdorff F, Bhai J, Billis K, Carvalho-Silva D, Cummins C, Clapham P, et al. (2017) Ensembl 2017. Nucleic Acids Res 45: D635–D642. 10.1093/nar/gkw1104 PubMed DOI PMC

Ameres SL, Martinez J, Schroeder R (2007) Molecular basis for target RNA recognition and cleavage by human RISC. Cell 130: 101–112. 10.1016/j.cell.2007.04.037 PubMed DOI

Babiarz JE, Ruby JG, Wang Y, Bartel DP, Blelloch R (2008) Mouse ES cells express endogenous shRNAs, siRNAs, and other microprocessor-independent, Dicer-dependent small RNAs. Genes Dev 22: 2773–2785. 10.1101/gad.1705308 PubMed DOI PMC

Bartel DP. (2018) Metazoan MicroRNAs. Cell 173: 20–51. 10.1016/j.cell.2018.03.006 PubMed DOI PMC

Ben-Asouli Y, Banai Y, Pel-Or Y, Shir A, Kaempfer R (2002) Human interferon-gamma mRNA autoregulates its translation through a pseudoknot that activates the interferon-inducible protein kinase PKR. Cell 108: 221–232. 10.1016/s0092-8674(02)00616-5 PubMed DOI

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 USA 98: 14428–14433. 10.1073/pnas.261562698 PubMed DOI PMC

Cenik ES, Fukunaga R, Lu G, Dutcher R, Wang YM, Hall TMT, Zamore PD (2011) Phosphate and R2D2 restrict the substrate specificity of dicer-2, an ATP-driven ribonuclease. Mol Cell 42: 172–184. 10.1016/j.molcel.2011.03.002 PubMed DOI PMC

Chakravarthy S, Sternberg SH, Kellenberger CA, Doudna JA (2010) Substrate-specific kinetics of dicer-catalyzed RNA processing. J Mol Biol 404: 392–402. 10.1016/j.jmb.2010.09.030 PubMed DOI PMC

Chalupnikova K, Solc P, Sulimenko V, Sedlacek R, Svoboda P (2014) An oocyte-specific ELAVL2 isoform is a translational repressor ablated from meiotically competent antral oocytes. Cell Cycle 13: 1187–1200. 10.4161/cc.28107 PubMed DOI PMC

Chendrimada TP, Gregory RI, Kumaraswamy E, Norman J, Cooch N, Nishikura K, Shiekhattar R (2005) TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436: 740–744. 10.1038/nature03868 PubMed DOI PMC

Cullen BR, Cherry S, tenOever BR (2013) Is RNA interference a physiologically relevant innate antiviral immune response in mammals? Cell Host Microbe 14: 374–378. 10.1016/j.chom.2013.09.011 PubMed DOI

D'Angelo W, Gurung C, Acharya D, Chen B, Ortolano N, Gama V, Bai F, Guo YL (2017) The molecular basis for the lack of inflammatory responses in mouse embryonic stem cells and their differentiated cells. J Immunol 198: 2147–2155. 10.4049/jimmunol.1601068 PubMed DOI PMC

David M, Dzamba M, Lister D, Ilie L, Brudno M (2011) SHRiMP2: Sensitive yet practical SHort read mapping. Bioinformatics 27: 1011–1012. 10.1093/bioinformatics/btr046 PubMed DOI

Diallo M, Arenz C, Schmitz K, Sandhoff K, Schepers U (2003) Long endogenous dsRNAs can induce complete gene silencing in mammalian cells and primary cultures. Oligonucleotides 13: 381–392. 10.1089/154545703322617069 PubMed DOI

Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: Ultrafast universal RNA-seq aligner. Bioinformatics 29: 15–21. 10.1093/bioinformatics/bts635 PubMed DOI PMC

Farrell PJ, Sen GC, Dubois MF, Ratner L, Slattery E, Lengyel P (1978) Interferon action: Two distinct pathways for inhibition of protein synthesis by double-stranded RNA. Proc Natl Acad Sci USA 75: 5893–5897. 10.1073/pnas.75.12.5893 PubMed DOI PMC

Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806–811. 10.1038/35888 PubMed DOI

Flemr M, Malik R, Franke V, Nejepinska J, Sedlacek R, Vlahovicek K, Svoboda P (2013) A retrotransposon-driven dicer isoform directs endogenous small interfering RNA production in mouse oocytes. Cell 155: 807–816. 10.1016/j.cell.2013.10.001 PubMed DOI

Flemr M, Moravec M, Libova V, Sedlacek R, Svoboda P (2014) Lin28a is dormant, functional, and dispensable during mouse oocyte-to-embryo transition. Biol Reprod 90: 131 10.1095/biolreprod.114.118703 PubMed DOI

Fukunaga R, Zamore PD (2014) A universal small molecule, inorganic phosphate, restricts the substrate specificity of Dicer-2 in small RNA biogenesis. Cell Cycle (Georgetown, Tex) 13: 1671–1676. 10.4161/cc.29066 PubMed DOI PMC

Gan L, Anton KE, Masterson BA, Vincent VA, Ye S, Gonzalez-Zulueta M (2002) Specific interference with gene expression and gene function mediated by long dsRNA in neural cells. J Neurosci Methods 121: 151–157. 10.1016/s0165-0270(02)00230-3 PubMed DOI

Gantier MP. (2014) Processing of double-stranded RNA in mammalian cells: A direct antiviral role? J interferon Cytokine Res 34: 469–477. 10.1089/jir.2014.0003 PubMed DOI

Gantier MP, Baugh JA, Donnelly SC (2007) Nuclear transcription of long hairpin RNA triggers innate immune responses. J Interferon Cytokine Res 27: 789–797. 10.1089/jir.2006.0152 PubMed DOI

Geiss G, Jin G, Guo J, Bumgarner R, Katze MG, Sen GC (2001) A comprehensive view of regulation of gene expression by double-stranded RNA-mediated cell signaling. J Biol Chem 276: 30178–30182. 10.1074/jbc.c100137200 PubMed DOI

Haase AD, Jaskiewicz L, Zhang HD, Laine S, Sack R, Gatignol A, Filipowicz W (2005) TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing. EMBO Rep 6: 961–967. 10.1038/sj.embor.7400509 PubMed DOI PMC

Hampf M, Gossen M (2006) A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays. Anal Biochem 356: 94–99. 10.1016/j.ab.2006.04.046 PubMed DOI

Kaufman RJ, Davies MV, Pathak VK, Hershey JW (1989) The phosphorylation state of eucaryotic initiation factor 2 alters translational efficiency of specific mRNAs. Mol Cell Biol 9: 946–958. 10.1128/mcb.9.3.946 PubMed DOI PMC

Kennedy EM, Whisnant AW, Kornepati AV, Marshall JB, Bogerd HP, Cullen BR (2015) Production of functional small interfering RNAs by an amino-terminal deletion mutant of human Dicer. Proc Natl Acad Sci USA 112: E6945–E6954. 10.1073/pnas.1513421112 PubMed DOI PMC

Kozomara A, Griffiths-Jones S (2014) miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res 42: D68–D73. 10.1093/nar/gkt1181 PubMed DOI PMC

Kristiansen H, Gad HH, Eskildsen-Larsen S, Despres P, Hartmann R (2011) The oligoadenylate synthetase family: An ancient protein family with multiple antiviral activities. J Interferon Cytokine Res 31: 41–47. 10.1089/jir.2010.0107 PubMed DOI

Kuhn RM, Haussler D, Kent WJ (2013) The UCSC genome browser and associated tools. Brief Bioinformatics 14: 144–161. 10.1093/bib/bbs038 PubMed DOI PMC

Laraki G, Clerzius G, Daher A, Melendez-Pena C, Daniels S, Gatignol A (2008) Interactions between the double-stranded RNA-binding proteins TRBP and PACT define the Medipal domain that mediates protein-protein interactions. RNA Biol 5: 92–103. 10.4161/rna.5.2.6069 PubMed DOI

Lassig C, Hopfner KP (2017) Discrimination of cytosolic self and non-self RNA by RIG-I-like receptors. J Biol Chem 292: 9000–9009. 10.1074/jbc.r117.788398 PubMed DOI PMC

Li Y, Basavappa M, Lu J, Dong S, Cronkite DA, Prior JT, Reinecker HC, Hertzog P, Han Y, Li WX, et al. (2016) Induction and suppression of antiviral RNA interference by influenza A virus in mammalian cells. Nat Microbiol 2: 16250 10.1038/nmicrobiol.2016.250 PubMed DOI PMC

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. 10.1126/science.1241911 PubMed DOI PMC

Liddicoat BJ, Piskol R, Chalk AM, Ramaswami G, Higuchi M, Hartner JC, Li JB, Seeburg PH, Walkley CR (2015) RNA editing by ADAR1 prevents MDA5 sensing of endogenous dsRNA as nonself. Science 349: 1115–1120. 10.1126/science.aac7049 PubMed DOI PMC

Linsen SE, de Wit E, Janssens G, Heater S, Chapman L, Parkin RK, Fritz B, Wyman SK, de Bruijn E, Voest EE, et al. (2009) Limitations and possibilities of small RNA digital gene expression profiling. Nat Methods 6: 474–476. 10.1038/nmeth0709-474 PubMed DOI

Ma E, MacRae IJ, Kirsch JF, Doudna JA (2008) Autoinhibition of human dicer by its internal helicase domain. J Mol Biol 380: 237–243. 10.1016/j.jmb.2008.05.005 PubMed DOI PMC

Ma J, Flemr M, Stein P, Berninger P, Malik R, Zavolan M, Svoboda P, Schultz RM (2010) MicroRNA activity is suppressed in mouse oocytes. Curr Biol 20: 265–270. 10.1016/j.cub.2009.12.042 PubMed DOI PMC

MacRae IJ, Ma E, Zhou M, Robinson CV, Doudna JA (2008) In vitro reconstitution of the human RISC-loading complex. Proc Natl Acad Sci USA 105: 512–517. 10.1073/pnas.0710869105 PubMed DOI PMC

Maillard PV, Ciaudo C, Marchais A, Li Y, Jay F, Ding SW, Voinnet O (2013) Antiviral RNA interference in mammalian cells. Science 342: 235–238. 10.1126/science.1241930 PubMed DOI PMC

Maillard PV, Van der Veen AG, Deddouche-Grass S, Rogers NC, Merits A, Reis e Sousa C (2016) Inactivation of the type I interferon pathway reveals long double-stranded RNA-mediated RNA interference in mammalian cells. EMBO J 35: 2505–2518. 10.15252/embj.201695086 PubMed DOI PMC

Malik R, Svoboda P (2012) Transgenic RNAi in mouse oocytes: The first decade. Anim Reprod Sci 134: 64–68. 10.1016/j.anireprosci.2012.08.012 PubMed DOI

Martens JH, O'Sullivan RJ, Braunschweig U, Opravil S, Radolf M, Steinlein P, Jenuwein T (2005) The profile of repeat-associated histone lysine methylation states in the mouse epigenome. EMBO J 24: 800–812. 10.1038/sj.emboj.7600545 PubMed DOI PMC

Martin M. (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 17: 3 10.14806/ej.17.1.200 DOI

Meurs E, Chong K, Galabru J, Thomas NS, Kerr IM, Williams BR, Hovanessian AG (1990) Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell 62: 379–390. 10.1016/0092-8674(90)90374-n PubMed DOI

Morse DP, Bass BL (1999) Long RNA hairpins that contain inosine are present in Caenorhabditis elegans poly(A)(+) RNA. America 96: 6048–6053. 10.1073/pnas.96.11.6048 PubMed DOI PMC

Murchison EP, Stein P, Xuan Z, Pan H, Zhang MQ, Schultz RM, Hannon GJ (2007) Critical roles for Dicer in the female germline. Genes Dev 21: 682–693. 10.1101/gad.1521307 PubMed DOI PMC

Murphy D, Dancis B, Brown JR (2008) The evolution of core proteins involved in microRNA biogenesis. BMC Evol Biol 8: 92 10.1186/1471-2148-8-92 PubMed DOI PMC

Nejepinska J, Flemr M, Svoboda P (2012a) The canonical RNA interference pathway in animals. In Regulatory RNAs, Mallick B, Ghosh Z (eds), pp 111–149 Springer: Berlin, Heidelberg

Nejepinska J, Malik R, Filkowski J, Flemr M, Filipowicz W, Svoboda P (2012b) dsRNA expression in the mouse elicits RNAi in oocytes and low adenosine deamination in somatic cells. Nucleic Acids Res 40: 399–413. 10.1093/nar/gkr702 PubMed DOI PMC

Nejepinska J, Malik R, Moravec M, Svoboda P (2012c) Deep sequencing reveals complex spurious transcription from transiently transfected plasmids. PLoS One 7: e43283 10.1371/journal.pone.0043283 PubMed DOI PMC

Nejepinska J, Malik R, Wagner S, Svoboda P (2014) Reporters transiently transfected into mammalian cells are highly sensitive to translational repression induced by dsRNA expression. PLoS One 9: e87517 10.1371/journal.pone.0087517 PubMed DOI PMC

Nishida KM, Miyoshi K, Ogino A, Miyoshi T, Siomi H, Siomi MC (2013) Roles of R2D2, a cytoplasmic D2 body component, in the endogenous siRNA pathway in Drosophila. Mol Cell 49: 680–691. 10.1016/j.molcel.2012.12.024 PubMed DOI

Paddison PJ, Caudy AA, Hannon GJ (2002) Stable suppression of gene expression by RNAi in mammalian cells. Proc Natl Acad Sci USA 99: 1443–1448. 10.1073/pnas.032652399 PubMed DOI PMC

Pfeffer S, Sewer A, Lagos-Quintana M, Sheridan R, Sander C, Grasser FA, van Dyk LF, Ho CK, Shuman S, Chien M, et al. (2005) Identification of microRNAs of the herpesvirus family. Nat Methods 2: 269–276. 10.1038/nmeth746 PubMed DOI

Provost P, Dishart D, Doucet J, Frendewey D, Samuelsson B, Radmark O (2002) Ribonuclease activity and RNA binding of recombinant human Dicer. EMBO J 21: 5864–5874. 10.1093/emboj/cdf578 PubMed DOI PMC

Qiu Y, Xu Y, Zhang Y, Zhou H, Deng YQ, Li XF, Miao M, Zhang Q, Zhong B, Hu Y, et al. (2017) Human virus-derived small RNAs can confer antiviral immunity in mammals. Immunity 46: 992–1004 e1005. 10.1016/j.immuni.2017.05.006 PubMed DOI

Reich DP, Tyc KM, Bass BL (2018) C. elegans ADARs antagonize silencing of cellular dsRNAs by the antiviral RNAi pathway. Genes Dev 32: 271–282. 10.1101/gad.310672.117 PubMed DOI PMC

Sadler AJ, Williams BR (2007) Structure and function of the protein kinase R. Current Top Microbiol Immunol 316: 253–292. 10.1007/978-3-540-71329-6_13 PubMed DOI

Shinagawa T, Ishii S (2003) Generation of Ski-knockdown mice by expressing a long double-strand RNA from an RNA polymerase II promoter. Genes Dev 17: 1340–1345. 10.1101/gad.1073003 PubMed DOI PMC

Smalheiser NR, Lugli G, Thimmapuram J, Cook EH, Larson J (2011) Endogenous siRNAs and noncoding RNA-derived small RNAs are expressed in adult mouse hippocampus and are up-regulated in olfactory discrimination training. RNA 17: 166–181. 10.1261/rna.2123811 PubMed DOI PMC

Smit AFA, Hubley R, Green P (2013–2015) RepeatMasker Open-4.0

Stein P, Svoboda P, Anger M, Schultz RM (2003a) RNAi: Mammalian oocytes do it without RNA-dependent RNA polymerase. RNA 9: 187–192. 10.1261/rna.2860603 PubMed DOI PMC

Stein P, Svoboda P, Schultz RM (2003b) Transgenic RNAi in mouse oocytes: A simple and fast approach to study gene function. Dev Biol 256: 187–193. 10.1016/s0012-1606(02)00122-7 PubMed DOI

Stein P, Zeng F, Pan H, Schultz RM (2005) Absence of non-specific effects of RNA interference triggered by long double-stranded RNA in mouse oocytes. Dev Biol 286: 464–471. 10.1016/j.ydbio.2005.08.015 PubMed DOI

Suh N, Baehner L, Moltzahn F, Melton C, Shenoy A, Chen J, Blelloch R (2010) MicroRNA function is globally suppressed in mouse oocytes and early embryos. Curr Biol 20: 271–277. 10.1016/j.cub.2009.12.044 PubMed DOI PMC

Suk K, Choi J, Suzuki Y, Ozturk SB, Mellor JC, Wong KH, MacKay JL, Gregory RI, Roth FP (2011) Reconstitution of human RNA interference in budding yeast. Nucleic Acids Res 39: e43 10.1093/nar/gkq1321 PubMed DOI PMC

Svoboda P. (2014) Renaissance of mammalian endogenous RNAi. FEBS Lett 588: 2550–2556. 10.1016/j.febslet.2014.05.030 PubMed DOI

Svobodova E, Kubikova J, Svoboda P (2016) Production of small RNAs by mammalian Dicer. Pflugers Arch 468: 1089–1102. 10.1007/s00424-016-1817-6 PubMed DOI PMC

Tam OH, Aravin AA, Stein P, Girard A, Murchison EP, Cheloufi S, Hodges E, Anger M, Sachidanandam R, Schultz RM, et al. (2008) Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 453: 534–538. 10.1038/nature06904 PubMed DOI PMC

Tang F, Kaneda M, O'Carroll D, Hajkova P, Barton SC, Sun YA, Lee C, Tarakhovsky A, Lao K, Surani MA (2007) Maternal microRNAs are essential for mouse zygotic development. Genes Dev 21: 644–648. 10.1101/gad.418707 PubMed DOI PMC

Todaro GJ, Green H (1963) Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol 17: 299–313. 10.1083/jcb.17.2.299 PubMed DOI PMC

Tsai K, Courtney DG, Kennedy EM, Cullen BR (2018) Influenza A virus-derived siRNAs increase in the absence of NS1 yet fail to inhibit virus replication. RNA 24: 1172–1182. 10.1261/rna.066332.118 PubMed DOI PMC

van der Veen AG, Maillard PV, Schmidt JM, Lee SA, Deddouche-Grass S, Borg A, Kjaer S, Snijders AP, Reis e Sousa C (2018) The RIG-I-like receptor LGP2 inhibits Dicer-dependent processing of long double-stranded RNA and blocks RNA interference in mammalian cells. EMBO J 37: e97479 10.15252/embj.201797479 PubMed DOI PMC

Vermeulen A, Behlen L, Reynolds A, Wolfson A, Marshall WS, Karpilow J, Khvorova A (2005) The contributions of dsRNA structure to Dicer specificity and efficiency. RNA 11: 674–682. 10.1261/rna.7272305 PubMed DOI PMC

Wang J, Tekle E, Oubrahim H, Mieyal JJ, Stadtman ER, Chock PB (2003) Stable and controllable RNA interference: Investigating the physiological function of glutathionylated actin. Proc Natl Acad Sci USA 100: 5103–5106. 10.1073/pnas.0931345100 PubMed DOI PMC

Wang Y, Mercier R, Hobman TC, LaPointe P (2013) Regulation of RNA interference by Hsp90 is an evolutionarily conserved process. Biochim Biophys Acta 1833: 2673–2681. 10.1016/j.bbamcr.2013.06.017 PubMed DOI

Watanabe T, Totoki Y, Toyoda A, Kaneda M, Kuramochi-Miyagawa S, Obata Y, Chiba H, Kohara Y, Kono T, Nakano T, et al. (2008) Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453: 539–543. 10.1038/nature06908 PubMed DOI

Wu C, Jin X, Tsueng G, Afrasiabi C, Su AI (2016) BioGPS: Building your own mash-up of gene annotations and expression profiles. Nucleic Acids Res 44: D313–D316. 10.1093/nar/gkv1104 PubMed DOI PMC

Yang S, Tutton S, Pierce E, Yoon K (2001) Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cells. Mol Cell Biol 21: 7807–7816. 10.1128/mcb.21.22.7807-7816.2001 PubMed DOI PMC

Yi CE, Bekker JM, Miller G, Hill KL, Crosbie RH (2003) Specific and potent RNA interference in terminally differentiated myotubes. J Biol Chem 278: 934–939. 10.1074/jbc.m205946200 PubMed DOI

Zhang HD, Kolb FA, Brondani V, Billy E, Filipowicz W (2002) Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J 21: 5875–5885. 10.1093/emboj/cdf582 PubMed DOI PMC

Enhanced RNAi does not provide efficient innate antiviral immunity in mice

Functional canonical RNAi in mice expressing a truncated Dicer isoform and long dsRNA

Structural and functional basis of mammalian microRNA biogenesis by Dicer

Physiologically relevant miRNAs in mammalian oocytes are rare and highly abundant

CRISPR-Induced Expression of N-Terminally Truncated Dicer in Mouse Cells

Key Mechanistic Principles and Considerations Concerning RNA Interference

The most abundant maternal lncRNA Sirena1 acts post-transcriptionally and impacts mitochondrial distribution

Restricted and non-essential redundancy of RNAi and piRNA pathways in mouse oocytes