Exogenous and endogenous dsRNAs perceived by plant Dicer-like 4 protein in the RNAi-depleted cellular context.
Antisense transcript
DCL4
Plant viruses
RNAi
Short RNAs
Yeast
dsRNAs
Journal
Cellular & molecular biology letters
ISSN: 1689-1392
Titre abrégé: Cell Mol Biol Lett
Pays: England
ID NLM: 9607427
Informations de publication
Date de publication:
07 Aug 2023
07 Aug 2023
Historique:
received:
02
05
2023
accepted:
24
06
2023
medline:
9
8
2023
pubmed:
8
8
2023
entrez:
7
8
2023
Statut:
epublish
Résumé
In plants, RNase III Dicer-like proteins (DCLs) act as sensors of dsRNAs and process them into short 21- to 24-nucleotide (nt) (s)RNAs. Plant DCL4 is involved in the biogenesis of either functional endogenous or exogenous (i.e. viral) short interfering (si)RNAs, thus playing crucial antiviral roles. In this study we expressed plant DCL4 in Saccharomyces cerevisiae, an RNAi-depleted organism, in which we could highlight the role of dicing as neither Argonautes nor RNA-dependent RNA polymerase is present. We have therefore tested the DCL4 functionality in processing exogenous dsRNA-like substrates, such as a replicase-assisted viral replicon defective-interfering RNA and RNA hairpin substrates, or endogenous antisense transcripts. DCL4 was shown to be functional in processing dsRNA-like molecules in vitro and in vivo into 21- and 22-nt sRNAs. Conversely, DCL4 did not efficiently process a replicase-assisted viral replicon in vivo, providing evidence that viral RNAs are not accessible to DCL4 in membranes associated in active replication. Worthy of note, in yeast cells expressing DCL4, 21- and 22-nt sRNAs are associated with endogenous loci. We provide new keys to interpret what was studied so far on antiviral DCL4 in the host system. The results all together confirm the role of sense/antisense RNA-based regulation of gene expression, expanding the sense/antisense atlas of S. cerevisiae. The results described herein show that S. cerevisiae can provide insights into the functionality of plant dicers and extend the S. cerevisiae tool to new biotechnological applications.
Sections du résumé
BACKGROUND
BACKGROUND
In plants, RNase III Dicer-like proteins (DCLs) act as sensors of dsRNAs and process them into short 21- to 24-nucleotide (nt) (s)RNAs. Plant DCL4 is involved in the biogenesis of either functional endogenous or exogenous (i.e. viral) short interfering (si)RNAs, thus playing crucial antiviral roles.
METHODS
METHODS
In this study we expressed plant DCL4 in Saccharomyces cerevisiae, an RNAi-depleted organism, in which we could highlight the role of dicing as neither Argonautes nor RNA-dependent RNA polymerase is present. We have therefore tested the DCL4 functionality in processing exogenous dsRNA-like substrates, such as a replicase-assisted viral replicon defective-interfering RNA and RNA hairpin substrates, or endogenous antisense transcripts.
RESULTS
RESULTS
DCL4 was shown to be functional in processing dsRNA-like molecules in vitro and in vivo into 21- and 22-nt sRNAs. Conversely, DCL4 did not efficiently process a replicase-assisted viral replicon in vivo, providing evidence that viral RNAs are not accessible to DCL4 in membranes associated in active replication. Worthy of note, in yeast cells expressing DCL4, 21- and 22-nt sRNAs are associated with endogenous loci.
CONCLUSIONS
CONCLUSIONS
We provide new keys to interpret what was studied so far on antiviral DCL4 in the host system. The results all together confirm the role of sense/antisense RNA-based regulation of gene expression, expanding the sense/antisense atlas of S. cerevisiae. The results described herein show that S. cerevisiae can provide insights into the functionality of plant dicers and extend the S. cerevisiae tool to new biotechnological applications.
Identifiants
pubmed: 37550627
doi: 10.1186/s11658-023-00469-2
pii: 10.1186/s11658-023-00469-2
pmc: PMC10405411
doi:
Substances chimiques
Plant Proteins
0
Ribonuclease III
EC 3.1.26.3
RNA, Double-Stranded
0
RNA, Small Interfering
0
Types de publication
Letter
Langues
eng
Sous-ensembles de citation
IM
Pagination
64Informations de copyright
© 2023. University of Wroclav.
Références
Pelechano V, Steinmetz LM. Gene regulation by antisense transcription. Nat Rev Genet. 2013;14:880–93.
pubmed: 24217315
Safi A, Saberiyan M, Sanaei M-J, Adelian S, Davarani Asl F, Zeinaly M, et al. The role of noncoding RNAs in metabolic reprogramming of cancer cells. Cell Mol Biol Lett. 2023;28:37.
pubmed: 37161350
pmcid: 10169341
Tatsuke T, Sakashita K, Masaki Y, Lee J, Kawaguchi Y, Kusakabe T. The telomere-specific non-LTR retrotransposons SART1 and TRAS1 are suppressed by Piwi subfamily proteins in the silkworm, Bombyx mori. Cell Mol Biol Lett. 2010. https://doi.org/10.2478/s11658-009-0038-9 .
doi: 10.2478/s11658-009-0038-9
pubmed: 19943120
Guo Z, Li Y, Ding SW. Small RNA-based antimicrobial immunity. Nat Rev Immunol. 2019;19:31–44.
pubmed: 30301972
Tan H, Li B, Guo H. The diversity of post-transcriptional gene silencing mediated by small silencing RNAs in plants. Essays Biochem. 2020;64:919–30.
Cuperus JT, Carbonell A, Fahlgren N, Garcia-Ruiz H, Burke RT, Takeda A, et al. Unique functionality of 22-nt miRNAs in triggering RDR6-dependent siRNA biogenesis from target transcripts in Arabidopsis. Nat Struct Mol Biol. 2010. https://doi.org/10.1038/nsmb.1866 .
doi: 10.1038/nsmb.1866
pubmed: 20562854
pmcid: 2916640
Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP. MicroRNAs in plants. Genes Dev. 2002;16:1616–26.
pubmed: 12101121
pmcid: 186362
Vazquez F, Blevins T, Ailhas J, Boller T, Meins F. Evolution of Arabidopsis MIR genes generates novel microRNA classes. Nucleic Acids Res. 2008;36:6429–38.
pubmed: 18842626
pmcid: 2582634
Parent JS, Bouteiller N, Elmayan T, Vaucheret H. Respective contributions of Arabidopsis DCL2 and DCL4 to RNA silencing. Plant J. 2015;81:223–32.
Mlotshwa S, Pruss GJ, Peragine A, Endres MW, Li J, Chen X, et al. DICER-LIKE2 plays a primary role in transitive silencing of transgenes in Arabidopsis. PLoS ONE. 2008;3: e1755.
pubmed: 18335032
pmcid: 2262140
Xia R, Chen C, Pokhrel S, Ma W, Huang K, Patel P, et al. 24-nt reproductive phasiRNAs are broadly present in angiosperms. Nat Commun. 2019;10:627.
pubmed: 30733503
pmcid: 6367383
Xie Z, Allen E, Wilken A, Carrington JC. DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana. Proc Natl Acad Sci USA. 2005;102:12984–9.
pubmed: 16129836
pmcid: 1200315
Allen E, Xie Z, Gustafson AM, Carrington JC. microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell. 2005;121:207–21.
pubmed: 15851028
Leonetti P, Stuttmann J, Pantaleo V. Regulation of plant antiviral defense genes via host RNA-silencing mechanisms. Virol J. 2021;18:194.
pubmed: 34565394
pmcid: 8474839
Burroughs AM, Ando Y, Aravind L. New perspectives on the diversification of the RNA interference system: insights from comparative genomics and small RNA sequencing: New perspectives on the diversification of the RNAi system. WIREs RNA. 2014;5:141–81.
pubmed: 24311560
Drinnenberg IA, Weinberg DE, Xie KT, Mower JP, Wolfe KH, Fink GR, et al. RNAi in budding yeast. Science. 2009;326:544–50.
pubmed: 19745116
pmcid: 3786161
Lamontagne B, Ghazal G, Lebars I, Yoshizawa S, Fourmy D, Abou ES. Sequence dependence of substrate recognition and cleavage by yeast RNase III. J Mol Biol. 2003;327:985–1000.
pubmed: 12662924
Elela SA, Igel H, Ares M. RNase III cleaves eukaryotic preribosomal RNA at a U3 snoRNP-dependent site. Cell. 1996;85:115–24.
pubmed: 8620530
Price BD, Rueckert RR, Ahlouist P. Complete replication of an animal virus and maintenance of expression vectors derived from it in Saccharomyces cerevisiae. Proc Natl Acad Sci USA. 1996;93:9465–70.
pubmed: 8790353
pmcid: 38451
Restrepo-Hartwig MA, Ahlquist P. Brome mosaic virus helicase- and polymerase-like proteins colocalize on the endoplasmic reticulum at sites of viral RNA synthesis. J Virol. 1996;70:8908–16.
pubmed: 8971020
pmcid: 190988
Pantaleo V, Rubino L, Russo M. Replication of Carnation Italian ringspot virus defective interfering RNA in Saccharomyces cerevisiae. J Virol. 2003;77:2116–23.
pubmed: 12525646
pmcid: 140986
Panaviene Z, Baker JM, Nagy PD. The overlapping RNA-binding domains of p33 and p92 replicase proteins are essential for tombusvirus replication. Virology. 2003;308:191–205.
pubmed: 12706102
Suk K, Choi J, Suzuki Y, Ozturk SB, Mellor JC, Wong KH, et al. Reconstitution of human RNA interference in budding yeast. Nucleic Acids Res. 2011;39: e43.
pubmed: 21252293
pmcid: 3074155
Deleris A, Gallego-Bartolome J, Bao J, Kasschau KD, Carrington JC, Voinnet O. Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. Science. 2006;313:68–71.
pubmed: 16741077
Hiraguri A, Itoh R, Kondo N, Nomura Y, Aizawa D, Murai Y, et al. Specific interactions between Dicer-like proteins and HYL1/DRB-family dsRNA-binding proteins in Arabidopsis thaliana. Plant Mol Biol. 2005;57:173–88.
pubmed: 15821876
Papp I, Mette MF, Aufsatz W, Daxinger L, Schauer SE, Ray A, et al. Evidence for nuclear processing of plant micro RNA and short interfering RNA precursors. Plant Physiol. 2003;132:1382–90.
pubmed: 12857820
pmcid: 167078
Ausubel FM. Current protocols in molecular biology. Brooklyn: Wiley; 1987.
Navarro B, Russo M, Pantaleo V, Rubino L. Cytological analysis of Saccharomyces cerevisiae cells supporting cymbidium ringspot virus defective interfering RNA replication. J Gen Virol. 2006;87:705–14.
pubmed: 16476994
Burgyan J, Dalmay T, Rubino L, Russo M. The replication of cymbidium ringspot tombusvirus defective interfering-satellite RNA hybrid molecules. Virology. 1992;190:579–86.
pubmed: 1381535
Mascorro-Gallardo JO, Covarrubias AA, Gaxiola R. Construction of a CUP1 promoter-based vector to modulate gene expression in Saccharomyces cerevisiae. Gene. 1996;172:169–70.
pubmed: 8654982
Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003;31:3406–15.
pubmed: 12824337
pmcid: 169194
Kushnirov VV. Rapid and reliable protein extraction from yeast. Yeast. 2000;16:857–60.
pubmed: 10861908
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55:611–22.
pubmed: 19246619
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–8.
pubmed: 11846609
Gursinsky T, Schulz B, Behrens SE. Replication of Tomato bushy stunt virus RNA in a plant in vitro system. Virology. 2009;390:250–60.
pubmed: 19520410
Leonetti P, Ghasemzadeh A, Consiglio A, Gursinsky T, Behrens S, Pantaleo V. Endogenous activated small interfering RNAs in virus-infected Brassicaceae crops show a common host gene-silencing pattern affecting photosynthesis and stress response. New Phytol. 2021;229:1650–64.
pubmed: 32945560
Ewels P, Magnusson M, Lundin S, Käller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016;32:3047–8.
pubmed: 27312411
pmcid: 5039924
Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21.
pubmed: 23104886
Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011;12:323.
pubmed: 21816040
pmcid: 3163565
Consiglio A, Mencar C, Grillo G, Marzano F, Caratozzolo MF, Liuni S. A fuzzy method for RNA-Seq differential expression analysis in presence of multireads. BMC Bioinformatics. 2016;17:345.
pubmed: 28185579
Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550.
pubmed: 25516281
pmcid: 4302049
Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet j. 2011;17:10.
Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10:R25.
pubmed: 19261174
pmcid: 2690996
Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, et al. The human genome browser at UCSC. Genome Res. 2002;12:996–1006.
pubmed: 12045153
pmcid: 186604
Wu H, Li B, Iwakawa HO, Pan Y, Tang X, Ling-Hu Q, et al. Plant 22-nt siRNAs mediate translational repression and stress adaptation. Nature. 2020;581:89–93.
pubmed: 32376953
Szittya G, Molnar A, Silhavy D, Hornyik C, Burgyan J. Short defective interfering RNAs of tombusviruses are not targeted but trigger post-transcriptional gene silencing against their helper virus. Plant Cell. 2002;14:359–72.
pubmed: 11884680
pmcid: 152918
Szittya G, Silhavy D, Molnar A, Havelda Z, Lovas A, Lakatos L, et al. Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation. EMBO J. 2003;22:633–40.
pubmed: 12554663
pmcid: 140757
Schuck J, Gursinsky T, Pantaleo V, Burgyan J, Behrens SE. AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system. Nucleic Acids Res. 2013;41:5090–103.
pubmed: 23535144
pmcid: 3643602
Yoshikawa M, Peragine A, Park MY, Poethig RS. A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev. 2005;19:2164–75.
pmcid: 1221887
Gasciolli V, Mallory AC, Bartel DP, Vaucheret H. Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs. Curr Biol. 2005;15:1494–500.
pubmed: 16040244
Nagy PD. Yeast as a model host to explore plant virus-host interactions. Annu Rev Phytopathol. 2008;46:217–42.
pubmed: 18422427
Pantaleo V, Rubino L, Russo M. The p36 and p95 replicase proteins of Carnation Italian ringspot virus cooperate in stabilizing defective interfering RNA. J Gen Virol. 2004;85:2429–33.
pubmed: 15269385
Nagy PD, Feng Z. Tombusviruses orchestrate the host endomembrane system to create elaborate membranous replication organelles. Curr Opin Virol. 2021;48:30–41.
pubmed: 33845410
Eddy SR. Computational genomics of noncoding RNA genes. Cell. 2002;109:137–40.
pubmed: 12007398
Sickmann A, Reinders J, Wagner Y, Joppich C, Zahedi R, Meyer HE, et al. The proteome of Saccharomyces cerevisiae mitochondria. Proc Natl Acad Sci USA. 2003;100:13207–12.
pubmed: 14576278
pmcid: 263752
Dean N, Zhang YB, Poster JB. The VRG4 gene is required for GDP-mannose transport into the lumen of the golgi in the yeast, Saccharomyces cerevisiae. J Biol Chem. 1997;272:31908–14.
pubmed: 9395539
Kruis AJ, Levisson M, Mars AE, Van Der Ploeg M, Garcés Daza F, Ellena V, et al. Ethyl acetate production by the elusive alcohol acetyltransferase from yeast. Metab Eng. 2017;41:92–101.
pubmed: 28356220
Ma J, Dobry CJ, Krysan DJ, Kumar A. Unconventional genomic architecture in the budding yeast Saccharomyces cerevisiae Masks the nested antisense gene NAG1. Eukaryot Cell. 2008;7:1289–98.
pubmed: 18310357
pmcid: 2519765
Weinhandl K, Winkler M, Glieder A, Camattari A. Carbon source dependent promoters in yeasts. Microb Cell Fact. 2014;13:5.
pubmed: 24401081
pmcid: 3897899
Yassour M, Pfiffner J, Levin JZ, Adiconis X, Gnirke A, Nusbaum C, et al. Strand-specific RNA sequencing reveals extensive regulated long antisense transcripts that are conserved across yeast species. Genome Biol. 2010;11:R87.
pubmed: 20796282
pmcid: 2945789
Kakiyama S, Tabara M, Nishibori Y, Moriyama H, Fukuhara T. Long DCL4-substrate dsRNAs efficiently induce RNA interference in plant cells. Sci Rep. 2019;9:6920.
pubmed: 31061468
pmcid: 6502814
Fukudome A, Kanaya A, Egami M, Nakazawa Y, Hiraguri A, Moriyama H, et al. Specific requirement of DRB4, a dsRNA-binding protein, for the in vitro dsRNA-cleaving activity of Arabidopsis Dicer-like 4. RNA. 2011;17:750–60.
pubmed: 21270136
pmcid: 3062185
MacRae IJ, Doudna JA. Ribonuclease revisited: structural insights into ribonuclease III family enzymes. Curr Opin Struct Biol. 2007;17:138–45.
pubmed: 17194582
Nakazawa Y, Hiraguri A, Moriyama H, Fukuhara T. The dsRNA-binding protein DRB4 interacts with the Dicer-like protein DCL4 in vivo and functions in the trans-acting siRNA pathway. Plant Mol Biol. 2007;63:777–85.
pubmed: 17221360
Faitova J, Krekac D, Hrstka R, Vojtesek B. Endoplasmic reticulum stress and apoptosis. Cell Mol Biol Lett. 2006. https://doi.org/10.2478/s11658-006-0040-4/html .
doi: 10.2478/s11658-006-0040-4/html
pubmed: 16977377
pmcid: 6275750
Cao M, Du P, Wang X, Yu YQ, Qiu YH, Li W, et al. Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs in Arabidopsis. Proc Natl Acad Sci USA. 2014. https://doi.org/10.1073/pnas.1407131111 .
doi: 10.1073/pnas.1407131111
pubmed: 25468977
pmcid: 4273407
Maillard PV, Van der Veen AG, Deddouche-Grass S, Rogers NC, Merits A, Reis e Sousa C. Inactivation of the type I interferon pathway reveals long double-stranded RNA variant:small-RNA interference in mammalian cells. EMBO J. 2016;35:2505–18.
pubmed: 27815315
pmcid: 5167344
Kattan SW, Hobani YH, Shaheen S, Mokhtar SH, Hussein MH, Toraih EA, et al. Association of cyclin-dependent kinase inhibitor 2B antisense RNA 1 gene expression and rs2383207 variant with breast cancer risk and survival. Cell Mol Biol Lett. 2021;26:14.
pubmed: 33849428
pmcid: 8045214
Li W-X, Ding S-W. Mammalian viral suppressors of RNA interference. Trends Biochem Sci. 2022;47:978–88.
pubmed: 35618579
pmcid: 10281742
Tombusviridae RD. In: King AMQAMJ, editor. Virus taxonomy classification and nomenclature of viruses. Amsterdam: Elsevier; 2012. p. 1111–38.
Russo M, Burgyan J, Martelli GP. Molecular biology of tombusviridae. Adv Virus Res. 1994;44:381–428.
pubmed: 7817878
Rubino L, Di Franco A, Russo M. Expression of a plant virus non-structural protein in Saccharomyces cerevisiae causes membrane proliferation and altered mitochondrial morphology. Microbiology. 2000;81:279–86.
Schwartz M, Chen J, Lee W-M, Janda M, Ahlquist P. Alternate, virus-induced membrane rearrangements support positive-strand RNA virus genome replication. Proc Natl Acad Sci USA. 2004;101:11263–8.
pubmed: 15280537
pmcid: 509192
Szittya G, Moxon S, Pantaleo V, Toth G, Rusholme Pilcher RL, Moulton V, et al. Structural and functional analysis of viral siRNAs. PLoS Pathog. 2010;6: e1000838.
pubmed: 20368973
pmcid: 2848561
Bartoszewski R, Sikorski AF. Editorial focus: understanding off-target effects as the key to successful RNAi therapy. Cell Mol Biol Lett. 2019;24:69.
pubmed: 31867046
pmcid: 6902517