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Quelling: post-transcriptional gene silencing guided by small RNAs in Neurospora crassa
Quelling: post-transcriptional gene silencing guided by small RNAs in Neurospora crassa Valerio Fulci and Giuseppe Macino The filamentous fungus Neurospora crassa is a model organism for the study of gene silencing. The most characterized gene silencing mechanism in this ascomycete is quelling, which occurs at the post-transcriptional level. Quelling is triggered by the introduction of transgenes and results in silencing of both transgenes and cognate endogenous mRNAs. Quelling is related to co-suppression, observed in plants, and RNA interference in animals; it requires an Argonaute protein and acts by generating small RNA molecules (about 25 nt long), which in turn target mRNAs to be silenced. It has been recently shown that quelling is needed for the taming of transposons but, unlike other model organisms, does not seem to play any role in heterochromatin assembly and maintenance. Addresses Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Genetica Molecolare, Universita` di Roma La Sapienza, Viale Regina Elena, 324, 00161, Roma, Italy Corresponding author: Macino, Giuseppe ([email protected])
Current Opinion in Microbiology 2007, 10:199–203 This review comes from a themed issue on Cell regulation Edited by Gisela Storz and Dieter Haas Available online 28th March 2007 1369-5274/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2007.03.016
Introduction: homology-dependent post-transcriptional gene silencing mechanisms The term ‘quelling’ refers to a post-transcriptional gene silencing (PTGS) phenomenon observed in Neurospora crassa [1] and related to co-suppression in plants and RNA interference (RNAi) in animals. Co-suppression in plants was the very first of these events to be observed: in 1990 Napoli et al. [2] attempted to overexpress the gene encoding the enzyme chalcone synthase (chs), which is responsible for pigmentation of flowers in petunia. To this end, they introduced extra copies of the chs gene in their plants. However, instead of obtaining plants bearing darker flowers, they isolated plants bearing white flowers, reminiscent of the chs mutant phenotype. Two years later, a similar phenomenon was observed in N. crassa by Romano and Macino [1]. They aimed to overexpress the albino-1 (al-1) gene, needed for the biosynthesis of the carotenoids, which confer to N. crassa its typical www.sciencedirect.com
orange pigmentation. Nevertheless, by introducing extra al-1 copies within the N. crassa genome they obtained about 30% of colonies displaying a white phenotype, identical to the one of al-1 mutants. This phenomenon was termed quelling. Extensive characterization revealed that the proteins involved in these mechanisms are evolutionarily conserved, although each system has its own peculiarities. Quelling, co-suppression and RNAi rely on small RNAs 22–25 nucleotides long, called small interfering RNAs (siRNAs), to direct degradation of target mRNAs and result in the silencing of genes at post-transcriptional level.
N. crassa and quelling Quelling is one of several gene silencing mechanisms acting in N. crassa; a transcriptional gene silencing (TGS) mechanism called repeat induced point-mutation (RIP; see [3] for a review) and a PTGS mechanism homologous to quelling, but only active during meiosis, called meiotic silencing of unpaired DNA (MSUD) [4], have been characterized. Quelling results in the specific inactivation of genes which are homologous to sequences of DNA introduced by transformation (transgenes). Inactivation occurs at a post-transcriptional level; in fact, the amounts of unspliced transcripts are not affected, whereas the mature quelled mRNAs undergo dramatic decrease detectable by northern blot [5]. The experimental system set up by Macino and co-workers [1] took advantage of the typical non-orange phenotype associated with the impairment of al-1 gene. N. crassa was transformed using a plasmid containing the entire sequence of the al-1 gene; a simple visual inspection of the N. crassa colonies that resulted from the transformation revealed that about 30% of them were not orange but rather white or pale yellow, indicating silencing of the al-1 gene [1]. This system, which relies on the analysis of a simple, qualitative and easily detectable phenotype, has been extensively used to characterize quelling [5–12]. The transformation of N. crassa with several different constructs revealed that the minimum length of the region of homology to induce quelling is 132 nt [5], that different sequences induce quelling with slightly different efficiencies (defined as the percentage of colonies displaying a non-orange phenotype) [1], and that the introduction of genomic regions which are not transcribed (i.e. promoter sequences) is not sufficient to induce quelling [5]. Moreover, it was soon realized that quelling is a reversible phenomenon: strains with a white phenotype, occasionally Current Opinion in Microbiology 2007, 10:199–203
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give rise to orange colonies; the reversion frequency varies greatly from one silenced strain to another [1,11]. The isolation of a stably quelled strain enabled a classical forward genetics approach by UV-mutation and subsequent characterization of strains which had lost ability to silence the expression of the al-1 reporter, identified by the orange phenotype. Mutational analysis led to the isolation of three classes of quelling defective (qde) mutants, called qde-1, qde-2 and qde-3 [11]. qde-1 encodes for an RNA-dependent RNA polymerase (RdRP) [10], qde3 for a RecQ helicase [9] and qde-2 for a protein belonging to the Argonaute family [7]. Two other proteins required for quelling, DCL-1 (Dicerlike-1) and DCL-2 escaped mutational screening because of their redundancy in the quelling pathway; however, a reverse genetics approach demonstrated their role in quelling in N. crassa [8]. Recently, Maiti and colleagues [13] identified a novel component of quelling, qip.
A mechanism for quelling The genetic identification of genes involved in quelling has been followed by the biochemical characterization of the corresponding proteins in N. crassa and their homologues in other model organisms. qde-1 was found to share homology with the sgs-2 and ego-1 genes, needed for RNAi in plants and Caenorhabditis elegans, respectively [14–16]; its biochemical activity has been described in detail, confirming its RdRP activity [17]. The structure of QDE-1 has been resolved recently [18,19]. QDE-2 belongs to the probably best characterized and most studied family of proteins in RNAi, the Argonaute family. Following genetic evidences linking proteins of this family to RNAi in fungi, nematodes and plants [20], a protein complex responsible for RNAi and containing a member of the Argonaute family was isolated in Drosophila melanogaster [21] and further characterized in humans [22]. The Argonaute proteins are the catalytic engines which provide the slicer (RNA degrading) activity to the RNA-induced silencing complex (RISC) [23]. A dicer enzyme was first identified in D. melanogaster [24] and subsequently found to be needed for RNAi in all organisms. These proteins belong to the family of the RNAseIII and cut double-stranded RNAs into fragments of about 21 nucleotides, with typical staggered ends. Dicer is also involved in maturation of miRNAs in animals and plants [25]. The current model for quelling postulates that the transcription of a transgene generates an RNA which is sensed by N. crassa and used as a substrate by the QDE-1 protein, yielding a double-stranded RNA molecule. Such molecule is a substrate for degradation by the two Dicerlike enzymes [8], which cut it into siRNAs, about 25 Current Opinion in Microbiology 2007, 10:199–203
nucleotides long [6]. These latter molecules are then incorporated into RISC as duplexes; one of the siRNA strands is retained by the RISC, the other is nicked by the slicer activity of QDE-2 and then removed by the QIP exonuclease [13]. The siRNA which remains associated to RISC is used as a molecular guide to detect homologous RNA molecules by base complementarity. Following identification, RNA molecules complementary to siRNAs are degraded, most likely by the QDE-2 enzyme (Figure 1) [7,13].
This model is supported by much experimental evidence; however, some aspects are still unclear One step that has not yet been fully understood is how the transgenic RNA triggers quelling. It has been suggested that some intrinsic property of the transgenic transcript, therefore referred to as an aberrant RNA, could be the activator of silencing in plants [26,27]. Some experimental evidences in plants have shown that de-capped mRNAs could be the molecules that trigger silencing [28]. However, recent reports point to mis-spliced and/or mis-terminated transcripts as putative aberrant RNAs [29]. Other models could be suggested, which do not require postulation on the transcription of an aberrant Figure 1
A model for quelling. Quelling is triggered by the introduction of a transgene (grey) homologous to an endogenous gene (green). Following transcription by RNA polymerase II (RNA pol II)of a transgenic RNA (red) QDE-1 converts it to a dsRNA molecule, which is then processed by either DCL-1 or DCL-2 yielding siRNAs about 25 nt long. siRNAs are loaded onto the RISC complex the QDE-2 slicer activity nicks a strand of the siRNA, which is then removed by QIP. The other siRNA strand is used as a guide by QDE-2 to degrade homologous endogenous transcripts (green). As suggested by analysis in other model organisms it is likely that other proteins are part of the RISC. QDE-3 could play a role in the transcription of the transgenic locus and/or recruit QDE-1 to the nascent transgenic RNA. www.sciencedirect.com
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RNA; for example, QDE-1 could be recruited to transgenic transcripts directly during transcription in the nucleus. This could be the result of intrinsic properties of the transgenic locus itself, rather than of the transgenic mRNA. A likely candidate for the recruitment of QDE-1 to the nascent transgenic locus is QDE-3. Indeed, the role of qde-3 in quelling is still unknown. Moreover, it is likely that qde-3 acts upstream of the formation of the dsRNA intermediate. In fact, the introduction of an inverted repeat construct in N. crassa, which is transcribed yielding a dsRNA that is homologous to the gene al-1, can induce silencing of endogenous al-1, circumventing not only the need for qde-1 but also qde-3 expression [30]. The predicted activity of a RecQ DNA helicase suggested for QDE-3 by homology analysis does not obviously correlate with the quelling mechanism. It has been suggested that QDE-3 could play a role in the resolution of complex DNA structures which are produced at the transgenic locus upon tandem integration, and thus enable proper transcription of the transgene itself [9], perhaps recruiting QDE-1 on the nascent transcript as discussed above. It is worth noting that QDE-3 shares homology with the RecQ helicase domain of the human protein WRN, involved in DNA repair and linked to the Werner syndrome. Indeed, qde-3 has been shown also to play a role in DNA repair, in conjunction with its homolog, RecQ-2 [31]. Even more interestingly, genes sharing some homology with the RNase D domain of wrn have been shown to be required for RNAi both in plants (wex-1) [32] and in Caenorhabditis elegans (mut-7) [33]. Moreover, a close homologue of qde-3, rRecQ-1, has been found to be associated with a novel class of small RNA, called piRNAs, recently isolated from rat testes [34].
Quelling efficiency and maintenance The fact that quelling occurs in a small percentage (about 30%) of the transformed strains [1] suggests that the introduction of transgenes is not sufficient per se to induce quelling. Molecular analysis revealed that the copy number of the transgene is an important factor (the more the copies integrated within the genome, the more the likelihood of quelling to occur) [1,35]. This suggests that a threshold should be reached to trigger quelling. One might then ask which the limiting factor in the establishment of quelling is. An answer comes from some experiments in which quelling efficiency has been increased by overexpressing the qde-1 gene, and thus presumably increasing the levels of dsRNA accumulating within the cell [12]. This is in agreement with an independent result showing that quelling efficiency is greatly increased (up to 80% of transformants quelled) when induced by an inverted-repeat construct rather than a transgene [30]. Both of these approaches led to an increase of dsRNA within the cell, suggesting that the www.sciencedirect.com
limiting factor of quelling might be the production of a dsRNA. It has also been shown that quelled strains have a tendency to revert to a wild type phenotype. The frequency of reversion varies and seems to be correlated with the copy number of the transgene [1]. The methylation of Lys9 of Histone 3 (H3K9) is required for maintenance of the transgene. In fact, knockout of the dim5 gene, responsible for H3K9 methylation in N. crassa, results in increased reversion frequency because of progressive loss of transgene copies [36].
The physiological role of quelling Quelling has most probably evolved as a mechanism of defence against viruses and transposons. In N. crassa, quelling co-operates with RIP and MSUD in maintaining control of the expansion of transposons within the genome. Indeed the efficiency of these three mechanisms is consistent with the fact that not a single active transposon can be found into the N. crassa genome [37], the only reported exception being the Tad element, occurring in the Adiopodoume` strain [38]. Nevertheless, remnants of past transposons are present throughout the N. crassa genome. This is mainly because RIP, occurring at premeiotic phase, has progressively saturated all of the ancient transposons with mutations [37]. However, several lines of evidence provide proof that quelling acts to silence transposons in N. crassa: on the one hand it has been shown that a functional qde-2, dcl-1 and dcl-2 genes are needed to tame the expansion of a transposable Tad element in N. crassa, whilst on the other hand siRNAs targeting the RIPed transposons relics have been found in N. crassa [36,39]. It has been shown in several organisms that RNAi is related to transcriptional gene silencing by guiding heterochromatin assembly [40]. In Saccharomyces pombe, for example, an Argonaute protein and a Dicer gene are required for the assembly of heterochromatin at centromeres and the mating-type locus [41,42]. Strikingly, N. crassa appears to differ from all other model organisms under this respect. Indeed, unlike other organisms, quelling-impairment does not affect the methylation of H3K9 and consequently chromatin configuration of quelled genes [36,43]. Moreover, the quelling is not needed for the maintenance of the heterochromatic state of RIPed transposon relics and centromeric regions [44]. In plants and animals the RNAi mechanism has been shown to play an important role in modulating endogenous genes expression at post transcriptional level. In fact, a novel class of small RNAs, called microRNA (miRNA) directs degradation and/or inhibition of translation of target mRNAs in a RISC-dependent mechanism in plants and animals [45,46]. However, miRNAs have not been found in fungi despite extensive analysis. Moreover, a Current Opinion in Microbiology 2007, 10:199–203
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translational repression has never been highlighted in quelling. Finally, it has been proposed that the quelling machinery could regulate some endogenous genes. In particular the locus frequency ( frq) is transcribed in both sense and antisense orientation, suggesting that a putative dsRNA molecule could form in vivo and be a substrate for the DCL-1 and/or DCL-2 enzymes [47]. However, a widespread role of quelling on the physiology of N. crassa seems to be unlikely because none of the mutants impaired in quelling has been reported to display major defects.
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Cogoni C, Irelan JT, Schumacher M, Schmidhauser TJ, Selker EU, Macino G: Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation. EMBO J 1996, 15:3153-3163.
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Catalanotto C, Pallotta M: ReFalo P, Sachs MS, Vayssie L, Macino G, Cogoni C: Redundancy of the two dicer genes in transgene-induced posttranscriptional gene silencing in Neurospora crassa. Mol Cell Biol 2004, 24:2536-2545.
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Conclusions Forward and reverse genetics approaches enabled the isolation of the genes involved in quelling. Although some aspects of the quelling mechanisms are still obscure, its characterization in N. crassa represented a valuable tool to shed light on the basics of RNAi. This is exemplified by the recent discovery and detailed description of the role of the protein QIP, an exonuclease, the activity of which is required for quelling, which suggests a role for other exonucleases involved in RNAi in other systems, but whose role remains unclear [13]. The recent discovery in rats of a close homologue of qde-3 in association with a novel class of small RNAs [34] suggests that this model organism could still offer some interesting hints and a model to test hypotheses. From a biotechnological point of view the precise characterization of some of the steps leading to quelling offers valuable tools for the manipulation of gene expression in fungi. From a biological point of view, the only examples of quelling occurring in physiological conditions are the production of siRNAs derived from transposon relics [44] and the silencing of the Tad element in the Adiopodoume` strain [39]. It can therefore be concluded that, in opposition with most other model organisms, where RNAi is tightly connected to gene expression regulation by miRNAs, the role of quelling is limited to the preservation of the genome integrity.
Acknowledgements We wish to thank Germano Cecere, Gianluca Azzalin, Marina Goldoni and Leandro Castellano for suggestions and discussion. We thank Prof Carlo Cogoni for reading the manuscript. This work was founded by Fondazione Cenci-Bolognetti.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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10. Cogoni C, Macino G: Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase. Nature 1999, 399:166-169. 11. Cogoni C, Macino G: Isolation of quelling-defective (qde) mutants impaired in posttranscriptional transgene-induced gene silencing in Neurospora crassa. Proc Natl Acad Sci USA 1997, 94:10233-10238. 12. Forrest EC, Cogoni C, Macino G: The RNA-dependent RNA polymerase, QDE-1, is a rate-limiting factor in posttranscriptional gene silencing in Neurospora crassa. Nucleic Acids Res 2004, 32:2123-2128. 13. Maiti M, Lee HC, Liu Y: QIP, a putative exonuclease, interacts with the Neurospora Argonaute protein and facilitates conversion of duplex siRNA into single strands. Genes Dev 2007. Through a biochemical approach the authors demonstrate the slicer activity of QDE-2 and isolate a novel factor, QIP, an exonuclease which is needed for quelling; QIP is responsible for the degradation of the passenger strand of the siRNA duplex. The authors suggest that other exonucleases involved in RNAi could play a similar role in other systems. 14. Dalmay T, Hamilton A, Rudd S, Angell S, Baulcombe DC: An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 2000, 101:543-553. 15. Mourrain P, Beclin C, Elmayan T, Feuerbach F, Godon C, Morel JB, Jouette D, Lacombe AM, Nikic S, Picault N et al.: Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell 2000, 101:533-542. 16. Smardon A, Spoerke JM, Stacey SC, Klein ME, Mackin N, Maine EM: EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans. Curr Biol 2000, 10:169-178. 17. Makeyev EV, Bamford DH: Cellular RNA-dependent RNA polymerase involved in posttranscriptional gene silencing has two distinct activity modes. Mol Cell 2002, 10:1417-1427. 18. Salgado PS, Koivunen MR, Makeyev EV, Bamford DH, Stuart DI, Grimes JM: The structure of an RNAi polymerase links RNA silencing and transcription. PLoS Biol 2006, 4:e434. The structure of QDE-1 RdRP suggests a relationship between RdRP involved in RNAi and RNA polymerases. 19. Laurila MR, Salgado PS, Makeyev EV, Nettelship J, Stuart DI, Grimes JM, Bamford DH: Gene silencing pathway RNAdependent RNA polymerase of Neurospora crassa: yeast expression and crystallization of selenomethionated QDE-1 protein. J Struct Biol 2005, 149:111-115. www.sciencedirect.com
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20. Fagard M, Boutet S, Morel JB, Bellini C, Vaucheret H: AGO1, QDE-2, and RDE-1 are related proteins required for posttranscriptional gene silencing in plants, quelling in fungi, and RNA interference in animals. Proc Natl Acad Sci USA 2000, 97:11650-11654. 21. Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ: Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 2001, 293:1146-1150. 22. Martinez J, Patkaniowska A, Urlaub H, Luhrmann R, Tuschl T: Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 2002, 110:563-574. 23. Parker JS, Barford D: Argonaute: A scaffold for the function of short regulatory RNAs. Trends Biochem Sci 2006, 31:622-630. 24. Bernstein E, Caudy AA, Hammond SM, Hannon GJ: Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001, 409:363-366. 25. Zeng Y: Principles of micro-RNA production and maturation. Oncogene 2006, 25:6156-6162. 26. Lindbo J, Silva-Rosales L, Proebsting W, Dougherty W: Induction of a highly specific antiviral state in transgenic plants: implications for regulation of gene expression and virus resistance. The Plant Cell Online 1993, 5:1749-1759. 27. English J, Mueller E, Baulcombe D: Suppression of virus accumulation in transgenic plants exhibiting silencing of nuclear genes. The Plant Cell Online 1996, 8:179-188. 28. Gazzani S, Lawrenson T, Woodward C, Headon D, Sablowski R: A link between mRNA turnover and RNA interference in Arabidopsis. Science 2004, 306:1046-1048. 29. Herr AJ, Molnar A, Jones A, Baulcombe DC: Inaugural article: defective RNA processing enhances RNA silencing and influences flowering of Arabidopsis. Proc Natl Acad Sci USA 2006, 103:14994-15001. 30. Goldoni M, Azzalin G, Macino G, Cogoni C: Efficient gene silencing by expression of double stranded RNA in Neurospora crassa. Fungal Genet Biol 2004, 41:1016-1024. 31. Pickford A, Braccini L, Macino G, Cogoni C: The QDE-3 homologue RecQ-2 co-operates with QDE-3 in DNA repair in Neurospora crassa. Curr Genet 2003, 42:220-227. 32. Glazov E, Phillips K, Budziszewski GJ, Schob H, Meins F Jr, Levin JZ: A gene encoding an RNase D exonuclease-like protein is required for post-transcriptional silencing in Arabidopsis. Plant J 2003, 35:342-349. 33. Ketting RF, Haverkamp TH, van Luenen HG, Plasterk RH: Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD. Cell 1999, 99:133-141. 34. Lau NC, Seto AG, Kim J, Kuramochi-Miyagawa S, Nakano T, Bartel DP, Kingston RE: Characterization of the piRNA complex from rat testes. Science 2006, 313:363-367.
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The authors show that a novel class of small RNAs isolated from rat testes is associated with a RecQ helicase homologous to QDE-3 and a protein of the Argonaute family. 35. Cogoni C, Romano N, Macino G: Suppression of gene expression by homologous transgenes. Antonie Van Leeuwenhoek 1994, 65:205-209. 36. Chicas A, Forrest EC, Sepich S, Cogoni C, Macino G: Small interfering RNAs that trigger posttranscriptional gene silencing are not required for the histone H3 Lys9 methylation necessary for transgenic tandem repeat stabilization in Neurospora crassa. Mol Cell Biol 2005, 25:3793-3801. The authors show that, whereas on the one hand quelling is not required for the methylation of H3Lys9, on the other hand, knock-down of the dim5 gene, required for H3 Lys9 methylation, results in loss of transgene copies and release of quelling. 37. Galagan JE, Calvo SE, Borkovich KA, Selker EU, Read ND, Jaffe D, FitzHugh W, Ma LJ, Smirnov S, Purcell S et al.: The genome sequence of the filamentous fungus Neurospora crassa. Nature 2003, 422:859-868. 38. Kinsey JA, Helber J: Isolation of a transposable element from Neurospora crassa. Proc Natl Acad Sci USA 1989, 86:1929-1933. 39. Nolan T, Braccini L, Azzalin G, De Toni A, Macino G, Cogoni C: The post-transcriptional gene silencing machinery functions independently of DNA methylation to repress a LINE1-like retrotransposon in Neurospora crassa. Nucleic Acids Res 2005, 33:1564-1573. In this paper the authors show that the qde-2 and the two dicer-like genes of N. crassa play a role in repression of retrotransposons in N. crassa. 40. Wassenegger M: The role of the RNAi machinery in heterochromatin formation. Cell 2005, 122:13-16. 41. Volpe TA, Kidner C, Hall IM, Teng G, Grewal SI, Martienssen RA: Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 2002, 297:1833-1837. 42. Hall IM, Shankaranarayana GD, Noma K, Ayoub N, Cohen A, Grewal SI: Establishment and maintenance of a heterochromatin domain. Science 2002, 297:2232-2237. 43. Freitag M, Lee DW, Kothe GO, Pratt RJ, Aramayo R, Selker EU: DNA methylation is independent of RNA interference in Neurospora. Science 2004, 304:1939. 44. Chicas A, Cogoni C, Macino G: RNAi-dependent and RNAiindependent mechanisms contribute to the silencing of RIPed sequences in Neurospora crassa. Nucleic Acids Res 2004, 32:4237-4243. 45. Valencia-Sanchez MA, Liu J, Hannon GJ, Parker R: Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev 2006, 20:515-524. 46. Massirer KB, Pasquinelli AE: The evolving role of microRNAs in animal gene expression. Bioessays 2006, 28:449-452. 47. Kramer C, Loros JJ, Dunlap JC, Crosthwaite SK: Role for antisense RNA in regulating circadian clock function in Neurospora crassa. Nature 2003, 421:948-952.
Current Opinion in Microbiology 2007, 10:199–203
Current Opinion in Microbiology 2007, 10:199–203 This review comes from a themed issue on Cell regulation Edited by Gisela Storz and Dieter Haas Available online 28th March 2007 1369-5274/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2007.03.016
Introduction: homology-dependent post-transcriptional gene silencing mechanisms The term ‘quelling’ refers to a post-transcriptional gene silencing (PTGS) phenomenon observed in Neurospora crassa [1] and related to co-suppression in plants and RNA interference (RNAi) in animals. Co-suppression in plants was the very first of these events to be observed: in 1990 Napoli et al. [2] attempted to overexpress the gene encoding the enzyme chalcone synthase (chs), which is responsible for pigmentation of flowers in petunia. To this end, they introduced extra copies of the chs gene in their plants. However, instead of obtaining plants bearing darker flowers, they isolated plants bearing white flowers, reminiscent of the chs mutant phenotype. Two years later, a similar phenomenon was observed in N. crassa by Romano and Macino [1]. They aimed to overexpress the albino-1 (al-1) gene, needed for the biosynthesis of the carotenoids, which confer to N. crassa its typical www.sciencedirect.com
orange pigmentation. Nevertheless, by introducing extra al-1 copies within the N. crassa genome they obtained about 30% of colonies displaying a white phenotype, identical to the one of al-1 mutants. This phenomenon was termed quelling. Extensive characterization revealed that the proteins involved in these mechanisms are evolutionarily conserved, although each system has its own peculiarities. Quelling, co-suppression and RNAi rely on small RNAs 22–25 nucleotides long, called small interfering RNAs (siRNAs), to direct degradation of target mRNAs and result in the silencing of genes at post-transcriptional level.
N. crassa and quelling Quelling is one of several gene silencing mechanisms acting in N. crassa; a transcriptional gene silencing (TGS) mechanism called repeat induced point-mutation (RIP; see [3] for a review) and a PTGS mechanism homologous to quelling, but only active during meiosis, called meiotic silencing of unpaired DNA (MSUD) [4], have been characterized. Quelling results in the specific inactivation of genes which are homologous to sequences of DNA introduced by transformation (transgenes). Inactivation occurs at a post-transcriptional level; in fact, the amounts of unspliced transcripts are not affected, whereas the mature quelled mRNAs undergo dramatic decrease detectable by northern blot [5]. The experimental system set up by Macino and co-workers [1] took advantage of the typical non-orange phenotype associated with the impairment of al-1 gene. N. crassa was transformed using a plasmid containing the entire sequence of the al-1 gene; a simple visual inspection of the N. crassa colonies that resulted from the transformation revealed that about 30% of them were not orange but rather white or pale yellow, indicating silencing of the al-1 gene [1]. This system, which relies on the analysis of a simple, qualitative and easily detectable phenotype, has been extensively used to characterize quelling [5–12]. The transformation of N. crassa with several different constructs revealed that the minimum length of the region of homology to induce quelling is 132 nt [5], that different sequences induce quelling with slightly different efficiencies (defined as the percentage of colonies displaying a non-orange phenotype) [1], and that the introduction of genomic regions which are not transcribed (i.e. promoter sequences) is not sufficient to induce quelling [5]. Moreover, it was soon realized that quelling is a reversible phenomenon: strains with a white phenotype, occasionally Current Opinion in Microbiology 2007, 10:199–203
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give rise to orange colonies; the reversion frequency varies greatly from one silenced strain to another [1,11]. The isolation of a stably quelled strain enabled a classical forward genetics approach by UV-mutation and subsequent characterization of strains which had lost ability to silence the expression of the al-1 reporter, identified by the orange phenotype. Mutational analysis led to the isolation of three classes of quelling defective (qde) mutants, called qde-1, qde-2 and qde-3 [11]. qde-1 encodes for an RNA-dependent RNA polymerase (RdRP) [10], qde3 for a RecQ helicase [9] and qde-2 for a protein belonging to the Argonaute family [7]. Two other proteins required for quelling, DCL-1 (Dicerlike-1) and DCL-2 escaped mutational screening because of their redundancy in the quelling pathway; however, a reverse genetics approach demonstrated their role in quelling in N. crassa [8]. Recently, Maiti and colleagues [13] identified a novel component of quelling, qip.
A mechanism for quelling The genetic identification of genes involved in quelling has been followed by the biochemical characterization of the corresponding proteins in N. crassa and their homologues in other model organisms. qde-1 was found to share homology with the sgs-2 and ego-1 genes, needed for RNAi in plants and Caenorhabditis elegans, respectively [14–16]; its biochemical activity has been described in detail, confirming its RdRP activity [17]. The structure of QDE-1 has been resolved recently [18,19]. QDE-2 belongs to the probably best characterized and most studied family of proteins in RNAi, the Argonaute family. Following genetic evidences linking proteins of this family to RNAi in fungi, nematodes and plants [20], a protein complex responsible for RNAi and containing a member of the Argonaute family was isolated in Drosophila melanogaster [21] and further characterized in humans [22]. The Argonaute proteins are the catalytic engines which provide the slicer (RNA degrading) activity to the RNA-induced silencing complex (RISC) [23]. A dicer enzyme was first identified in D. melanogaster [24] and subsequently found to be needed for RNAi in all organisms. These proteins belong to the family of the RNAseIII and cut double-stranded RNAs into fragments of about 21 nucleotides, with typical staggered ends. Dicer is also involved in maturation of miRNAs in animals and plants [25]. The current model for quelling postulates that the transcription of a transgene generates an RNA which is sensed by N. crassa and used as a substrate by the QDE-1 protein, yielding a double-stranded RNA molecule. Such molecule is a substrate for degradation by the two Dicerlike enzymes [8], which cut it into siRNAs, about 25 Current Opinion in Microbiology 2007, 10:199–203
nucleotides long [6]. These latter molecules are then incorporated into RISC as duplexes; one of the siRNA strands is retained by the RISC, the other is nicked by the slicer activity of QDE-2 and then removed by the QIP exonuclease [13]. The siRNA which remains associated to RISC is used as a molecular guide to detect homologous RNA molecules by base complementarity. Following identification, RNA molecules complementary to siRNAs are degraded, most likely by the QDE-2 enzyme (Figure 1) [7,13].
This model is supported by much experimental evidence; however, some aspects are still unclear One step that has not yet been fully understood is how the transgenic RNA triggers quelling. It has been suggested that some intrinsic property of the transgenic transcript, therefore referred to as an aberrant RNA, could be the activator of silencing in plants [26,27]. Some experimental evidences in plants have shown that de-capped mRNAs could be the molecules that trigger silencing [28]. However, recent reports point to mis-spliced and/or mis-terminated transcripts as putative aberrant RNAs [29]. Other models could be suggested, which do not require postulation on the transcription of an aberrant Figure 1
A model for quelling. Quelling is triggered by the introduction of a transgene (grey) homologous to an endogenous gene (green). Following transcription by RNA polymerase II (RNA pol II)of a transgenic RNA (red) QDE-1 converts it to a dsRNA molecule, which is then processed by either DCL-1 or DCL-2 yielding siRNAs about 25 nt long. siRNAs are loaded onto the RISC complex the QDE-2 slicer activity nicks a strand of the siRNA, which is then removed by QIP. The other siRNA strand is used as a guide by QDE-2 to degrade homologous endogenous transcripts (green). As suggested by analysis in other model organisms it is likely that other proteins are part of the RISC. QDE-3 could play a role in the transcription of the transgenic locus and/or recruit QDE-1 to the nascent transgenic RNA. www.sciencedirect.com
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RNA; for example, QDE-1 could be recruited to transgenic transcripts directly during transcription in the nucleus. This could be the result of intrinsic properties of the transgenic locus itself, rather than of the transgenic mRNA. A likely candidate for the recruitment of QDE-1 to the nascent transgenic locus is QDE-3. Indeed, the role of qde-3 in quelling is still unknown. Moreover, it is likely that qde-3 acts upstream of the formation of the dsRNA intermediate. In fact, the introduction of an inverted repeat construct in N. crassa, which is transcribed yielding a dsRNA that is homologous to the gene al-1, can induce silencing of endogenous al-1, circumventing not only the need for qde-1 but also qde-3 expression [30]. The predicted activity of a RecQ DNA helicase suggested for QDE-3 by homology analysis does not obviously correlate with the quelling mechanism. It has been suggested that QDE-3 could play a role in the resolution of complex DNA structures which are produced at the transgenic locus upon tandem integration, and thus enable proper transcription of the transgene itself [9], perhaps recruiting QDE-1 on the nascent transcript as discussed above. It is worth noting that QDE-3 shares homology with the RecQ helicase domain of the human protein WRN, involved in DNA repair and linked to the Werner syndrome. Indeed, qde-3 has been shown also to play a role in DNA repair, in conjunction with its homolog, RecQ-2 [31]. Even more interestingly, genes sharing some homology with the RNase D domain of wrn have been shown to be required for RNAi both in plants (wex-1) [32] and in Caenorhabditis elegans (mut-7) [33]. Moreover, a close homologue of qde-3, rRecQ-1, has been found to be associated with a novel class of small RNA, called piRNAs, recently isolated from rat testes [34].
Quelling efficiency and maintenance The fact that quelling occurs in a small percentage (about 30%) of the transformed strains [1] suggests that the introduction of transgenes is not sufficient per se to induce quelling. Molecular analysis revealed that the copy number of the transgene is an important factor (the more the copies integrated within the genome, the more the likelihood of quelling to occur) [1,35]. This suggests that a threshold should be reached to trigger quelling. One might then ask which the limiting factor in the establishment of quelling is. An answer comes from some experiments in which quelling efficiency has been increased by overexpressing the qde-1 gene, and thus presumably increasing the levels of dsRNA accumulating within the cell [12]. This is in agreement with an independent result showing that quelling efficiency is greatly increased (up to 80% of transformants quelled) when induced by an inverted-repeat construct rather than a transgene [30]. Both of these approaches led to an increase of dsRNA within the cell, suggesting that the www.sciencedirect.com
limiting factor of quelling might be the production of a dsRNA. It has also been shown that quelled strains have a tendency to revert to a wild type phenotype. The frequency of reversion varies and seems to be correlated with the copy number of the transgene [1]. The methylation of Lys9 of Histone 3 (H3K9) is required for maintenance of the transgene. In fact, knockout of the dim5 gene, responsible for H3K9 methylation in N. crassa, results in increased reversion frequency because of progressive loss of transgene copies [36].
The physiological role of quelling Quelling has most probably evolved as a mechanism of defence against viruses and transposons. In N. crassa, quelling co-operates with RIP and MSUD in maintaining control of the expansion of transposons within the genome. Indeed the efficiency of these three mechanisms is consistent with the fact that not a single active transposon can be found into the N. crassa genome [37], the only reported exception being the Tad element, occurring in the Adiopodoume` strain [38]. Nevertheless, remnants of past transposons are present throughout the N. crassa genome. This is mainly because RIP, occurring at premeiotic phase, has progressively saturated all of the ancient transposons with mutations [37]. However, several lines of evidence provide proof that quelling acts to silence transposons in N. crassa: on the one hand it has been shown that a functional qde-2, dcl-1 and dcl-2 genes are needed to tame the expansion of a transposable Tad element in N. crassa, whilst on the other hand siRNAs targeting the RIPed transposons relics have been found in N. crassa [36,39]. It has been shown in several organisms that RNAi is related to transcriptional gene silencing by guiding heterochromatin assembly [40]. In Saccharomyces pombe, for example, an Argonaute protein and a Dicer gene are required for the assembly of heterochromatin at centromeres and the mating-type locus [41,42]. Strikingly, N. crassa appears to differ from all other model organisms under this respect. Indeed, unlike other organisms, quelling-impairment does not affect the methylation of H3K9 and consequently chromatin configuration of quelled genes [36,43]. Moreover, the quelling is not needed for the maintenance of the heterochromatic state of RIPed transposon relics and centromeric regions [44]. In plants and animals the RNAi mechanism has been shown to play an important role in modulating endogenous genes expression at post transcriptional level. In fact, a novel class of small RNAs, called microRNA (miRNA) directs degradation and/or inhibition of translation of target mRNAs in a RISC-dependent mechanism in plants and animals [45,46]. However, miRNAs have not been found in fungi despite extensive analysis. Moreover, a Current Opinion in Microbiology 2007, 10:199–203
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translational repression has never been highlighted in quelling. Finally, it has been proposed that the quelling machinery could regulate some endogenous genes. In particular the locus frequency ( frq) is transcribed in both sense and antisense orientation, suggesting that a putative dsRNA molecule could form in vivo and be a substrate for the DCL-1 and/or DCL-2 enzymes [47]. However, a widespread role of quelling on the physiology of N. crassa seems to be unlikely because none of the mutants impaired in quelling has been reported to display major defects.
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Conclusions Forward and reverse genetics approaches enabled the isolation of the genes involved in quelling. Although some aspects of the quelling mechanisms are still obscure, its characterization in N. crassa represented a valuable tool to shed light on the basics of RNAi. This is exemplified by the recent discovery and detailed description of the role of the protein QIP, an exonuclease, the activity of which is required for quelling, which suggests a role for other exonucleases involved in RNAi in other systems, but whose role remains unclear [13]. The recent discovery in rats of a close homologue of qde-3 in association with a novel class of small RNAs [34] suggests that this model organism could still offer some interesting hints and a model to test hypotheses. From a biotechnological point of view the precise characterization of some of the steps leading to quelling offers valuable tools for the manipulation of gene expression in fungi. From a biological point of view, the only examples of quelling occurring in physiological conditions are the production of siRNAs derived from transposon relics [44] and the silencing of the Tad element in the Adiopodoume` strain [39]. It can therefore be concluded that, in opposition with most other model organisms, where RNAi is tightly connected to gene expression regulation by miRNAs, the role of quelling is limited to the preservation of the genome integrity.
Acknowledgements We wish to thank Germano Cecere, Gianluca Azzalin, Marina Goldoni and Leandro Castellano for suggestions and discussion. We thank Prof Carlo Cogoni for reading the manuscript. This work was founded by Fondazione Cenci-Bolognetti.
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