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EMBO Rep. Author manuscript; available in PMC 2010 October 18.
Published in final edited form as:
PMCID: PMC2828748
EMSID: UKMS32197

Hairpin RNA induces secondary siRNAs synthesis and silencing in trans in fission yeast

Abstract

RNA interference (RNAi) is widespread in eukaryotes and regulates gene expression transcriptionally or post-transcriptionally. In fission yeast, RNAi is tightly coupled to template transcription and chromatin modifications that establish heterochromatin in cis. Exogenous dsRNA triggers appear to induce heterochromatin formation in trans only when certain silencing proteins are overexpressed. We show that GFP hairpin dsRNA allows production of high levels of Ago1-associated small interfering RNAs (siRNAs) that can induce heterochromatin formation at a remote locus. This silencing does not require any manipulations apart from hairpin expression. In cells expressing a ura4+-GFP fusion gene, production of GFP siRNAs cause the appearance of ura4 siRNAs from the target gene. Production of these secondary siRNAs depends on RDRPRdp1 function and other RNAi pathway components. This demonstrates that transitivity occurs in fission yeast and implies that RDRPRdp1 can synthesize RNA from targeted RNA templates in vivo, generating siRNAs not homologous to the hairpin.

Introduction

Small-RNA mediated silencing mechanisms have been observed in the vast majority of eukaryotes. The RNAi machinery frequently reduces gene expression by cleaving homologous transcripts or by inhibiting their translation. However, RNAi can also act at the transcriptional level, leading to chromatin modification and heterochromatin formation.

The key features of RNA silencing include the production of 21–25 nt small RNAs by the ribonuclease III enzyme Dicer and the formation of an Argonaute-containing complex into which these small RNAs are incorporated (Farazi et al, 2008). The Ago complex is guided to transcripts homologous to the encapsulated small RNA in order to block their translation or utilize its inherent endonuclease activity (known as ‘slicing’) to cleave the cognate transcripts.

In fission yeast, Schizosaccharomyces pombe, the RNAi machinery is required for the formation of heterochromatin at pericentromeric repeats, the mating type locus and subtelomeric repeats. Within these regions chromatin is underacetylated and methylated on lysine 9 of histone H3 (H3K9me) and the transcription of underlying genes is repressed [reviewed by (Buhler & Moazed, 2007; Grewal & Jia, 2007)]. The formation of heterochromatin at these loci is a multi-step process triggered by double strand (ds) RNA production. Non-coding transcripts derived from repetitive elements form dsRNA and are processed into small RNAs (siRNAs) by Dicer (Dcr1). siRNAs are loaded into Argonaute (Ago1) which directs the RNA-induced initiation of transcriptional gene silencing complex (RITS) to homologous repeats. RITS is composed of Ago1, Tas3 and Chp1 and binds H3K9me chromatin via the chromo-domain of Chp1 allowing the recruitment of the RNA-dependent RNA polymerase complex (RDRC) to chromatin. The RDRC complex which contains Rdp1 (RNA-dependent RNA polymerase - RDRP) is thought to synthesize dsRNA via the RDRP activity of Rdp1 and thereby amplify the siRNA pool. The action of the RNAi machinery recruits the key histone methyltransferase Clr4 to siRNA homologous loci. H3K9 methylation by Clr4 allows binding of the chromo-domain proteins Swi6, Chp1, Chp2 and Clr4 itself to chromatin, forming a nucleation site from which heterochromatin components can spread outwards along the chromatin fibre, repressing underlying genes.

In most organisms, the expression of exogenous double stranded hairpin RNAs is sufficient to generate siRNAs which in turn silence target genes by inducing post-transcriptional RNA cleavage and degradation, demonstrating that the RNAi machinery can act in trans to inhibit expression of any gene (Kennerdell & Carthew, 2000; Paddison et al, 2002; Smith et al, 2000; Tavernarakis et al, 2000). In plants, hairpin RNA triggered trans-silencing is well known to be converted into transcriptional silencing due to modification of homologous DNA and chromatin that mediates repression (Baulcombe, 2004). In contrast, in fission yeast several observations indicate that the RNAi machinery is much more constrained and unable to efficiently promote transcriptional silencing via heterochromatin formation in trans. siRNAs derived from a silenced marker gene can occasionally repress a second copy of the same gene only if the nuclease Eri1 is deleted (Buhler et al, 2006). Moreover, expression of a long GFP hairpin RNA can reduce expression of a homologous target post-transcriptionally, without heterochromatin associated chromatin modifications (Sigova et al, 2004). Finally, ura4-based hairpins were shown to silence ura4+ gene expression only when the chromo-domain protein Swi6 (HP1) was overexpressed and when antisense transcription occurred across the target gene (Iida et al, 2008).

Here we show that exogenous hairpin RNAs can in fact induce heterochromatin formation in wild-type fission yeast. In contrast with previous reports, silencing does not necessarily require Swi6 over-expression and/or antisense transcription. Analysis of the hairpin mediated trans-silencing reveals two important features of RNAi mediated gene silencing in S. pombe. First, upon silencing, a class of siRNAs, not derived directly from the hairpin, but corresponding to the target gene, are detected; RDRPRdp1 is required for the production of these additional siRNAs in vivo. Second, the presence of Ago1 associated siRNAs does not guarantee robust silencing; Ago1 mediated slicing and degradation of a target RNA may only be a marginal activity in fission yeast.

Results and Discussion

A ura4+-hairpin silences a homologous target gene when adjacent to heterochromatin

To assess whether the fission yeast RNAi machinery can induce heterochromatin formation in trans, we constructed a hairpin complementary to 200 bp of ura4+ expressed from the nmt1 promoter (U-HP), and integrated at ars1 on chromosome 1 (Fig 1A). The hairpin generates detectable ura4 homologous siRNAs (Fig 1B). To assess the influence of chromatin context on the ability of U-HP to repress homologous target genes, we tested if the hairpin silences the normal ura4+ gene, or, ura4+ when placed within close proximity to pericentromeric heterochromatin of cen1 (otr1L Xho:ura4+). Silencing of ura4+ results in restricted growth on plates lacking uracil (-URA) and good growth on counter-selective plates containing 5-fluorootic acid (5-FOA) (Allshire et al, 1995). Expression of U-HP did not affect the growth of cells with ura4+ at its normal chromosomal location. However, growth on 5-FOA plates revealed that ura4+ adjacent to centromeric repeats is more sensitive and silenced by this same hairpin (Fig 1C). This observation raises the possibility that in S. pombe, exogenous siRNAs only efficiently silence in trans when the target locus is nearby endogenous sites of heterochromatin. In fact, H3K9me was detected on ura4+ at this site, but does not result in transcriptional repression (Allshire et al, 1995; Trewick et al, 2007). Expression of U-HP in these cells clearly tips the balance in favour of robust heterochromatin formation and silencing. Other ura4-hairpins have also been shown to tighten weak silencing at other locations within heterochromatin domains (Iida et al, 2008).

Figure 1
A ura4+-hairpin silences ura4+ inserted close to centromere 1, but not the endogenous ura4+ gene

Primary GFP siRNAs allow the production of secondary siRNAs from a target locus

It has been reported previously that a GFP hairpin (GFP-HP) can silence a target gene post-transcriptionally, resulting in decreased levels of GFP-mRNA independently of the RITS components Tas3 and Chp1 and the heterochromatin associated protein Swi6 (Sigova et al, 2004). However, in that study the target GFP gene was expressed from the exceptionally strong adh1 promoter and the GFP hairpin RNA was expressed from a high copy, mitotically unstable plasmid. These features may not be conducive to heterochromatin formation at the target locus. To overcome these issues we developed a moderately expressed target for GFP-hairpin generated siRNAs. A ura4+-GFP C-terminal fusion gene was constructed, expressed from the ura4 promoter, and integrated at the arg3 locus. The GFP-HP construct, expressed from the nmt1 promoter, is composed of an RNA with two GFP ORFs arranged in an inverted orientation around the first intron from the rad9 gene (Sigova et al, 2004). To promote uniform expression, the GFP-HP construct was integrated at ars1; it clearly produces detectable siRNAs which are incorporated into Ago1 (Fig 2A, B). Equivalent levels of cen siRNAs are present in cells with and without the hairpin, suggesting that GFP siRNA production does not interfere with cen siRNA production. Massively parallel sequencing of FLAG-Ago1 associated siRNAs showed that both sense and anti-sense GFP homologous siRNAs are generated which cover most of the length of GFP (Fig 2C). GFP siRNAs were calculated to be approximately 4 fold more abundant than siRNAs originating from centromeric dg repeats but at equivalent levels to the centromeric dh element siRNAs (see Supplementary Information).

Figure 2
Detection of primary and secondary siRNAs

In plants it is known that the production of exogenous siRNAs to one part of a target gene allows the generation of secondary siRNA homologous to regions 5′ and 3′ to the initial primary siRNA target (Baulcombe, 2007). Northern analyses with a ura4 probe allowed weak detection of siRNAs homologous to ura4 5′ to the GFP target (data not shown). To increase sensitivity, siRNAs were concentrated by gel purification and subjected to Northern analyses. Small RNAs homologous to both the ura4 and cen probes were clearly detected in wild-type cells but not in dcr1Δ cells. Detection of these ura4 siRNAs was hairpin dependent indicating that, as in other systems, secondary siRNAs are generated in fission yeast (Fig 2D).

Sequencing of gel purified small RNA confirmed the presence of ura4 siRNAs in wild-type cells. In total 1276 reads for ura4 were obtained in wild-type cells and only 14 in cells lacking the Rdp1 (Fig 3A, Table SIII), even though 70% more reads were obtained for rdp1Δ (16.42M versus 11.63M). Furthermore, siRNA homologous to the 5′ and 3′ UTR regions of the ura4-GFP transcript were prevalent in wild-type but absent in rdp1Δ. Similarly, fewer small RNAs corresponding to the 5′UTR and 3′UTR of the GFP-HP transcript were detected in rdp1Δ. In contrast, substantial GFP siRNAs were detected in rdp1Δ cells (Fig 3 A, B, Table SIII).

Figure 3
Secondary siRNAs are dependent on Rdp1

It has been shown in vitro that Rdp1 (RDRP) can mediate the synthesis of dsRNA from a single stranded RNA template without a complementary primer (Motamedi et al, 2004; Sugiyama et al, 2005). The analyses we present provide the first evidence for Rdp1-mediated secondary siRNAs generation in vivo in fission yeast.

GFP siRNAs induce unstable silencing of target genes

To assess the ability of the GFP-hairpin to promote gene silencing we tested if it affects the expression of different target genes and furthermore if it leads to chromatin modification indicative of heterochromatin formation. Similar to wild-type ura4+ cells, cells expressing the ura4+-GFP fusion gene grow on medium lacking uracil (-URA), but not on plates containing 5-FOA. In contrast, wild-type cells that express both the GFP-HP and the ura4+-GFP target gene grow on 5-FOA indicating that the GFP hairpin mediates repression of ura4+-GFP (wt: Fig 4A). We also combined this chromosomally expressed GFP-HP with cells expressing an ade6+-GFP fusion gene. On limiting adenine indicator plates, ade6 cells form red colonies whereas ade6+ cells form white colonies. Red colonies were clearly visible in cells expressing GFP-HP indicating that the ade6+-GFP gene can be silenced (Fig 4B - arrowheads). Thus, the GFP hairpin appears to mediate RNAi induced silencing of target genes.

Figure 4
GFP-HP induces trans silencing of target genes

To further characterise how the GFP-HP mediates repression of ura4+-GFP,levels were assessed by RT-PCR (qRT-PCR). Surprisingly, transcript levels were not significantly reduced when grown in non-selective medium compared with control cells (data not shown). However, when the silenced population was selected by growing cells in counter selective 5-FOA medium, a reduction in ura4-GFP transcripts was apparent (Fig 4C). Therefore, although GFP siRNAs were produced at a high level and are loaded into Ago1 (Fig 2B), it appears that target GFP transcripts were not efficiently reduced. Consistent with this observation, GFP-HP only silences ade6+-GFP in 19% of colonies (Fig 4B), and re-plating assays of cells from red colonies resulted in a variegated population indicating that silencing is unstable (data not shown). This suggests that classical post-transcriptional mediated RNAi knockdown of transcripts is not particularly prevalent in fission yeast. S.pombe Ago1 may not efficiently slice engaged transcripts and perhaps, in association with Chp1 and Tas3 in RITS, Ago1 is more tailored to affect transcription of the template by inducing heterochromatin formation. In agreement with this, we found that silencing of ura4+-GFP by the GFP hairpin was not only dependent on the RNAi factors Dcr1, Ago1, Arb1, and Rdp1 but also on the H3K9 methyltransferase Clr4, on the chromo-domain proteins Chp1, Swi6, Chp2 and on the histone deacetylases Sir2 and Clr3 (Fig 4A and S1).

Heterochromatin is formed in response to GFP hairpin expression

Loss of GFP-HP mediated silencing in clr4Δ and swi6Δ cells suggests that heterochromatin may be formed on the ura4+-GFP target. This was confirmed by Chromatin Immunoprecipitation assays (ChIP) which indicated that H3K9me2 and Swi6 were present on the ura4+-GFP fusion gene in cells expressing GFP-HP (Fig 5A). The GFP hairpin itself is expressed from the nmt1 promoter at the ars1 locus; it might also be a target for the GFP siRNAs generated from its hairpin RNA. ChIP analyses indicated that higher levels of H3K9me2 could be detected on the GFP-HP locus than on the ura4+-GFP target gene and this was also dependent on Dcr1 and Clr4 (Fig 5B). H3K9me2 at this locus recruits Swi6 (Fig S2) and therefore probably allows the formation of intact heterochromatin. This high level of H3K9me2 might be related to the production of sense and anti-sense GFP RNA at the same locus allowing the formation of dsRNA and its processing in cis. Previously it was found that ura4 hairpin RNAs induced heterochromatin formation on a ura4+ target gene when antisense transcripts traverse the target gene (Iida et al, 2008). However, anti-sense transcripts across the GFP portion of ura4+-GFP could not be detected (Fig 5C). This suggests that antisense transcription is not an absolute requirement for RNAi-directed chromatin modification and silencing of a target gene by exogenous siRNAs.

Figure 5
GFP-HP induces chromatin modifications

The fact that the locus expressing the GFP hairpin itself attracts heterochromatin presumably also influences the levels of hairpin RNA and thus the resulting siRNAs. This may cause the levels of siRNAs, and thus silencing itself, to oscillate. This could explain the clear instability of the silent state observed with the GFP hairpin both with the ura4+-GFP and ade6+-GFP targets

Conclusion

These analyses demonstrate that GFP siRNAs generated by the expression of a GFP hairpin can act in trans to establish heterochromatin on target genes bearing homology to GFP siRNAs and silence their expression. This silencing does not require other manipulations such as deletion of eri1+ or increased expression of the heterochromatin component Swi6HP1, which have previously been shown to promote RNAi-mediated silencing in trans (Buhler et al, 2006; Iida et al, 2008). Interestingly, the silencing we report only involves a marginal decrease in the levels of target RNA. In addition, upon silencing of ura4+-GFP by the GFP hairpin, we detected ura4 siRNAs, thus providing clear evidence for the production of secondary siRNAs in S. pombe.

Our data show that GFP hairpin derived siRNAs are sufficient to induce chromatin modification (H3K9me2, and Swi6 recruitment) on an homologous gene. The GFP siRNAs clearly also act in cis inducing heterochromatin formation on the GFP-HP construct itself. Thus centromeres, telomeres and mating type loci have no inherent special properties for being selected as targets for the RNAi-directed chromatin modification machinery in fission yeast.

Our analyses indicate that the ability of a hairpin RNA to silence in trans depends on the chromosomal location of the target genes and it suggests that the presence of a patch of heterochromatin can provide a foothold for hairpin-mediated silencing. In fact, the ura4 hairpin utilised here only induced silencing of the ura4+ gene when it was placed in close proximity to heterochromatin and known to have some associated H3K9me2 (Noma et al, 2006; Trewick et al, 2007). Non-coding RNAs and siRNAs generated from this region may also contribute to the HP-mediated silencing (Cam et al, 2005; Wilhelm et al, 2008). The GFP hairpin allowed heterochromatin formation at the arg3:ura4+-GFP locus. It is possible that convergent transcripts from the adjacent cmb1+ gene might provide sufficient transient H3K9 methylation (Gullerova & Proudfoot, 2008; Zofall et al, 2009) to promote and enable heterochromatin formation upon production of GFP siRNAs. Alternatively, other still undefined features of this region, such as the acetylation status of the surrounding chromatin, may allow heterochromatin formation by the GFP-HP. Insertion of the URA-GFP target in other regions such as ‘gene-poor’ domains or regions with distinctive chromatin will determine which feature promotes or prevents RNAi-directed heterochromatin formation.

Methods

Yeast strains

All S. pombe strains used are listed in Table S I. Standard procedures were used for growth and genetic manipulations (Moreno et al, 1991). Detailed descriptions of constructs and strains are presented in Supp. Material and Methods.

RNA analysis

Small RNA was prepared as described in Supp. Material and Methods.

For Solexa/Illumina sequencing, 5′ and 3′-adapters were ligated to the purified RNA, cDNA was then synthesized by reverse transcription and sequenced with Illumina/Solexa 1G Sequencing System.

For Northern blotting RNAs were run on denaturing polyacrylamide gels, electro transferred onto Hybond-NX membranes (GE Healthcare) and UV crosslinked. Membranes were probed with P32-labelled oligos or PCR products (see Supp. Methods).

For RT-PCR, RNA prepared with the RNeasy kit (Qiagen) was treated with Turbo DNase (Ambion) and reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen).

ChIP

ChIP was performed as described (Pidoux et al, 2004). In brief, cells were fixed in 1% PFA for 15 min (H3K9me2) or in 3% PFA for 30 min (Swi6) at RT. Immunoprecipitation was performed with monoclonal H3K9me2 or a polyclonal Swi6 antibody and the sample analysed by qPCR.

Analysis of sequencing data

Individual sequence reads were produced by The GenePool, University of Edinburgh, next-generation genomics and bioinformatics facility (genepool.bio.ed.ac.uk). After trimming the adaptor sequences, the sequence reads were mapped onto the S. pombe reference genome and our GFP constructs using SOAP2 (Li et al 2009). The following SOAP2 parameters were used: “-M 0 -r 2 -m 0 -x 0 -v 0” thus allowing for no mismatches or insertions and reporting all repeats. In order to compare regions of the genome, the number of mapped sequences was calculated per base and per region using in-house perl scripts (available on request to the authors).

Supplementary Material

Supplementary Information

Acknowledgements

We thank G.Hamilton and A. Pidoux for comments and the GenePool, University of Edinburgh, for siRNA sequencing. We are grateful to the following for strains and reagents: N. Rhind, P. Zamore. S. I. Grewal, D. Moazed. This research was supported by: EMBO-LTF (to F.S. and A.B.), Wellcome Trust Programme grant (065061/Z to RCA). R.C.A is a Wellcome Trust Principal Research Fellow.

Footnotes

Supplementary information is available at EMBO reports online

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