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. 2002 Jun 1;30(11):2546-54.
doi: 10.1093/nar/30.11.2546.

Dominant genetic screen for cofactors that enhance antisense RNA-mediated gene silencing in fission yeast

Mitch Raponi  1 , Greg M Arndt
Affiliations

Dominant genetic screen for cofactors that enhance antisense RNA-mediated gene silencing in fission yeast

Mitch Raponi et al. Nucleic Acids Res. .

Abstract

Specific gene silencing has been demonstrated in a number of organisms by the introduction of antisense RNA. Mutagenesis of host-encoded factors has begun to unravel the mechanism of several forms of RNA-mediated gene silencing and has suggested that it may have been conserved through evolution. This has led to the identification of certain host genes, which, when mutated, abrogate this phenomenon. Conversely, the identification of other factors that, when co-expressed or overexpressed, can enhance gene inhibition is equally important for both elucidating the mechanism of this process and enhancing gene silencing in recalcitrant systems. We have taken such a dominant genetic approach to identify several host-encoded factors that dramatically enhance target gene silencing when co-expressed with antisense RNA in fission yeast. The transcription factor thi1 and, surprisingly, the ATP-dependent RNA helicase ded1 were initially shown to enhance gene silencing in this system. Additionally, screening of a Schizosaccharomyces pombe cDNA library identified four novel antisense-enhancing sequences (aes factors) all of which are homologous to genes encoding proteins with natural affinities for nucleic acids. These findings demonstrate the utility of this strategy in identifying host-encoded factors that can modulate gene silencing when co-expressed with antisense RNA and possibly other forms of gene-silencing activators.

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Figures

Figure 1
Figure 1
Overexpression of the transcriptional activator thi1. (A) The long (pGT2), short 5′ (pGT59) and short 3′ (pGT61) lacZ antisense constructs are shown in relation to the target lacZ cassette. The adh1-driven target lacZ gene is integrated at the ura4 locus on chromosome III of the fission yeast strain RB3-2. (B) thi1 was co-expressed with the long lacZ antisense gene in the strain RB3-2 (long antisense + thi1) and co-transformants were assayed for β-galactosidase activity. RB3-2 expressing the antisense lacZ (long antisense) or thi1 (thi1) genes only were also analysed. The control strain was RB3-2 transformed with pREP2 and pREP4 (control). Three independent colonies were assayed in triplicate for each strain. (C) Northern blot analysis of RB3-2 containing the pREP2 and pREP4 (–) or pREP2 and pREP4-thi1 (+) plasmids. RNA was probed with the nmt1 promoter fragment to detect all transcripts driven by the nmt1 promoter including the endogenously expressed nmt1 gene (1.3 kb) and the episomally expressed nmt1 promoter and terminator cassette (0.25 kb). This latter cassette is transcribed from the control vectors pREP2 and pREP4. The ethidium bromide-stained gel showing the rRNA bands indicates equal RNA loading.
Figure 2
Figure 2
Co-expression of antisense lacZ genes and ded1. (A) β-Galactosidase assay of antisense lacZ and ded1 co-transformants. The pREP4-ded1 plasmid was co-expressed with the pREP2 plasmid containing different antisense lacZ constructs. Three colonies were assayed in triplicate for each strain. Strains were co-transformed with the appropriate control plasmid to complement auxotrophy. (B) Light microscopic analysis of ded1 transformants. The strain RB3-2 was co-transformed with the plasmids indicated and grown to mid-logarithmic phase before examination.
Figure 3
Figure 3
Overexpression screening strategy for antisense RNA-modulating factors. A target strain containing the integrated lacZ gene under control of the adh1 promoter and the episomal vector containing the nmt1-driven lacZ antisense gene was transformed with a S.pombe cDNA library. Library fragments were driven by the nmt1 promoter. Transformants were individually screened for a change in the lacZ-encoded blue-colour colony phenotype and then transformants of interest were further characterised by quantitative β-galactosidase assay and sequence analysis of antisense enhancing sequences.
Figure 4
Figure 4
Co-expression screen using a S.pombe cDNA library. (A) Transformants were grown on minimal medium plates and overlaid with X-gal-containing medium. Those that showed a reduced blue-colour phenotype (white arrow) were analysed further. Transformants demonstrating an enhanced blue-colour phenotype were also identified (black arrow). (B) Transformants that showed a visual reduction in the blue phenotype were assayed for β-galactosidase activity in liquid culture in the absence of thiamine (black histograms). Thiamine was added to the medium to repress expression of the antisense and cDNA cassettes (white histograms). Transformants were again assayed for β-galactosidase activity following antisense vector segregation (grey histograms). Asterisks indicate transformants showing an antisense-dependent enhancement of gene silencing. One colony was assayed in triplicate for each transformant.
Figure 5
Figure 5
Schematic alignment of aes factors with known nucleotide and protein sequences. Regions of identity are shaded black. The length of protein sequences is followed by aa (amino acids). (A) Protein alignment of aes1 with C.albicans hypothetical protein and S.cerevisiae TS protein. (B) Region of S.pombe EFTu that aligns with the aes2-encoded protein. (C) Nucleotide sequence of aes3 aligns with the antisense strand of S.pombe sna41 (indicated by an arrow) and the sense strand of a S.pombe hypothetical protein (partial 3′ sequence shown). The putative aes3-encoded protein is indicated. The absence of complete homology to the sna41 sequence indicates that this factor may be a chimera. (D) Nucleotide alignment of aes4 with the antisense strand of S.pombe L7a (arrow). Possible aes4-encoded protein alignment with S.cerevisiae hypothetical protein. Accession numbers are shown in brackets.
Figure 6
Figure 6
Sequence alignment of the aes1 protein with related proteins. Sequences displayed are S.pombe (aes1), C.albicans (AJ390519) and S.cerevisiae (NP_011894.1). Identical residues are shown in black and conservative substitutions are indicated in grey. The Clusta1W algorithm was used for the alignment and the PrettyBox program (Wisconsin Package v.10.0; Genetics Computer Group, Madison, WI) was used for display.
Figure 7
Figure 7
Expression of aes factors. (A) Co-expression of the unique aes factors with the long antisense lacZ plasmid in RB3-2. Three colonies were assayed in triplicate for each transformant. In this tertiary assay data were normalized to account for plasmid segregation. (B) Microscopic analysis of transformants expressing aes factors. Transformants were examined at mid-logarithmic phase. (C) Northern analysis of aes-containing strains. RNA was fractionated on a 1% MOPS/formaldehyde agarose gel and transferred to a nylon membrane. RNA was then probed with the nmt1 fragment. The endogenous nmt1 fragment fractionates at 1.3 kb. W30 contains aes2 (∼1.2 kb), W21 contains aes3 (∼0.8 kb) and W27 contains aes1 (∼1.4 kb).
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