Multiple Transcriptional and Post-transcriptional Pathways Collaborate to Control Sense and Antisense RNAs of Tf2 Retroelements in Fission Yeast

Abstract

Retrotransposons are mobile genetic elements that colonize eukaryotic genomes by replicating through an RNA intermediate. As retrotransposons can move within the host genome, defense mechanisms have evolved to repress their potential mutagenic activities. In the fission yeast Schizosaccharomyces pombe, the mRNA of Tf2 long terminal repeat retrotransposons is targeted for degradation by the 3'-5' exonucleolytic activity of the exosome-associated protein Rrp6. Here, we show that the nuclear poly(A)-binding protein Pab2 functions with Rrp6 to negatively control Tf2 mRNA accumulation. Furthermore, we found that Pab2/Rrp6-dependent RNA elimination functions redundantly to the transcriptional silencing mediated by the CENP-B homolog, Abp1, in the suppression of antisense Tf2 RNA accumulation. Interestingly, the absence of Pab2 attenuated the derepression of Tf2 transcription and the increased frequency of Tf2 mobilization caused by the deletion of abp1 Our data also reveal that the expression of antisense Tf2 transcripts is developmentally regulated and correlates with decreased levels of Tf2 mRNA. Our findings suggest that transcriptional and post-transcriptional pathways cooperate to control sense and antisense RNAs expressed from Tf2 retroelements.

Keywords: Abp1; Pab2; S. pombe; Tf2; retrotransposon.

Figure 1

Pab2 represses expression of Tf2 mRNA. (A) Northern blot analysis of total RNA prepared from wild-type (lane 1) and pab2Δ (lane 2) cells. The blot was hybridized using probes complementary to Tf2 open reading frame and 18s rRNA. (B) Quantification of Northern blots data for Tf2 mRNA levels. The data and error bars represent the average and SD from at least three independent experiments. *** P < 0.001; Student’s t-test. (C) Schematic representation of an individual ura4⁺-tagged Tf2 element. (D) Northern blot analysis of total RNA prepared from wild-type (lanes 1, 3, and 5) and pab2Δ (lanes 2, 4, and 6) cells expressing Tf2-1 (lanes 1 and 2), Tf2-10 (lanes 3 and 4), and Tf2-11 (lanes 5 and 6) ura4⁺-tagged constructs. The blot was hybridized using probes complementary to ura4⁺ antisense sequence and the rps2 mRNA. (E) Quantification of Northern blots data for ura4⁺ antisense RNA levels. The data and errors bars represent the average and SEM from at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001; two-way ANOVA including Sidak’s multiple comparison test.

Figure 2

Deletion of pab2 and rrp6 attenuates the Tf2 derepression observed in the absence of Abp1. (A) Northern blot analysis of total RNA prepared from wild-type (lane 1), pab2Δ (lane 2), rrp6Δ (lane 3), abp1Δ (lane 4), abp1Δ/pab2Δ (lane 5), and abp1Δ/rrp6Δ (lane 6) cells. The blot was hybridized using probes complementary to the Tf2 open reading frame and 18s rRNA. (B) Quantification of Northern blots data for Tf2 mRNA levels. The data and error bars represent the average and SD from at least three independent experiments. *** P < 0.001; Student’s t-test. (C) Schematic representation of Tf2 genes. Bars above the Tf2 retrotransposon show the positions of PCR products used for the ChIP analysis. Arrows indicates transcriptional start site (TSS) and polyadenylation site (Poly(A)). (D) ChIP assays measuring RNAPII density along Tf2 loci. The input and coprecipitated DNA were quantified by qPCR along the Tf2 loci using specific primer as shown in (C). ChIP data are presented as the mean of the relative level of RNAPII density normalized to a control intergenic region. Error bars represent the SEM for at least three independent experiments. ** P < 0.01, *** P < 0.001; two-way ANOVA including Tukey’s multiple comparison test.

Figure 3

The absence of Pab2 reduces the mobilization frequency of Tf2 elements in the abp1Δ mutant. (A) Diagram of the system used for calculating the Tf2 retrotransposition rates. The endogenous Tf2-12 element was marked with a neoAI cassette, which is interrupted by an artificial intron. Mobilization of Tf2-12 mRNA with spliced neoAI cassette will allow expression of the drug resistance gene, thereby allowing growth on media containing G418. (B) Quantification of Tf2-12 retrotransposition rate by fluctuation analysis. The data and error bars represent the average and 95% confidence interval from 40 independent experiments. (C) Northern blot analysis of total RNA prepared from wild-type (lanes 1 and 5), pab2Δ (lanes 2 and 6), abp1Δ (lane 3), and abp1Δ/pab2Δ (lane 4) cells expressing Tf2-12 neoAI. The blots were hybridized using probes complementary to neoAI antisense RNA and the 25S rRNA. (D) Quantification of Northern blots data for Tf2-12 mRNA levels. The data and error bars represent the average and SD from at least three independent experiments.(* P < 0.05, ** P < 0.01, *** P < 0.001; Student’s t-test. (E) Western blot analysis of total proteins prepared from wild-type (lane 1), pab2Δ (lane 2), abp1Δ (lane 3), and abp1Δ/pab2Δ (lane 4) cells expressing Tf2-12 neoAI. The blot was analyzed using antibodies specific for the reverse transcriptase (RT) and tubulin. (F) Quantification of Western blots data for Tf2 protease-reverse transcriptase (PR-RT) levels. The data and errors bars represent the average and SD from at least three independent experiments. * P < 0.05; Student’s t-test.

Figure 4

Pab2 and Rrp6 function in a pathway redundant to Abp1 to suppress the accumulation of antisense Tf2 RNAs. (A) Northern blot analysis of total RNA prepared from wild-type (lanes 1 and 7), pab2Δ (lanes 2 and 8), rrp6Δ (lane 3), abp1Δ (lanes 4 and 9), abp1Δ/pab2Δ (lanes 5 and 10), and abp1Δ/rrp6Δ (lane 6) cells. The blots were hybridized using probes complementary to Tf2 antisense RNA and 18s rRNA. (B) Quantification of Northern blots data for Tf2 antisense RNA levels. The data and error bars represent the average and SD from at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001; Student’s t-test. (C) Schematization of antisense Tf2 RNA. Arrows indicate transcriptional start site (TSS) and polyadenylation site (Poly(A)), as determined by 5′ and 3′ RACE, respectively.

Figure 5

Ago1-dependent repression of Tf2 mRNA up-regulation in the pab2Δ mutant by exogenous expression of antisense Tf2 RNAs. (A) Northern blot analysis of total RNA prepared from wild-type (lanes 1–2), pab2Δ (lanes 3–4), ago1Δ (lanes 5–6), and pab2Δ/ago1Δ (lanes 7–8) cells expressing exogenous Tf2AS in trans (lanes 2, 4, 6, and 8) or transformed with the empty vector (lanes 1, 3, 5, and 7). The blot was hybridized using probes complementary to Tf2 antisense RNA, Tf2 mRNA, and 18s rRNA. (B) Quantification of Northern blots data for Tf2 mRNA levels. The data and error bars represent the average and SEM from at least three independent experiments. * P < 0.05; two-way ANOVA including Sidak’s multiple comparison test. (C) Northern blot analysis of total RNA prepared from wild-type (lanes 1–3) and pab2Δ (lanes 4–6) cells expressing exogenous Tf2AS RNA (lanes 2 and 5), gfp mRNA (lanes 3 and 6), or transformed with the empty vector (lanes 1 and 4). The blot was hybridized using probes complementary to Tf2 antisense RNA, gfp mRNA, and 18s rRNA. (D) Quantification of Northern blots data for Tf2 antisense RNA and gfp mRNA levels. The data and error bars represent the average and SEM from at least three independent experiments. * P < 0.05; two-way ANOVA including Sidak’s multiple comparison test.

Figure 6

The expression of antisense Tf2 transcripts is developmentally regulated. (A) Meiosis progression in synchronized diploid h⁺/h⁺ pat1-114/pat1-114 cells. The data and error bars represent the average and SD from three independent experiments. At least 100 cells were counted by fluorescence microscopy for each time point. (B) Northern blot analysis of total RNA prepared from synchronized diploid h⁺/h⁺ pat1-114/pat1-114 cells at each hour from 0 to 8 hr (lanes 1–9), 10 hr (lane 10), and 12 hr (lane 11) after meiotic induction. The blot was hybridized using probes complementary to Tf2 antisense RNA, Tf2 mRNA, and 25s rRNA. The asterisk indicates a shorter antisense transcript mapping to the 3′ end of the Tf2 ORF. (C and D) Quantification of Northern blots data for antisense Tf2 RNA (C) and the Tf2 mRNA (D) levels. The data and error bars represent the average and SD from at least three independent experiments. * P < 0.05, ** P < 0.01; Student’s t-test.

Figure 7

Regulation of Tf2 expression by Pab2 and Abpl in fission yeast. Suggested model for how the absence of Pab2/Rrp6 attenuates the derepression of Tf2 transcription detected in the abplΔ single mutant. In wild-type cells (top panel), Abpl facilitates the recruitment of histone deacetylases such as Clr3 and Clr6 at Tf2 loci, repressing transcriptional activity at LTRs and Tf2 internal cryptic promoters. Concurrently, Pab2 collaborates with Rrp6 to promote degradation of Tf2 mRNA and antisense (Tf2AS) transcripts at the site of transcription. In the abplΔ/pab2Δ double mutant (bottom panel), the absence of Abp1 allows increased transcriptional activity from LTRs and internal cryptic promoters, whereas the absence of Pab2 reduces the capacity of Rrp6 to degrade Tf2 mRNA and Tf2AS transcripts. The accumulating levels Tf2AS at the site of transcription may act in cis to promote Tf2 repression via RNAi or redundant mechanisms that prevent the accumulation of dsRNA.