Importance of polyadenylation in the selective elimination of meiotic mRNAs in growing S. pombe cells

Abstract

A number of meiosis-specific mRNAs are initially weakly transcribed, but then selectively removed during fission yeast mitotic growth. These mRNAs harbour a region termed DSR (determinant of selective removal), which is recognized by the YTH family RNA-binding protein Mmi1p. Mmi1p directs the destruction of these mRNAs in collaboration with nuclear exosomes. However, detailed molecular mechanisms underlying this process of selective mRNA elimination have remained elusive. In this study, we demonstrate the critical role of polyadenylation in this process. Two-hybrid and genetic screens revealed potential interactions between Mmi1p and proteins involved in polyadenylation. Additional investigations showed that destruction of DSR-containing mRNAs by exosomes required polyadenylation by a canonical poly(A) polymerase. The recruitment of Pab2p, a poly(A)-binding protein, to the poly(A) tail was also necessary for mRNA destruction. In cells undergoing vegetative growth, Mmi1p localized with exosomes, Pab2p, and components of the polyadenylation complex in several patchy structures in the nucleoplasm. These patches may represent the sites for degradation of meiosis-specific mRNAs with untimely expression.

Figure 1

Components of the polyadenylation complex and the poly(A)-binding protein Pab2p contribute to the elimination of DSR-containing mRNAs. (A) JY450 (WT), JV564 (mmi1-ts3), JT452 (pla1-ts37) and JT453 (rna15-ts10) cells were cultured at 25°C in YE liquid medium and then shifted to 37°C. After the indicated times, total RNA was extracted from each sample and processed for northern blot analysis (lanes 1–12). For JT454 (pab2Δ), total RNA was extracted from cells cultured at 30°C (lane 13). A total of 5μg of RNA was loaded in each lane. (B) Suppression of sme2Δ by pla1-ts37, rna15-ts10 and pab2Δ. JY450 (WT), JZ464 (sme2Δ), JT452 (pla1-ts37), JT453 (rna15-ts10), JT455 (pla1-ts37 sme2Δ) and JT456 (rna15-ts10 sme2Δ) cells were cultured in YE medium at 25°C and spotted onto an SSA plate. Incubation was continued at 25°C for 4 days, and then cells were stained with iodine and the sporulation efficiency of each cell line was calculated. JT454 (pab2Δ) and JT457 (pab2Δ sme2Δ) cells and control JY450 and JZ464 cells were cultured at 30°C and after 2 days the sporulation efficiency was measured at 30°C.

Figure 2

DSR-containing mRNAs suffer Pla1p-dependent excessive polyadenylation in the rrp6-ts32 strain at the restrictive temperature. (A) Northern blot analysis of the expression of meiosis-specific genes mei4, rec8, meu1, meu2 and spo5 in exosome mutants. Cells were cultured in YE liquid medium at 25°C, shifted to the restrictive temperature 37°C and then sampled for RNA extraction after incubation for the indicated times. Lanes 1–3, JY450 (WT; wild-type); lanes 4–6, JV564 (mmi1-ts3); lanes 7–9, JT432 (rrp6-ts32); and lanes 10–12, JT449 (dis3-ts4). Total RNA (5 μg) was loaded in each lane. (B) JT432 (rrp6-ts32) and JT458 (rrp6-ts32 pla1-ts37) cells were cultured at 25°C, and then shifted to 37°C. At the indicated times, total RNA was extracted from each sample and processed for northern blot analysis as in (A). (C) Northern blots of RNase H-treated rec8 and spo5 transcripts derived from growing mutant strains as indicated. RNase H treatment was performed in the absence (lanes 1 and 2) and presence (lanes 3 and 4) of poly(dT). (D) JV564 (mmi1-ts3) and JT496 (mmi1-ts3 rrp6-ts32) cells were cultured at 25°C, and then shifted to 37°C. At the indicated times, total RNA was extracted from each sample and processed for northern blot analysis as in (A).

Figure 3

A poly(A) tail is required for the elimination of DSR-containing RNA. (A) Schematic illustration of the reporter constructs used in (B). A detailed explanation of each construct is provided in the text. adh1 pr., the promoter for the adh1 gene; GFP, the ORF for jellyfish green fluorescent protein; DSR, the DSR sequence of the spo5 gene; DSR(M10), a defective form of DSR; adh1 ter., the terminator for the adh1 gene; and snu2 ter., the terminator for the snu2 gene. (B) Constructs 1–4, as shown in (A), were integrated at the lys1 locus of the parental strain JY333. The resulting four strains were cultured in YE at 30°C. Total RNA was extracted from each culture and analysed by northern blot analysis. RNA (5 μg) was loaded in each lane and the GFP sequence was used as the probe. (C) Comparison of the band intensity in (B) between lanes 1 and 2 (adh1 term.) and lanes 3 and 4 (snu2 term.). (D) Schematic illustration of the reporter constructs used in (E). Either a poly(dA) tract or a poly(dT) tract was inserted after the DSR sequence in Construct 3 and Construct 4. The tract length was 10, 50 or 100 bases. (E) Constructs carrying a poly(dA) or poly(dT) tract, as shown in (D), were integrated at the lys1 locus of JY333. The resulting strains were examined for GFP expression by northern blot analysis as described in (B). The intensity of each band was measured and normalized by the amount of rRNA as the loading control, which is graphically shown under the northern blot panel. (F) Comparison of the band intensity in (E) between lanes 1 and 5 (A-0), lanes 2 and 6 (A-10), lanes 3 and 7 (A-50), lanes 4 and 8 (A-100), lanes 9 and 13 (T-0), lanes 10 and 14 (T-10), lanes 11 and 15 (T-50) and lanes 12 and 16 (T-100). (G) Evaluation of the necessity of Pab2p for the elimination of DSR-containing RNA with an artificial poly(A) tract. The expression of GFP was monitored by northern blot analysis in wild-type (JY450) and pab2Δ (JT454) cells carrying either Construct 3-A50 or 4-A50.

Figure 4

Cid14p is most likely not involved in the Mmi1-dependent selective elimination of the meiotic mRNAs (A) JY450 (WT), JT432 (rrp6-ts32) and JT654 (cid14Δ) cells were cultured at 25°C in YE medium, and then shifted to 37°C. At the indicated times, total RNA was extracted from each sample and processed for northern blot analysis. (B) JY450 (WT), JZ464 (sme2Δ), JT654 (cid14Δ) and JT655 (cid14Δ sme2Δ) cells were cultured in YE medium at 30°C and spotted onto an SPA plate. Incubation was continued at 30°C for 1 day, and then cells were stained with iodine and the sporulation efficiency was calculated. (C) JT432 (rrp6-ts32) and JT656 (rrp6-ts32 cid14Δ) cells were cultured at 25°C in YE medium, and then shifted to 37°C. At the indicated times, total RNA was extracted from each sample and processed for northern blot analysis.

Figure 5

Factors involved in selective elimination of DSR-containing mRNA form patchy structures in the nucleoplasm. (A–D) GFP-tagged Mmi1p (Mmi1–GFP) was co-expressed with either (A) Rrp6–mCherry, (B) Dis3–mCherry, (C) Pla1–mCherry or (D) Pab2–mCherry from the respective endogenous promoters. Three independent cells (numbered 1–3) were examined for each combination. An image of the nuclear region, stacked along the z-axis, is shown.

Figure 6

A putative model for the cooperation of Mmi1p, the polyadenylation complex, Pab2p and the exosome in the induction of selective elimination of a DSR-containing mRNA. In Step 1, Mmi1p bound to the DSR sequence on the transcript interacts with a conventional polyadenylation complex and promotes polyadenylation of apparently unusual nature. In Step 2, the poly(A)-binding protein Pab2p is recruited to the produced poly(A) tail. The affinity of Mmi1p for Pab2p may facilitate this recruitment. In Step 3, Pab2p, likely in collaboration with Mmi1p, recruits a nuclear exosome to the target transcript, which then digests it from the 3′ end.