SAD-2 is required for meiotic silencing by unpaired DNA and perinuclear localization of SAD-1 RNA-directed RNA polymerase

Abstract

A gene unpaired during the meiotic homolog pairing stage in Neurospora generates a sequence-specific signal that silences the expression of all copies of that gene. This process is called Meiotic Silencing by Unpaired DNA (MSUD). Previously, we have shown that SAD-1, an RNA-directed RNA polymerase (RdRP), is required for MSUD. We isolated a second gene involved in this process, sad-2. Mutated Sad-2 (RIP) alleles, like those of Sad-1, are dominant and suppress MSUD. Crosses homozygous for Sad-2 are blocked at meiotic prophase. SAD-2 colocalizes with SAD-1 in the perinuclear region, where small interfering RNAs have been shown to reside in mammalian cells. A functional sad-2(+) gene is necessary for SAD-1 localization, but the converse is not true. The data suggest that SAD-2 may function to recruit SAD-1 to the perinuclear region, and that the proper localization of SAD-1 is important for its activity.

Fig. 1.

The strategy for the identification of strains carrying dominant mutations that affect meiotic silencing. The expression of asm-1⁺ is required for ascospore maturation, whereas the expression of r⁺ is required for the production of spindle-shaped ascospores. Thus, crosses involving unpaired copies of the asm-1⁺ and r⁺ lead to the production of white, round ascospores (Upper). However, a dominant mutant of meiotic silencing (Sad-x) can bypass the pairing requirements of asm-1⁺ and r⁺, thus allowing the recovery of black spindle-shaped ascospores (Lower).

Fig. 2.

Meiotic arrest in asci of Sad-2 × Sad-2. Acriflavin staining is shown. (A) Ascus development is arrested in meiotic prophase, following an apparently normal karyogamy in the young asci. (B) Chromosome pairing is grossly abnormal in early pachytene stage asci; arrow shows two unpaired segments of a chromosome. (C) An arrested ascus at 5–6 days after fertilization contains a single nucleus with diffuse chromatin. (D) Normally paired pachytene chromosomes in a wild-type ascus at 4 days after fertilization. (Scale bars, 10 μm.)

Fig. 3.

Suppression of meiotic silencing of GFP-tagged histone H1 (hH1) by Sad-2. (A) The expression of unpaired hH1-GFP is completely silenced during meiosis and postmeiotic mitosis by meiotic silencing. Consequently, the nuclei do not fluoresce. (B) Meiotic silencing of hH1-GFP is suppressed by Sad-2, and the nuclei fluoresce throughout meiosis and until after ascospore delimitation. (Scale bars, 50 μm.)

Fig. 4.

Perinuclear localization of SAD-2. (A–C) Before karyogamy, SAD-2 is seen as from 1 to >20 foci (arrows) around the two fusing nuclei (arrowheads). (D–F) After karyogamy, foci (arrows) fuse around the nucleus (arrowhead). (G) At leptotene, SAD-2 is seen as crescents (arrows) and cytoplasmic foci (arrowheads). (H) Corresponding DAPI showing the nucleolus (arrow; nu) and mitochondria (arrowhead; m). (I) Nucleolus faces the upper crescent (arrow); the cytoplasmic spots of SAD-2 (arrowheads) do not coincide with mitochondria. (J–N) Four Z sections through a pachytene nucleus and the corresponding DAPI. Arrows point to SAD-2-GFP around the nucleus in J and above nucleus in M. (O and P) SAD-2 forms 20–100 small, irregular foci (O) around the diffuse stage nucleus (P). (Q and R) Two ascospores with SAD-2 foci (arrows) located in the vicinity of their nuclei (arrowheads). (S–U) RFP is covalently attached to SAD-2 (S). The overlay (U) between DAPI (T) and red signals shows that the ring formed by SAD-2-RFP (S) is located outside of the nucleus. (Scale bar, 3 μm in A–Q and S–U, and 10 μm in R.)

Fig. 5.

Colocalization of SAD-1 and SAD-2. (A–C) Colocalization of SAD-1-GFP and SAD-2-RFP in both the nuclear periphery (arrows) and the two cytoplasmic spots (arrowheads). (E–G) In some asci, the two proteins do not completely colocalize (arrows). (D and H) Corresponding DAPI of A and E, respectively. (Scale bars, 3 μm.)

Fig. 6.

The effect of sad-2 mutation on the localization of SAD-1 and the converse. (A–G) SAD-1-GFP is not localized to the perinuclear region in a Sad-2 × Sad-2 cross. (A and B) SAD-1-GFP (arrows) appears at the correct time in croziers, when the two prekaryogamy nuclei (arrowheads) are not yet fused. (C and D) In 5% of the asci, SAD-1-GFP (arrow) is seen near the nucleus. (E–G) In 95% of the asci, SAD-1-GFP (arrow) is seen in 1–20 spots distributed throughout the cytoplasm. (H–J) SAD-2-GFP is correctly localized in a Sad-1 × Sad-1 cross. During early pachytene, brighter spots are formed (arrows in H). These face the telomeres, which are grouped in a restricted area at the bouquet stage (delimited by arrowheads in I and J). (Scale bars, 3 μm.)