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.epigenetics ͉ meiosis ͉ MSUD ͉ Neurospora ͉ RNA interference C oenocytic organisms, in which nuclei coexist in a common cytoplasm, are probably at especially high risk from proliferation of detrimental retrotransposons. In the haploid ascomycete Neurospora crassa, several gene-silencing mechanisms exist to maintain its genome integrity. Quelling, which defends the organism during the vegetative phase, is an RNA interference (RNAi) system that suppresses the expression of transgenes occurring in more than one copy (1, 2). Another surveillance system, known as repeat-induced point mutation (RIP), is a premeiotic process that scans the genome for duplicated sequences and targets them for C to T mutations (3). A third silencing mechanism, named meiotic silencing by unpaired DNA, scans the genome and monitors the pairing of DNA segments with their homologs during meiotic prophase (4-6). This mechanism probably prevents the expression and transposition of invasive sequences, and serves the organism in its need to counter exogenous elements and perhaps to regulate endogenous elements. Deletion or extensive mutation in an RdRPencoding gene, sad-1 (suppressor of ascus dominance), reduces meiotic silencing to a low level. RdRP plays an important role in some RNAi systems (2). For example, if foreign nucleic acids trigger the production of aberrant RNA (aRNA), the singlestranded aRNA can be replicated into a double-stranded species (dsRNA) via the activity of an RdRP. The dsRNA is then processed into small interfering RNA (siRNA) duplexes by Dicer. The siRNAs subsequently guide the cleavage of mRNA via the RNA-induced silencing complex. The fact that an RdRP is required for meiotic silencing suggests that the synthesis of dsRNA, its amplification, or both, are essential for the process.We have now identified an additional gene, sad-2, which is also required for meiotic silencing. Dominant mutations (Sad-2) can suppress the meiotic silencing of unpaired loci with efficiency comparable to that of Sad-1. A Sad-2 mutation does not give any obvious abnormal phenotype during vegetative growth and, correspondingly, sad-2 ϩ mRNA can only...

Neurospora crassa, a Model System for Epigenetics Research

“…Intriguingly, all reported components of the meiotic silencing machinery identified so far have a perinuclear localization (Fig. 9) (Shiu et al 2006). Our working model (Fig.…”

Neurospora Spore KillersSk-2andSk-3Suppress Meiotic Silencing by Unpaired DNA

“…Other genes involved in the meiotic silencing process include sad-2, which encodes a novel protein that controls the perinuclear localization of SAD-1 (Shiu et al 2006), and sms-2, which encodes an argonaute-like protein (Lee et al 2003). We report here that the Spore killer strains Sk-2 and Sk-3 contain a previously unidentified element that suppresses MSUD.…”

Dominant Suppression of Repeat-Induced Point Mutation in Neurospora crassa by a Variant Catalytic Subunit of DNA Polymerase-ζ

“…Strains Sad-2 (RIP32) A (RLM 30-12) and Sad-2 (RIP32) a (RLM33-12) (Shiu et al 2006) were kindly provided by Robert L. Metzenberg (Department of Biology, California State University, Northridge, CA). They were used to construct the heterokaryons ½(leu-3 Srp; Sad-2 a) 1 (erg-3; his-1 a) and ½(Dp(IBj5); trp-1; Sad-2 a) 1 (erg-3; his-1 a) .…”