Neurospora spore killers Sk-2 and Sk-3 suppress meiotic silencing by unpaired DNA

In Neurospora crassa, pairing of homologous DNA segments is monitored during meiotic prophase I. Any genes not paired with a homolog, as well as any paired homologs of that gene, are silenced during the sexual phase by a mechanism known as meiotic silencing by unpaired DNA (MSUD). Two genes required for MSUD have been described previously: sad-1 (suppressor of ascus dominance), encoding an RNA-directed RNA polymerase, and sad-2, encoding a protein that controls the perinuclear localization of SAD-1. Inactivation of either sad-1 or sad-2 suppresses MSUD. We have now shown that MSUD is also suppressed by either of two Spore killer strains, Sk-2 and Sk-3. These were both known to contain a haplotype segment that behaves as a meiotic drive element in heterozygous crosses of killer x sensitive. Progeny ascospores not carrying the killer element fail to mature and are inviable. Crosses homozygous for either of the killer haplotypes suppress MSUD even though ascospores are not killed. The killer activity maps to the same 30-unit-long region within which recombination is suppressed in killer x sensitive crosses. We suggest that the region contains a suppressor of MSUD.     

Characterization of Interactions Between and Among Components of the Meiotic Silencing by Unpaired DNA Machinery in Neurospora crassa Using Bimolecular Fluorescence Complementation

Bimolecular fluorescence complementation (BiFC) is based on the complementation between two nonfluorescent fragments of the yellow fluorescent protein (YFP) when they are united by interactions between proteins covalently linked to them. We have successfully applied BiFC in Neurospora crassa using two genes involved in meiotic silencing by unpaired DNA (MSUD) and observed macromolecular complex formation involving only SAD-1 proteins, only SAD-2 proteins, and mixtures of SAD-1 and SAD-2 proteins.     

Efficient Detection of Unpaired DNA Requires a Member of the Rad54-Like Family of Homologous Recombination Proteins

Meiotic silencing by unpaired DNA (MSUD) is a process that detects unpaired regions between homologous chromosomes and silences them for the duration of sexual development. While the phenomenon of MSUD is well recognized, the process that detects unpaired DNA is poorly understood. In this report, we provide two lines of evidence linking unpaired DNA detection to a physical search for DNA homology. First, we have found that a putative SNF2-family protein (SAD-6) is required for efficient MSUD in Neurospora crassa. SAD-6 is closely related to Rad54, a protein known to facilitate key steps in the repair of double-strand breaks by homologous recombination. Second, we have successfully masked unpaired DNA by placing identical transgenes at slightly different locations on homologous chromosomes. This masking falls apart when the distance between the transgenes is increased. We propose a model where unpaired DNA detection during MSUD is achieved through a spatially constrained search for DNA homology. The identity of SAD-6 as a Rad54 paralog suggests that this process may be similar to the searching mechanism used during homologous recombination.     

Meiotic silencing by unpaired DNA.

The silencing of gene expression by segments of DNA present in excess of the normal number is called cosuppression in plants and quelling in fungi. We describe a related process, meiotic silencing by unpaired DNA (MSUD). DNA unpaired in meiosis causes silencing of all DNA homologous to it, including genes that are themselves paired. A semidominant Neurospora mutant, Sad-1, fails to perform MSUD. Sad-1 suppresses the sexual phenotypes of many ascus-dominant mutants. MSUD may provide insights into the function of genes necessary for meiosis, including genes for which ablation in vegetative life would be lethal. It may also contribute to reproductive isolation of species within the genus Neurospora. The wild-type allele, sad-1(+), encodes a putative RNA-directed RNA polymerase.     

Mutants defective in a Mucor circinelloides dicer-like gene are not compromised in siRNA silencing but display developmental defects

Dicer proteins are ribonuclease III enzymes that process double stranded RNA precursors into small RNAs categorized as small interfering RNAs (siRNAs) or microRNAs (miRNAs), which suppress gene expression through the RNA silencing mechanism. We have isolated a dicer-like gene (dcl-1) of Mucor circinelloides, the first gene of this family to be identified in zygomycetes. The dcl-1 mRNA occurred in multiple forms, including the truncated molecules that result from premature polyadenylation. Null dcl-1 mutants were not impaired as regards transgene-induced gene silencing, since they exhibited the same silencing frequency as the wild-type strain and accumulated the two size classes of siRNA associated with RNA silencing in M. circinelloides. However, dcl-1 mutants showed a reduced growth rate and a hyphal growth alteration, which suggests that the dcl-1 gene has some role in the control of endogenous functions.     

Complex Formation of RNA Silencing Proteins in the Perinuclear Region of Neurospora crassa

In Neurospora, genes not paired during meiosis are targeted by meiotic silencing by unpaired DNA (MSUD). Here, our bimolecular fluorescence complementation (BiFC) study suggests that RNA-directed RNA polymerase, Dicer, Argonaute, and others form a silencing complex in the perinuclear region, with intimate interactions among the majority of them. We have also shown that SAD-2 is likely the anchor for this assembly.     

RNA silencing in fungi: mechanisms and applications

Two RNA silencing-related phenomena, quelling and meiotic silencing by unpaired DNA (MSUD) have been identified in the fungus Neurospora crassa. Similar to the case with the siRNA and miRNA pathways in Drosophila, different sets of protein components including RNA-dependent RNA polymerase, argonaute and dicer, are used in the quelling and MSUD pathways. Orthologs of the RNA silencing components are found in most, but not all, fungal genomes currently available in the public databases, indicating that the majority of fungi possess the silencing machinery. Advantage and disadvantage of RNA silencing as a tool to explore gene function in fungi are discussed.     

Characterization of hypovirus-derived small RNAs generated in the chestnut blight fungus by an inducible DCL-2-dependent pathway

The disruption of one of two dicer genes, dcl-2, of the chestnut blight fungus Cryphonectria parasitica was recently shown to increase susceptibility to mycovirus infection (G. C. Segers, X. Zhang, F. Deng, Q. Sun, and D. L. Nuss, Proc. Natl. Acad. Sci. USA 104:12902-12906, 2007). We now report the accumulation of virus-derived small RNAs (vsRNAs) in hypovirus CHV1-EP713-infected wild-type and dicer gene dcl-1 mutant C. parasitica strains but not in hypovirus-infected dcl-2 mutant and dcl-1 dcl-2 double-mutant strains. The CHV1-EP713 vsRNAs were produced from both the positive and negative viral RNA strands at a ratio of 3:2 in a nonrandom distribution along the viral genome. We also show that C. parasitica responds to hypovirus and mycoreovirus infections with a significant increase (12- to 20-fold) in dcl-2 expression while the expression of dcl-1 is increased only modestly (2-fold). The expression of dcl-2 is further increased (~35-fold) following infection with a hypovirus CHV1-EP713 mutant that lacks the p29 suppressor of RNA silencing. The combined results demonstrate the biogenesis of mycovirus-derived small RNAs in a fungal host through the action of a specific dicer gene, dcl-2. They also reveal that dcl-2 expression is significantly induced in response to mycovirus infection by a mechanism that appears to be repressed by the hypovirus-encoded p29 suppressor of RNA silencing.     

Histone H3K56 Acetylation Is Required for Quelling-induced Small RNA Production through Its Role in Homologous Recombination

Quelling and DNA damage-induced small RNA (qiRNA) production are RNA interference (RNAi)-related phenomenon from repetitive genomic loci in Neurospora. We have recently proposed that homologous recombination from repetitive DNA loci allows the RNAi pathway to recognize repetitive DNA to produce small RNA. However, the mechanistic detail of this pathway remains largely unclear. By systematically screening the Neurospora knock-out library, we identified RTT109 as a novel component required for small RNA production. RTT109 is a histone acetyltransferase for histone H3 lysine 56 (H3K56) and H3K56 acetylation is essential for the small RNA biogenesis pathway. Furthermore, we showed that RTT109 is required for homologous recombination and H3K56Ac is enriched around double strand break, which overlaps with RAD51 binding. Taken together, our results suggest that H3K56 acetylation is required for small RNA production through its role in homologous recombination.     

Genetic interactions between DNA demethylation and methylation in Arabidopsis

DNA demethylation in Arabidopsis (Arabidopsis thaliana) is mediated by DNA glycosylases of the DEMETER family. Three DEMETER-LIKE (DML) proteins, REPRESSOR OF SILENCING1 (ROS1), DML2, and DML3, function to protect genes from potentially deleterious methylation. In Arabidopsis, much of the DNA methylation is directed by RNA interference (RNAi) pathways and used to defend the genome from transposable elements and their remnants, repetitive sequences. Here, we investigated the relationship between DML demethylation and RNAi-mediated DNA methylation. We found that genic regions demethylated by DML enzymes are enriched for small interfering RNAs and generally contain sequence repeats, transposons, or both. The most common class of small interfering RNAs was 24 nucleotides long, suggesting a role for an RNAi pathway that depends on RNA-DEPENDENT RNA POLYMERASE2 (RDR2). We show that ROS1 removes methylation that has multiple, independent origins, including de novo methylation directed by RDR2-dependent and -independent RNAi pathways. Interestingly, in rdr2 and drm2 mutant plants, we found that genes demethylated by ROS1 accumulate CG methylation, and we propose that this hypermethylation is due to the ROS1 down-regulation that occurs in these mutant backgrounds. Our observations support the hypothesis that DNA demethylation by DML enzymes is one mechanism by which Arabidopsis genes are protected from genome defense pathways.     

两种高效 RNA 干涉载体系统的构建及应用

在真核细胞基因功能研究中, RNA 干涉 (RNAi) 已成为一种强有力的选择性沉默基因表达的实验工具. 建立一套可在哺乳动物培养细胞中高效、经济地表达 siRNA 的载体系统是 RNA 干涉研究的必要前提之一. 从 HepG2 细胞基因组 DNA 中克隆得到 H1 全长启动子 (374 bp),以之为基础构建了两套 RNA 干涉载体系统, pSL 和带有绿色荧光蛋白 (EGFP) 标签的 pESL ,并对 p53 基因进行了相应的 RNA 干涉研究. 干涉质粒瞬时转染 HepG2 细胞后,分别利用半定量 RT-PCR 和蛋白质印迹检测 p53 表达水平. 与商品化载体 pSilencer^TM 3.1-H1 hygro 相比, pSL 和 pESL 对 p53 基因表达具有更高的干涉效率. 结果显示：干涉载体 pSL 和 pESL 能高效特异地下调目的基因表达,可作为哺乳动物中基因功能分析的有效工具.     

Neurospora crassa,a Model System for Epigenetics Research

The filamentous fungus Neurospora crassa has provided a rich source of knowledge on epigenetic phenomena that would have been difficult or impossible to gain from other systems. Neurospora sports features found in higher eukaryotes but absent in both budding and fission yeast, including DNA methylation and H3K27 methylation, and also has distinct RNA interference (RNAi)-based silencing mechanisms operating in mitotic and meiotic cells. This has provided an unexpected wealth of information on gene silencing systems. One silencing mechanism, named repeat-induced point mutation (RIP), has both epigenetic and genetic aspects and provided the first example of a homology-based genome defense system. A second silencing mechanism, named quelling, is an RNAi-based mechanism that results in silencing of transgenes and their native homologs. A third, named meiotic silencing, is also RNAi-based but is distinct from quelling in its time of action, targets, and apparent purpose.