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. MEIOSIS is fundamental to sexual reproduction. During meiosis, chromosomes are replicated, aligned, recombined, and segregated to nuclei that will develop into gametes. Two of these key processes, alignment and recombination, likely require a search for DNA homology between chromosomes (Barzel and Kupiec 2008;Moore and Shaw 2009). Such homology searching is necessary because sexual organisms inherit a copy of each chromosome from each of its parents. These chromosomes, referred to as homologs, must somehow find each other so that alignment, recombination, and segregation can occur.Although recent research has improved our understanding of homology search mechanisms (Forget and Kowalczykowski 2012;Renkawitz et al. 2013), there are many questions that remain unanswered. The filamentous fungus Neurospora crassa may be useful for investigating the unknowns of homology searching because it possesses a genetically tractable phenomenon called meiotic silencing by unpaired DNA (MSUD) (Aramayo and Selker 2013;Billmyre et al. 2013). MSUD scans pairs of homologs for segments of DNA that are not accurately paired between them. If improper pairing (i.e., unpairing) is identified, the offending sequences are silenced for the duration of sexual development. For example, if a hypothetical gene called "gene A" is on the left arm of one chromosome but on the right arm of its homolog, it will be silenced. The same holds true if gene A has been lost from one of the homologs.A functional MSUD response can be easily detected with alleles that affect ascospore (sexual spore) color or shape. Indeed, MSUD was discovered during studies of ascospore maturation-1 (asm-1), a gene required for the production of pigmented (black) ascospores . A cross between an asm-1 + strain and an asm-1 D strain produces mostly unpigmented (white) ascospores. This is because MSUD silences the unpaired asm-1 + all...

Neurospora fungi harbor a group of meiotic drive elements known as Spore killers (Sk). Spore killer-2 (Sk-2) and Spore killer-3 (Sk-3) are two Sk elements that map to a region of suppressed recombination. Although this recombination block is limited to crosses between Sk and Sk-sensitive (Sk S ) strains, its existence has hindered Sk characterization. Here we report the circumvention of this obstacle by combining a classical genetic screen with next-generation sequencing technology and three-point crossing assays. This approach has allowed us to identify a novel locus called rfk-1, mutation of which disrupts spore killing by Sk-2. We have mapped rfk-1 to a 45-kb region near the right border of the Sk-2 element, a location that also harbors an 11-kb insertion (Sk-2 INS1 ) and part of a .220-kb inversion (Sk-2 INV1 ). These are the first two chromosome rearrangements to be formally identified in a Neurospora Sk element, providing evidence that they are at least partially responsible for Sk-based recombination suppression. Additionally, the proximity of these chromosome rearrangements to rfk-1 (a critical component of the spore-killing mechanism) suggests that they have played a key role in the evolution of meiotic drive in Neurospora.G ENOMES typically consist of large groups of cooperating genes that encode information needed to maintain and reproduce themselves as well as their cellular environments. However, individual genes within genomes do not always cooperate (Dawkins 2006;Burt and Trivers 2008). For example, ranGAP in fruit flies and tcd1 in mice are selfish genes that collude with others to bias meiotic transmission ratios to their advantage (Kusano et al. 2003;Lyon 2003). While ranGAP works within a gene complex known as Segregation Distorter, tcd1 works within another called the t haplotype. Because these selfish gene complexes "drive" through meiosis, they are referred to as meiotic drive elements. Such elements are found throughout sexual organisms, with other well-known examples being X d in stalkeyed flies, Ab10 in maize, and the Spore killers in various fungi (Rhoades 1942;Raju 1994;Presgraves et al. 1997;Burt and Trivers 2008).Neurospora fungi spend most of their life cycle in a vegetative haploid phase. However, specific environmental cues (e.g., nitrogen limitation) can trigger initiation of the sexual cycle, which includes a brief diploid phase that allows meiotic drive to function in this group of fungi. Sexual reproduction in Neurospora begins when an asexual spore (conidium) from a male parent donates a nucleus to an immature fruiting body (protoperithecium) of a female parent. Pairs of nuclei, one from each parent, fuse within the meiotic cells (asci) of the fertilized fruiting body (perithecium) to form a diploid nucleus that passes through the standard stages of meiosis (Raju 1980). The four haploid meiotic products then undergo a postmeiotic mitosis to produce eight nuclei, each of which is incorporated into an American football-shaped ascospore (Raju 1980). Ascospores darken as...

Sk-2 is a meiotic drive element that was discovered in wild populations of Neurospora fungi over 40 years ago. While early studies quickly determined that Sk-2 transmits itself through sexual reproduction in a biased manner via spore killing, the genetic factors responsible for this phenomenon have remained mostly unknown. Here, we identify and characterize rfk-1, a gene required for Sk-2-based spore killing. The rfk-1 gene contains four exons, three introns, and two stop codons, the first of which undergoes RNA editing to a tryptophan codon during sexual development. Translation of an unedited rfk-1 transcript in vegetative tissue is expected to produce a 102-amino acid protein, whereas translation of an edited rfk-1 transcript in sexual tissue is expected to produce a protein with 130 amino acids. These findings indicate that unedited and edited rfk-1 transcripts exist and that these transcripts could have different roles with respect to the mechanism of meiotic drive by spore killing. Regardless of RNA editing, spore killing only succeeds if rfk-1 transcripts avoid silencing caused by a genome defense process called meiotic silencing by unpaired DNA (MSUD). We show that rfk-1's MSUD avoidance mechanism is linked to the genomic landscape surrounding the rfk-1 gene, which is located near the Sk-2 border on the right arm of chromosome III. In addition to demonstrating that the location of rfk-1 is critical to spore-killing success, our results add to accumulating evidence that MSUD helps protect Neurospora genomes from complex meiotic drive elements.

Aberrant alternative splicing of mRNAs results in dysregulated gene expression in multiple neurological disorders. Here, we show that hundreds of mRNAs are incorrectly expressed and spliced in white blood cells and brain tissues of individuals with fragile X syndrome (FXS). Surprisingly, the FMR1 (Fragile X Messenger Ribonucleoprotein 1) gene is transcribed in >70% of the FXS tissues. In all FMR1 -expressing FXS tissues, FMR1 RNA itself is mis-spliced in a CGG expansion–dependent manner to generate the little-known FMR1 -217 RNA isoform, which is comprised of FMR1 exon 1 and a pseudo-exon in intron 1. FMR1 -217 is also expressed in FXS premutation carrier–derived skin fibroblasts and brain tissues. We show that in cells aberrantly expressing mis-spliced FMR1 , antisense oligonucleotide (ASO) treatment reduces FMR1 -217, rescues full-length FMR1 RNA, and restores FMRP (Fragile X Messenger RibonucleoProtein) to normal levels. Notably, FMR1 gene reactivation in transcriptionally silent FXS cells using 5-aza-2′-deoxycytidine (5-AzadC), which prevents DNA methylation, increases FMR1 -217 RNA levels but not FMRP. ASO treatment of cells prior to 5-AzadC application rescues full-length FMR1 expression and restores FMRP. These findings indicate that misregulated RNA-processing events in blood could serve as potent biomarkers for FXS and that in those individuals expressing FMR1-217 , ASO treatment may offer a therapeutic approach to mitigate the disorder.

34Meiotic drive elements like Spore killer-2 (Sk-2) in Neurospora are transmitted through sexual 35 reproduction to the next generation in a biased manner. Sk-2 achieves this biased transmission 36 through spore killing. Here, we identify rfk-1 as a gene required for the spore killing mechanism. 37The rfk-1 gene is associated with a 1,481 bp DNA interval (called AH36) near the right border of 38 the 30 cM Sk-2 element, and its deletion eliminates the ability of Sk-2 to kill spores. The rfk-1 39 gene also appears to be sufficient for spore killing because its insertion into a non-Sk-2 isolate 40 disrupts sexual reproduction after the initiation of meiosis. Although the complete rfk-1 41 transcript has yet to be defined, our data indicate that rfk-1 encodes a protein of at least 39 amino 42 acids and that rfk-1 has evolved from a partial duplication of gene ncu07086. We also present 43 evidence that rfk-1's location near the right border of Sk-2 is critical for the success of spore 44 killing. Increasing the distance of rfk-1 from the right border of Sk-2 causes it to be inactivated 45 by a genome defense process called meiotic silencing by unpaired DNA (MSUD), adding to 46 accumulating evidence that MSUD exists, at least in part, to protect genomes from meiotic drive. 47 48 49 50In eukaryotic organisms, genetic loci are typically transmitted through sexual reproduction to the 51 next generation in a Mendelian manner. However, some loci possess the ability to improve their 52 own transmission rate through meiosis at the expense of a competing locus. These "selfish" loci 53 are often referred to as meiotic drive elements (Zimmering et al. 1970). The genomic conflict 54 caused by meiotic drive elements may impact processes ranging from gametogenesis to 55 speciation (Lindholm et al. 2016). Meiotic drive elements are found across the eukaryote tree of 56 life (Burt and Trivers 2008; Bravo Núñez et al. 2018) and classic examples include SD in fruit 57 flies (Larracuente and Presgraves 2012), the t-complex in mice (Lyon 2003; Sugimoto 2014), 58and Ab10 in Zea mays (Rhoades 1952; Kanizay et al. 2013). In the fungal kingdom, the known 59 meiotic drive elements achieve biased transmission through spore killing (Raju 1994) and a 60 handful of spore killer systems have been studied in detail. While the prion-based spore killing 61 mechanism of het-s in Podospora anserina is the best characterized (Dalstra et al. 2003; Saupe 62 2011), the mechanisms by which other fungal meiotic drive elements kill spores are mostly 63 unknown (e.g., see Grognet et al. 2014; Hu et al. 2017; Nuckolls et al. 2017). 65Two fungal meiotic drive elements have been identified in the fungus Neurospora 66 intermedia (Turner and Perkins, 1979). This species is closely related to the genetic model 67 Neurospora crassa (Davis 2000), and the mating processes in both fungi are essentially identical. 68Mating begins with fertilization of an immature fruiting body called a protoperithecium by a 69 mating partner of the opposite mating type. After ferti...