Involvement of RNA granule proteins in meiotic silencing by unpaired DNA

In Neurospora crassa, expression from an unpaired gene is suppressed by a mechanism known as meiotic silencing by unpaired DNA (MSUD). MSUD utilizes common RNA interference (RNAi) factors to silence target mRNAs. Here, we report that Neurospora CAR-1 and CGH-1, homologs of two Caenorhabditis elegans RNA granule components, are involved in MSUD. These fungal proteins are found in the perinuclear region and P-bodies, much like their worm counterparts. They interact with components of the meiotic silencing complex (MSC), including the SMS-2 Argonaute. This is the first time MSUD has been linked to RNA granule proteins.

Recombination-independent recognition of DNA homology for meiotic silencing in Neurospora crassa

The pairing of homologous chromosomes represents a critical step of meiosis in nearly all sexually reproducing species. In many organisms, pairing involves chromosomes that remain apparently intact. The mechanistic nature of homology recognition at the basis of such pairing is unknown. Using “meiotic silencing by unpaired DNA” (MSUD) as a model process, we demonstrate the existence of a cardinally different approach to DNA homology recognition in meiosis. The main advantage of MSUD over other experimental systems lies in its ability to identify any relatively short DNA fragment lacking a homologous allelic partner. Here, we show that MSUD does not rely on the canonical mechanism of meiotic recombination, yet it is promoted by REC8, a conserved component of the meiotic cohesion complex. We also show that certain patterns of interspersed homology are recognized as pairable during MSUD. Such patterns need to be colinear and must contain short tracts of sequence identity spaced apart at 21 or 22 base pairs. By using these periodicity values as a guiding parameter in all-atom molecular modeling, we discover that homologous DNA molecules can pair by forming quadruplex-based contacts with an interval of 2.5 helical turns. This process requires right-handed plectonemic coiling and additional conformational changes in the intervening double-helical segments. Our results 1) reconcile genetic and biophysical evidence for the existence of direct homologous double-stranded DNA (dsDNA)–dsDNA pairing, 2) identify a role for this process in initiating RNA interference, and 3) suggest that chromosomes can be cross-matched by a precise mechanism that operates on intact dsDNA molecules.

Meiotic Silencing in Pigs: A Case Study in a Translocated Azoospermic Boar

Carriers of balanced constitutional reciprocal translocations usually present a normal phenotype, but often show reproductive disorders. For the first time in pigs, we analyzed the meiotic process of an autosome–autosome translocation associated with azoospermia. Meiotic process analysis revealed the presence of unpaired autosomal segments with histone γH2AX accumulation sometimes associated with the XY body. Additionally, γH2AX signals were observed on apparently synapsed autosomes other than the SSC1 or SSC15, as previously observed in Ataxia with oculomotor apraxia type 2 patients or knock-out mice for the Senataxin gene. Gene expression showed a downregulation of genes selected on chromosomes 1 and 15, but no upregulation of SSCX genes. We hypothesized that the total meiotic arrest observed in this boar might be due to the silencing of crucial autosomal genes by the mechanism referred to as meiotic silencing of unsynapsed chromatin (MSUC).

An NCBP3-Domain Protein Mediates Meiotic Silencing by Unpaired DNA

In the filamentous fungus Neurospora crassa, genes unpaired during meiosis are silenced by a process known as meiotic silencing by unpaired DNA (MSUD). MSUD utilizes common RNA interference (RNAi) proteins, such as Dicer and Argonaute, to target homologous mRNAs for silencing. Previously, we demonstrated that nuclear cap-binding proteins NCBP1 and NCBP2 are involved in MSUD. We report here that SAD-8, a protein similar to human NCBP3, also mediates silencing. Although SAD-8 is not essential for either vegetative or sexual development, it is required for MSUD. SAD-8 localizes predominantly in the nucleus and interacts with both NCBP1 and NCBP2. Similar to NCBP1 and NCBP2, SAD-8 interacts with a component (Argonaute) of the perinuclear meiotic silencing complex (MSC), further implicating the involvement of cap-binding proteins in silencing.

Recombination-independent recognition of DNA homology for meiotic silencing inNeurospora crassa

ABSTRACTPairing of homologous chromosomes represents a key aspect of meiosis in nearly all sexually reproducing species. While meiotic pairing requires the formation of double-strand DNA breaks in some organisms, in many others it can proceed in the apparent absence of DNA breakage and recombination. The mechanistic nature of such recombination-independent pairing represents a fundamental question in molecular biology. Using “meiotic silencing by unpaired DNA” (MSUD) in the fungusNeurospora crassaas a model system, we demonstrate the existence of a cardinally new solution to the problem of inter-chromosomal homology recognition during meiosis. Here we take advantage of the unique ability of MSUD to efficiently detect and silence (by RNA interference) any relatively short DNA fragment lacking a homologous allelic partner. We show that MSUD does not require the function of eukaryotic RecA proteins and the type II topoisomerase-like protein Spo11. We further show that MSUD can recognize patterns of weak interspersed homology in which short units of sequence identity are arrayed with a periodicity of 11 base-pairs (bp). Taken together, these results reveal the function of a recombination-independent homology-directed process in guiding the expression of small interfering RNAs and suggest that meiotic chromosomes can be evaluated for sequence homology at base-pair resolution by a mechanism that operates on intact DNA molecules.

Suppressors of Meiotic Silencing by Unpaired DNA

Meiotic silencing by unpaired DNA (MSUD) is a gene silencing process that occurs within meiotic cells of Neurospora crassa and other fungi. We have previously developed a high-throughput screen to identify suppressors of this silencing pathway. Here, a list of MSUD suppressor candidates from a single pass of the first 84 plates of the Neurospora knockout library is provided.