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During the sexual phase of Neurospora crassa, unpaired genes are subject to a silencing mechanism known as meiotic silencing by unpaired DNA (MSUD). MSUD targets the transcripts of an unpaired gene and utilizes typical RNA interference (RNAi) factors for its process. Using a reverse genetic screen, we have identified a meiotic silencing gene called sad-9, which encodes a DEAD-box RNA helicase. While not essential for vegetative growth, SAD-9 plays a crucial role in both sexual development and MSUD. Our results suggest that SAD-9, with the help of the SAD-2 scaffold protein, recruits the SMS-2 Argonaute to the perinuclear region, the center of MSUD activity.

rs5758550 has been associated with enhanced transcription and suggested to be a useful marker of CYP2D6 activity. As there are limited and inconsistent data regarding the utility of this distant "enhancer" single nucleotide polymorphism (SNP), our goal was to further assess the impact of rs5758550 on CYP2D6 activity toward two probe substrates, atomoxetine (ATX) and dextromethorphan (DM), using in vivo urinary metabolite (DM; n = 188) and pharmacokinetic (ATX; n = 70) and in vitro metabolite formation (ATX and DM; n = 166) data. All subjects and tissues were extensively genotyped, the "enhancer" SNP phased with established CYP2D6 haplotypes either computationally or experimentally, and the impact on CYP2D6 activity investigated using several linear models of varying complexity to determine the proportion of variability in CYP2D6 activity captured by each model. For all datasets and models, the "enhancer" SNP had no or only a modest impact on CYP2D6 activity prediction. An increased effect, when present, was more pronounced for ATX than DM suggesting potential substate-dependency. In addition, CYP2D6*2 alleles with the "enhancer" SNP were associated with modestly higher metabolite formation rates in vitro, but not in vivo; no effect was detected for CYP2D6*1 alleles with "enhancer" SNP. In summary, it remains inconclusive whether the small effects detected in this investigation are indeed caused by the "enhancer" SNP or are rather due to its incomplete linkage with other variants within the gene. Taken together, there does not appear to be sufficient evidence to warrant the "enhancer" SNP be included in clinical CYP2D6 pharmacogenetic testing.

Cytochrome P450 2D6 (CYP2D6) copy number (CN) variation affects the metabolism of numerous prescribed drugs. Sequence variation within primer or probe target regions of hydrolysis probe CN assays can generate false-positive calls for CN loss. Furthermore, CYP2D6–CYP2D7 hybrids and gene conversions can cause difficult to interpret discordant CN calls. The identification of haplotypes with CN variations and structural arrangements is important to predict phenotype accurately. During clinical testing with hydrolysis probe assays targeting three CYP2D6 regions (intron 2, intron 6, and exon 9), samples with haplotypes causing inconsistent CN calls were identified. To resolve these cases, next-generation sequencing and allele-specific Sanger sequencing was performed. Sequence analysis of 16 samples, all but one from subjects of African descent, identified six novel suballeles containing single-nucleotide polymorphisms, which cause false-positive calls for CN loss in introns 2 and 6. Five samples with an exon 9 CN loss contained CYP2D6/CYP2D7 hybrids (∗13 or ∗36) and one sample was found to have a novel haplotype, CYP2D6∗141. Interestingly, CYP2D6∗141 contains a CYP2D7-derived exon 9 conversion and core single-nucleotide polymorphisms that are otherwise found in CYP2D6∗17 and ∗27. Although these variants are rare, they can cause inconsistent CN calls that typically are reported as no calls or indeterminant, and thus may deprive patients, particularly those of African descent, from taking full benefit of pharmacogenetic testing.

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.

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.

Background: The CYP2D6 gene locus has been extensively studied over decades, yet a portion of variability in CYP2D6 activity cannot be explained by known sequence variations within the gene, copy number variation or structural rearrangements. It was proposed that rs5758550 located 116 kb downstream of the CYP2D6 gene locus increases gene expression and thus contributes to variability in CYP2D6 activity. This finding has, however, not been validated. The purpose of the study was to address a major technological barrier, i.e. experimentally linking rs5758550, also referred to as the ‘enhancer’ SNP, to CYP2D6 haplotypes >100 kb away. To overcome this challenge is essential to ultimately determine the contribution of the ‘enhancer’ SNP to interindividual variability in CYP2D6 activity. Methods: A large ethnically mixed population sample (n=3162) was computationally phased to determine linkage between the ‘enhancer’ SNP and CYP2D6 haplotypes (or star alleles). To experimentally validate predicted linkages, DropPhase2D6, a digital droplet PCR (ddPCR)-based method was developed. 10X Genomics Linked-Reads were utilized as a proof of concept. Results: Phasing predicted that the ‘enhancer’ SNP can occur on numerous CYP2D6 haplotypes including CYP2D6 *1, *2, *5 and *41 and suggested that linkage is incomplete, i.e. a portion of these alleles do not have the ‘enhancer’ SNP. Phasing also revealed differences amongst the European and African ancestry data sets regarding the proportion of alleles with and without the ‘enhancer’ SNP. DropPhase2D6 was utilized to confirm or refute the predicted ‘enhancer’ SNP location for individual samples, e.g. of n=3 samples genotyped as *1/*41, rs5758550 was on the *41 allele of two samples and on *1 allele of one sample. Our findings highlights that the location of the ‘enhancer’ SNP must not be assigned by ‘default’. Furthermore, linkage between the ‘enhancer’ SNP and CYP2D6 star allele haplotypes was confirmed with 10X Genomics technology. We were unable, however, to verify selected haplotypes predicted by Ray et al (PMID 30520769). Conclusions: Since the ‘enhancer’ SNP can be present on a portion of normal, decreased, or no function alleles, the phase of the ‘enhancer’ SNP must be considered when investigating the impact of the ‘enhancer’ SNP on CYP2D6 activity.

In the filamentous fungus Neurospora crassa, cross walls between individual cells are normally incomplete, making the entire fungal network vulnerable to attack by viruses and selfish DNAs. Accordingly, several genome surveillance mechanisms are maintained to help the fungus combat these repetitive elements. One of these defense mechanisms is called meiotic silencing by unpaired DNA (MSUD), which identifies and silences unpaired genes during meiosis. Utilizing common RNA interference (RNAi) proteins, such as Dicer and Argonaute, MSUD targets mRNAs homologous to the unpaired sequence to achieve silencing. In this study, we have identified an additional silencing component, namely the cap-binding complex (CBC). Made up of cap-binding proteins CBP20 and CBP80, CBC associates with the 5′ cap of mRNA transcripts in eukaryotes. The loss of CBC leads to a deficiency in MSUD activity, suggesting its role in mediating silencing. As confirmed in this study, CBC is predominantly nuclear, although it is known to travel in and out of the nucleus to facilitate RNA transport. As seen in animals but not in plants, CBP20’s robust nuclear import depends on CBP80 in Neurospora. CBC interacts with a component (Argonaute) of the perinuclear meiotic silencing complex (MSC), directly linking the two cellular factors.

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.

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.

During meiosis in the filamentous fungus Neurospora crassa, unpaired genes are identified and silenced by a process known as meiotic silencing by unpaired DNA (MSUD). Previous work has uncovered six proteins required for MSUD, all of which are also essential for meiotic progression. Additionally, they all localize in the perinuclear region, suggesting that it is a center of MSUD activity. Nevertheless, at least a subset of MSUD proteins must be present inside the nucleus, as unpaired DNA recognition undoubtedly takes place there. In this study, we identified and characterized two new proteins required for MSUD, namely SAD-4 and SAD-5. Both are previously uncharacterized proteins specific to Ascomycetes, with SAD-4 having a range that spans several fungal classes and SAD-5 seemingly restricted to a single order. Both genes appear to be predominantly expressed in the sexual phase, as molecular study combined with analysis of publicly available mRNA-seq datasets failed to detect significant expression of them in the vegetative tissue. SAD-4, like all known MSUD proteins, localizes in the perinuclear region of the meiotic cell. SAD-5, on the other hand, is found in the nucleus (as the first of its kind). Both proteins are unique compared to previously identified MSUD proteins in that neither is required for sexual sporulation. This homozygous-fertile phenotype uncouples MSUD from sexual development and allows us to demonstrate that both SAD-4 and SAD-5 are important for the production of masiRNAs, which are the small RNA molecules associated with meiotic silencing.

RNA interference (RNAi) depends on the production of small RNA to regulate gene expression in eukaryotes. Two RNAi systems exist to control repetitive selfish elements in Neurospora crassa. Quelling targets transgenes during vegetative growth, whereas meiotic silencing by unpaired DNA (MSUD) silences unpaired genes during meiosis. The two mechanisms require common RNAi proteins, such as RNA-directed RNA polymerases, Dicers, and Argonaute slicers. We have previously demonstrated that, while Quelling depends on the redundant dicer activity of DCL-1 and DCL-2, only DCL-1 is required for MSUD. Here, we show that QDE-2-interacting protein (QIP), an exonuclease that is important for the production of single-stranded siRNA during Quelling, is also required for MSUD. QIP is crucial for sexual development and is shown to colocalize with other MSUD proteins in the perinuclear region.