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. E UKARYOTIC genomes are protected from viruses and transposons by a variety of defenses, many of which are based on RNA interference (RNAi). In a typical RNA silencing process, a double-stranded RNA is cleaved into small RNAs of 21-25 nt by an RNase III enzyme known as Dicer (Chang et al. 2012). An Argonaute-containing complex incorporates these small RNA species and uses them to guide transcriptional or post-transcriptional gene silencing.Neurospora crassa, a filamentous fungus, is protected by at least two RNA silencing processes. The first process, called quelling (Romano and Macino 1992), defends the N. crassa genome from repetitive elements such as transposons (Nolan et al. 2005). The quelling machinery may also play an important role in rDNA stability and DNA damage response (Cecere and Cogoni 2009;Lee et al. 2009). The second defense process, known as meiotic silencing by unpaired DNA (MSUD) (Shiu et al. 2001), works specifically in meiotic cells and silences genes that are not paired between homologous chromosomes (Kelly and Aramayo 2007;Chang et al. 2012). Because parental genomes are likely to have differentially located transposons, MSUD is well suited to protect an organism from their amplification during meiosis.In N. crassa, meiosis and sexual spore (ascospore) formation take place in specialized sac cells (asci). During homolog pairing, MSUD scans for the presence of unpaired DNA. If such unpaired DNA is detected, MSUD will silence...
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.
In Neurospora crassa, unpaired genes are silenced by a mechanism called meiotic silencing by unpaired DNA (MSUD). Although some RNA interference proteins are necessary for this process, its requirement of small RNAs has yet to be formally established. Here we report the characterization of small RNAs targeting an unpaired region, using Illumina sequencing.N EUROSPORA crassa, a filamentous fungus, is composed of a network of thread-like cells called hyphae. Like other coenocytic organisms, in which nuclei (and other organelles) share the same cytoplasm, it is especially susceptible to systemic invasion by viruses or transposons. Accordingly, several gene-silencing mechanisms are maintained in this fungus (Catalanotto et al. 2006;Dang et al. 2011). For example, quelling, an RNA interference (RNAi) mechanism, silences the expression of hyperhaploid transgenes during the vegetative phase (Romano and Macino 1992). Repeat-induced point mutation (RIP), on the other hand, is a premeiotic process that targets duplicated sequences for G:C to A:T mutations (Cambareri et al. 1989).We are interested in another genome surveillance system known as meiotic silencing by unpaired DNA (MSUD) (Aramayo and Metzenberg 1996;Shiu et al. 2001;Kelly and Aramayo 2007). In this system, genes unpaired during the pairing stage in meiotic prophase I, as well as any homologous copies found in the zygote, are transiently silenced during the sexual phase. Our current model suggests that an unpaired DNA triggers the production of sequence-specific single-stranded aberrant RNAs, which are converted into double strands by the SAD-1 RNA-directed RNA polymerase (RdRP) (Shiu and Metzenberg 2002). A double-stranded RNA (dsRNA) is processed into single-stranded small interfering (si)RNAs (by the DCL-1 Dicer and the QIP exonuclease) (Alexander et al. 2008;Lee et al. 2010a;Xiao et al. 2010), which subsequently direct the SMS-2 Argonaute slicer to cleave homologous mRNAs (Lee et al. 2003).Illumina sequencing, previously known as Solexa sequencing, is a high-throughput sequencing method driven by a proprietary parallel sequence-by-synthesis technology (Bennett et al. 2005;Bentley et al. 2008). The standard single-read length, although not ideal for resolving short sequence repeats (Morozova and Marra 2008), is extremely suitable for large-scale analyses of small RNAs (Czech et al. 2008;Qi et al. 2009). A variety of small RNA species have been profiled in N. crassa, including QDE-2-interacting small RNAs (qiRNAs), microRNA-like RNAs (milRNAs), and dicer-independent small interfering RNAs (disiRNAs) (Lee et al. 2009(Lee et al. , 2010b. Although MSUD depends on several proteins that are implicated in RNAi (Siomi and Siomi 2009), the involvement of small RNAs in the process has not been formally established. In this work, we set out to detect and characterize small RNAs targeting an unpaired region, using Illumina sequencing. Results Setup of a cross containing an unpaired regionUnpaired genes are targeted for silencing during sexual development in Neu...
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 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.
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