QIP, a Protein That Converts Duplex siRNA Into Single Strands, Is Required for Meiotic Silencing by Unpaired DNA

Xiao, Hua; Alexander, William G.; Hammond, Thomas M.; Boone, Erin C.; Perdue, Tony D.; Pukkila, Patricia J.; Shiu, Patrick

doi:10.1534/genetics.110.118273

Cited by 47 publications

(63 citation statements)

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“…Since the initial discovery of MSUD in the laboratory of R. L. Metzenberg, much has been learned about the latter process. Essentially, it appears to be mediated by an elaborate silencing complex that stations itself around the perimeter of the nucleus, (Catalanotto et al 2004;Maiti et al 2007;Alexander et al 2008;Lee et al 2010;Xiao et al 2010). …”

Section: Discussionmentioning

confidence: 99%

“…These seven include common RNA interference (RNAi) proteins such as an RNA-directed RNA polymerase called SAD-1 (Shiu et al 2001), an Argonaute protein called SMS-2 (Lee et al 2003), and a Dicer protein called DCL-1 (Alexander et al 2008). The four others are SAD-2, a presumptive scaffold protein (Shiu et al 2006); SAD-3, a putative helicase (Hammond et al 2011a); SAD-4, a protein required for MSUD-specific small RNA generation (Hammond et al 2013a,b); and QIP, an exonuclease (Lee et al 2010;Xiao et al 2010). The eighth characterized MSUD gene encodes a nuclear protein called SAD-5 (Hammond et al 2013b).…”

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

et al. 2014

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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...

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Section: Discussionmentioning

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

et al. 2014

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“…For fluorescent microscopy, Zeiss LSM710 and Olympus BX61 were used. Perithecial sample preparation and GFP/RFP/DAPI visualization were essentially as described (Alexander et al 2008;Xiao et al 2010). …”

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“…This aRNA is then transported to the perinuclear region, where it encounters at least six known MSUD proteins (five of which are related to other RNAi processes). These include SAD-1, an RNA-directed RNA polymerase thought to turn an aRNA into a doublestranded RNA (dsRNA) (Shiu and Metzenberg 2002); SAD-3, a helicase that may help SAD-1 form dsRNAs (Hammond et al 2011a); DCL-1, a Dicer protein that cleaves a dsRNA into small interfering RNAs (siRNAs) (Alexander et al 2008); QIP, an exonuclease that processes siRNAs into single strands (Lee et al 2010a;Xiao et al 2010); SMS-2, an Argonaute protein that uses siRNAs to target complementary mRNAs (Lee et al 2003); and SAD-2, which is the only uncommon RNA silencing protein listed here and may serve as a scaffold for other MSUD proteins in the perinuclear region (Shiu et al 2006). All six of these proteins colocalize in the nuclear periphery, suggesting that they are members of a silencing complex.…”

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Novel Proteins Required for Meiotic Silencing by Unpaired DNA and siRNA Generation inNeurospora crassa

et al. 2013

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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...

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“…Qip is required for the RNA-silencing machinery in quelling, which is vegetative-specific, meiotic silencing by unpaired DNA (MSUD) and normal sexual development (Lee et al 2010; Xiao et al 2010). Qip, together with QDE-2 and exosome, mediates the maturation of milR-1 milRNAs (Xue et al 2012).…”

Section: Introductionmentioning

confidence: 99%

Qipgene inFusarium oxysporumis required for normal hyphae morphology and virulence

et al. 2015

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Ribonucleic acid (RNA)-silencing mechanisms exist in many eukaryotes to regulate a variety of biological processes. The known molecular components are related to Dicers, Argonautes and RNA-dependent RNA polymerases. Previous biochemical studies have also suggested that Qip, with an exonuclease domain, facilitates the conversion of duplex small interfering RNAs into single strands. In our study, the Qip gene in Fusarium oxysporum was disrupted using homologous recombination technology. The deletion of the Qip gene resulted in a decrease in colony growth rates but increased the number of branches. Additionally, the ΔQip mutant had a reduced pathogenicity in cabbage. Our results show Qip gene in F. oxysporum is required for normal hyphae morphology and virulence. The mutant will be useful for elucidating the relationship between the RNA-silencing mechanism and hyphal growth and development in F. oxysporum.

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QIP, a Protein That Converts Duplex siRNA Into Single Strands, Is Required for Meiotic Silencing by Unpaired DNA

Cited by 47 publications

References 46 publications

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

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

Novel Proteins Required for Meiotic Silencing by Unpaired DNA and siRNA Generation inNeurospora crassa

Qipgene inFusarium oxysporumis required for normal hyphae morphology and virulence

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