An NCBP3-Domain Protein Mediates Meiotic Silencing by Unpaired DNA

Boone, Erin C.; Xiao, Hua; Vierling, Michael M; Decker, Logan M; Sy, Victor T; Kennedy, Rana F; Bonham, Marilyn A; Schmitz, Shannon F; John, Annie M; Hammond, Thomas M.; Shiu, Patrick

doi:10.1534/g3.120.401236

Cited by 5 publications

(6 citation statements)

References 47 publications

(84 reference statements)

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“…In Neurospora, ARS2 may indeed recruit NCBP3 (SAD-8), an essential MSUD factor, to the CBC. Also, ARS2 interacts with the SMS-2 Argonaute in the perinuclear region, once again establishing a connection between the CBC and the meiotic silencing complex (MSC) (DECKER et al 2017;BOONE et al 2020). This supports our ongoing hypothesis that the CBC and (especially) SAD-8 help SMS-2 to capture mRNAs as they are exported from the nucleus.…”

Section: V4 Discussionsupporting

confidence: 76%

“…When ARS2 (NCU02800) is removed from a strain, it exhibits a markedly reduced growth rate as compared to wild type (Figure V.1,A). This is different from the other cap-binding proteins studied, as strains without cbp20, cbp80, or sad-8 all exhibited relatively normal linear growth rates (DECKER et al 2017;BOONE et al 2020). This observation suggests that ARS2 plays a role in the vegetative cycle.…”

Section: V3i Ars2 Has a Role In Vegetative Growth And Sexual Developmentmentioning

confidence: 69%

“…Primers for DJ-PCR-based fluorescent tagging were designed as previously described (HAMMOND et al 2011b;BOONE et al 2020). It is possible that they do not interact in the perinuclear region at all or that their interaction there is indirect (e.g., through another protein).…”

Section: Tgctcaaaccgccgtaattgttgmentioning

confidence: 99%

“…This work is published in "Involvement of RNA granule proteins in meiotic BOONE et al 2020). CBC binds to a protein called arsenite resistance protein 2 (ARS2), which acts as a platform to recruit key RNA processing factors (LYKKE-ANDERSEN et al 2021).…”

Section: Iv5 Publicationmentioning

confidence: 99%

“…It has been proposed that ARS2 may help recruit NCBP3 to CBC (RAMBOUT AND MAQUAT 2021). Since CBC, NCBP3, and ARS2 have been shown to play a role in MSUD (Decker, 2015;Decker et al, 2017;Boone et al, 2020), we will explore the functions of ars2 and characterize the corresponding mutant phenotype.…”

Section: Iv5 Publicationmentioning

confidence: 99%

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Molecular dissection of the roles of RNA processing proteins in meiotic silencing by unpaired DNA

Vierling¹

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[EMBARGOED UNTIL 6/1/2023] The filamentous fungus Neurospora crassa is a model organism for the study of genome defense and epigenetic mechanisms. N. crassa utilizes meiotic silencing by unpaired DNA (MSUD) to silence unpaired genes during sexual development. MSUD requires two steps: a sequence is identified as unpaired during meiosis and any RNA homologous to that unpaired sequence is targeted for degradation. The mechanism accomplishing this degradation is RNA interference (RNAi), and many genes important to this pathway have been identified. Once a signal is produced from an unpaired sequence, most likely an RNA molecule, that RNA is converted into small interfering RNAs (siRNAs) that act as sequence specific guides for mRNA destruction. MSUD is presumed to be a strong deterrent of transposons and viruses, which can cause detrimental mutations as they proliferate and insert themselves randomly into the host's genome. The cap-binding complex (CBC) is composed of cap-binding protein 80 (CBP80) and cap-binding protein 20 (CBP20), which bind the 7-methylguanosine cap of mRNA. The CBC has been implicated in being important for steps of mRNA maturation such as splicing and for aiding mRNA export from the nucleus. Work from multiple model organisms has implicated the CBC in small RNA-mediated post transcriptional silencing of mRNA, and in Neurospora, removal of either or both CBC components has been shown to reduce the efficacy of MSUD. The CBC may mediate MSUD through its interaction with the Argonaute protein suppressor of meiotic silencing-2 (SMS-2) by delivering mRNA to SMS-2 for silencing. Elimination of either CBC component causes a reduction in sexual development but does not hamper vegetative growth. Another MSUD effector protein called SAD-8 has homology to a recently discovered mammalian protein, nuclear cap-binding protein 3 (NCBP3). Crosses in a sad-8-null background are almost completely deficient in MSUD activity, indicating that SAD-8 is a crucial player of MSUD. SAD-8 interacts with CBP20 and CBP80 (likely forming a trimer) and has a mostly nuclear localization. Also, SAD-8 and SMS-2 interact in the perinuclear region of spore sacs, which further supports a connection between the CBC and MSUD silencing complex (MSC). SAD-8, unlike many other MSUD proteins, is not important for sexual sporulation. P-bodies are cytoplasmic foci found in eukaryotic cells. They are implicated not only in normal turnover of mRNAs, but also targeted translational repression and destruction of specific mRNA. Two proteins that localize to P-bodies, CGH-1 and CAR-1, have been identified through our work as MSUD mediators. These proteins reside in both P-bodies and the perinuclear region of Neurospora spore sacs. CAR-1 and CGH-1 only interact directly in P-bodies, but they interact with several MSUD proteins (including SMS-2) in the perinuclear region. Both proteins are involved in vegetative growth and sexual development as both phases of the life cycle are affected by their removal. ARS2 is a direct binding partner of the CBC in many organisms and is theorized to be a sorting platform for diverse co-transcriptional complexes being recruited to the CBC. One such cofactor believed to be recruited to the CBC is NCBP3 in human cells. NCBP3's ortholog in Neurospora crassa, SAD-8, is a known MSUD mediator and previous results showed that ARS2 also mediates MSUD. Upon further investigation, ARS2 was shown to interact with both SAD-8 and CBC, supporting the notion that ARS2 helps recruit the former to the latter. ARS2 also interacts with SMS-2, supporting the idea that the CBC/ARS2/SAD-8 complex can help SMS-2 to more efficiently capture exported mRNAs. Besides silencing, ARS-2 also plays a role in linear growth and ascospore production.

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Section: V4 Discussionsupporting

confidence: 76%

Section: V3i Ars2 Has a Role In Vegetative Growth And Sexual Developmentmentioning

confidence: 69%

Section: Tgctcaaaccgccgtaattgttgmentioning

confidence: 99%

Section: Iv5 Publicationmentioning

confidence: 99%

Section: Iv5 Publicationmentioning

confidence: 99%

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Molecular dissection of the roles of RNA processing proteins in meiotic silencing by unpaired DNA

Vierling¹

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Gene drive by Fusarium SKC1 is dependent on its competing allele

Lohmar

Rhoades

Hammond

et al. 2022

Fungal Genetics and Biology

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Involvement of RNA granule proteins in meiotic silencing by unpaired DNA

Xiao

Vierling

Kennedy

et al. 2021

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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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An NCBP3-Domain Protein Mediates Meiotic Silencing by Unpaired DNA

Cited by 5 publications

References 47 publications

Molecular dissection of the roles of RNA processing proteins in meiotic silencing by unpaired DNA

Molecular dissection of the roles of RNA processing proteins in meiotic silencing by unpaired DNA

Gene drive by Fusarium SKC1 is dependent on its competing allele

Involvement of RNA granule proteins in meiotic silencing by unpaired DNA

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