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1.
Noncoding RNA ; 9(6)2023 Dec 14.
Article in English | MEDLINE | ID: mdl-38133210

ABSTRACT

We are delighted to share with you our thirteenth Journal Club and highlight some of the most interesting papers published recently [...].

3.
G3 (Bethesda) ; 13(8)2023 08 09.
Article in English | MEDLINE | ID: mdl-37052947

ABSTRACT

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 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.


Subject(s)
Meiosis , Neurospora crassa , Meiosis/genetics , DNA, Fungal/genetics , Fungal Proteins/genetics , Neurospora crassa/metabolism , DEAD-box RNA Helicases/genetics
4.
Noncoding RNA ; 8(3)2022 May 05.
Article in English | MEDLINE | ID: mdl-35645338

ABSTRACT

We are delighted to share with you our eleventh Journal Club and highlight some of the most interesting papers published recently [...].

6.
G3 (Bethesda) ; 11(10)2021 09 27.
Article in English | MEDLINE | ID: mdl-34568932

ABSTRACT

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.


Subject(s)
Gene Silencing , Neurospora crassa , DNA, Fungal , Meiosis , Neurospora crassa/genetics , RNA , RNA Interference
7.
Noncoding RNA ; 7(3)2021 Sep 16.
Article in English | MEDLINE | ID: mdl-34564320

ABSTRACT

We are delighted to share with you our ninth Journal Club and highlight some of the most interesting papers published recently [...].

8.
G3 (Bethesda) ; 10(6): 1919-1927, 2020 06 01.
Article in English | MEDLINE | ID: mdl-32291289

ABSTRACT

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.


Subject(s)
Gene Silencing , Neurospora crassa , DNA, Fungal , Fungal Proteins/genetics , Humans , Meiosis , Neurospora crassa/genetics , Neurospora crassa/metabolism
9.
Noncoding RNA ; 5(2)2019 Jun 11.
Article in English | MEDLINE | ID: mdl-31212658

ABSTRACT

We are delighted to share with you our seventh Journal Club and highlight some of the most interesting papers published recently [...].

10.
Genetics ; 212(1): 93-110, 2019 05.
Article in English | MEDLINE | ID: mdl-30918007

ABSTRACT

Sk-2 is a meiotic drive element that was discovered in wild populations of Neurospora fungi over 40 years ago. While early studies quickly determined that Sk-2 transmits itself through sexual reproduction in a biased manner via spore killing, the genetic factors responsible for this phenomenon have remained mostly unknown. Here, we identify and characterize rfk-1, a gene required for Sk-2-based spore killing. The rfk-1 gene contains four exons, three introns, and two stop codons, the first of which undergoes RNA editing to a tryptophan codon during sexual development. Translation of an unedited rfk-1 transcript in vegetative tissue is expected to produce a 102-amino acid protein, whereas translation of an edited rfk-1 transcript in sexual tissue is expected to produce a protein with 130 amino acids. These findings indicate that unedited and edited rfk-1 transcripts exist and that these transcripts could have different roles with respect to the mechanism of meiotic drive by spore killing. Regardless of RNA editing, spore killing only succeeds if rfk-1 transcripts avoid silencing caused by a genome defense process called meiotic silencing by unpaired DNA (MSUD). We show that rfk-1's MSUD avoidance mechanism is linked to the genomic landscape surrounding the rfk-1 gene, which is located near the Sk-2 border on the right arm of chromosome III. In addition to demonstrating that the location of rfk-1 is critical to spore-killing success, our results add to accumulating evidence that MSUD helps protect Neurospora genomes from complex meiotic drive elements.


Subject(s)
Fungal Proteins/metabolism , Meiosis , Neurospora/metabolism , RNA Editing , Spores, Fungal/metabolism , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Neurospora/genetics , Neurospora/physiology , Spores, Fungal/genetics
11.
Noncoding RNA ; 5(1)2019 Jan 28.
Article in English | MEDLINE | ID: mdl-30696000

ABSTRACT

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.

12.
Noncoding RNA ; 4(3)2018 Sep 18.
Article in English | MEDLINE | ID: mdl-30231579

ABSTRACT

We are delighted to share with you our sixth Journal Club and highlight some of the most interesting papers published recently [...].

13.
G3 (Bethesda) ; 7(8): 2871-2882, 2017 08 07.
Article in English | MEDLINE | ID: mdl-28667016

ABSTRACT

Meiotic silencing by unpaired DNA (MSUD) is a biological process that searches pairs of homologous chromosomes (homologs) for segments of DNA that are unpaired. Genes found within unpaired segments are silenced for the duration of meiosis. In this report, we describe the identification and characterization of Neurospora crassa sad-7, a gene that encodes a protein with an RNA recognition motif (RRM). Orthologs of sad-7 are found in a wide range of ascomycete fungi. In N. crassa, sad-7 is required for a fully efficient MSUD response to unpaired genes. Additionally, at least one parent must have a functional sad-7 allele for a cross to produce ascospores. Although sad-7-null crosses are barren, sad-7Δ strains grow at a wild-type (wt) rate and appear normal under vegetative growth conditions. With respect to expression, sad-7 is transcribed at baseline levels in early vegetative cultures, at slightly higher levels in mating-competent cultures, and is at its highest level during mating. These findings suggest that SAD-7 is specific to mating-competent and sexual cultures. Although the role of SAD-7 in MSUD remains elusive, green fluorescent protein (GFP)-based tagging studies place SAD-7 within nuclei, perinuclear regions, and cytoplasmic foci of meiotic cells. This localization pattern is unique among known MSUD proteins and raises the possibility that SAD-7 coordinates nuclear, perinuclear, and cytoplasmic aspects of MSUD.


Subject(s)
DNA, Fungal/genetics , Fungal Proteins/metabolism , Gene Silencing , Meiosis/genetics , RNA Recognition Motif , Alleles , Amino Acid Sequence , Cell Nucleus/metabolism , Fungal Proteins/chemistry , Gene Expression Regulation, Fungal , Genes, Suppressor , Green Fluorescent Proteins/metabolism , Neurospora crassa/cytology , Neurospora crassa/genetics , Neurospora crassa/growth & development , Spores, Fungal/genetics
14.
G3 (Bethesda) ; 7(4): 1149-1155, 2017 04 03.
Article in English | MEDLINE | ID: mdl-28179391

ABSTRACT

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.


Subject(s)
DNA, Fungal/metabolism , Gene Silencing , Meiosis , Neurospora/cytology , Neurospora/genetics , Nuclear Cap-Binding Protein Complex/metabolism , Cell Nucleus/metabolism , DNA, Fungal/genetics , Genes, Fungal , Protein Binding
15.
Genetics ; 199(4): 1017-21, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25644701

ABSTRACT

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.


Subject(s)
Argonaute Proteins/metabolism , Fungal Proteins/metabolism , Gene Silencing , Neurospora crassa/genetics , Ribonuclease III/metabolism , Argonaute Proteins/genetics , Fungal Proteins/genetics , Karyopherins/genetics , Karyopherins/metabolism , Protein Binding , Protein Transport , Ribonuclease III/genetics
16.
Genetics ; 198(3): 895-904, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25146971

ABSTRACT

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.


Subject(s)
DNA, Fungal/genetics , Fungal Proteins/metabolism , Homologous Recombination/genetics , Neurospora crassa/genetics , Cell Nucleus/metabolism , Chromatin Assembly and Disassembly , Crosses, Genetic , Gene Deletion , Gene Expression Regulation, Fungal , Genes, Fungal , Homozygote , Humans , Meiosis , Mutagenesis, Insertional , Neurospora crassa/cytology , Neurospora crassa/growth & development , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Homology, Nucleic Acid , Spores, Fungal/growth & development , Suppression, Genetic
17.
Genetics ; 197(4): 1165-74, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24931406

ABSTRACT

Neurospora fungi harbor a group of meiotic drive elements known as Spore killers (Sk). Spore killer-2 (Sk-2) and Spore killer-3 (Sk-3) are two Sk elements that map to a region of suppressed recombination. Although this recombination block is limited to crosses between Sk and Sk-sensitive (Sk(S)) strains, its existence has hindered Sk characterization. Here we report the circumvention of this obstacle by combining a classical genetic screen with next-generation sequencing technology and three-point crossing assays. This approach has allowed us to identify a novel locus called rfk-1, mutation of which disrupts spore killing by Sk-2. We have mapped rfk-1 to a 45-kb region near the right border of the Sk-2 element, a location that also harbors an 11-kb insertion (Sk-2(INS1)) and part of a >220-kb inversion (Sk-2(INV1)). These are the first two chromosome rearrangements to be formally identified in a Neurospora Sk element, providing evidence that they are at least partially responsible for Sk-based recombination suppression. Additionally, the proximity of these chromosome rearrangements to rfk-1 (a critical component of the spore-killing mechanism) suggests that they have played a key role in the evolution of meiotic drive in Neurospora.


Subject(s)
Chromosomes, Fungal/genetics , Gene Rearrangement , Genes, Fungal , Meiosis , Neurospora/genetics , Base Sequence , Chromosome Mapping , DNA, Fungal/genetics , Genetic Loci , Molecular Sequence Data , Mutation , Sequence Analysis, DNA , Spores, Fungal/genetics
18.
Genetics ; 194(1): 279-84, 2013 May.
Article in English | MEDLINE | ID: mdl-23502674

ABSTRACT

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.


Subject(s)
DNA, Fungal/metabolism , Gene Silencing , Meiosis/genetics , Neurospora crassa/cytology , Neurospora crassa/genetics , RNA, Fungal/metabolism , Base Sequence , Crosses, Genetic , Exons/genetics , GC Rich Sequence/genetics , Genes, Fungal , Genetic Loci/genetics , Introns/genetics , Nucleotides/genetics , RNA, Small Interfering/metabolism , Reproducibility of Results , Spores, Fungal/genetics
19.
Genetics ; 194(1): 91-100, 2013 May.
Article in English | MEDLINE | ID: mdl-23502675

ABSTRACT

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.


Subject(s)
DNA, Fungal/metabolism , Fungal Proteins/metabolism , Gene Silencing , Meiosis , Neurospora crassa/cytology , Neurospora crassa/metabolism , RNA, Small Interfering/metabolism , Cell Nucleus/metabolism , Crosses, Genetic , Gene Deletion , Gene Expression Regulation, Fungal , Genes, Dominant/genetics , Genes, Fungal/genetics , Genes, Suppressor , Homozygote , Neurospora crassa/genetics , Phylogeny , RNA, Fungal/metabolism , Reproduction/genetics
20.
Proc Natl Acad Sci U S A ; 109(30): 12093-8, 2012 Jul 24.
Article in English | MEDLINE | ID: mdl-22753473

ABSTRACT

Meiotic drive is a non-Mendelian inheritance phenomenon in which certain selfish genetic elements skew sexual transmission in their own favor. In some cases, progeny or gametes carrying a meiotic drive element can survive preferentially because it causes the death or malfunctioning of those that do not carry it. In Neurospora, meiotic drive can be observed in fungal spore killing. In a cross of Spore killer (Sk) × WT (Sk-sensitive), the ascospores containing the Spore killer allele survive, whereas the ones with the sensitive allele degenerate. Sk-2 and Sk-3 are the most studied meiotic drive elements in Neurospora, and they each theoretically contain two essential components: a killer element and a resistance gene. Here we report the identification and characterization of the Sk resistance gene, rsk (resistant to Spore killer). rsk seems to be a fungal-specific gene, and its deletion in a killer strain leads to self-killing. Sk-2, Sk-3, and naturally resistant isolates all use rsk for resistance. In each killer system, rsk sequences from an Sk strain and a resistant isolate are highly similar, suggesting that they share the same origin. Sk-2, Sk-3, and sensitive rsk alleles differ from each other by their unique indel patterns. Contrary to long-held belief, the killer targets not only late but also early ascospore development. The WT RSK protein is dispensable for ascospore production and is not a target of the spore-killing mechanism. Rather, a resistant version of RSK likely neutralizes the killer element and prevents it from interfering with ascospore development.


Subject(s)
Chromosome Segregation/genetics , Genes, Fungal/genetics , Inheritance Patterns/genetics , Neurospora/genetics , Spores, Fungal/genetics , Base Sequence , Crosses, Genetic , Genetic Vectors/genetics , Molecular Sequence Data , Mutagenesis , Polymerase Chain Reaction , Sequence Analysis, DNA
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