Biology subjects

Rhoades, N.

Publications and source records attributed to Rhoades, N..

5 recordsLinked to original sources

bioRxiv2026

Molecular characterization of required for killing-2: a gene required for spore killing by Neurospora Sk-3.

The Sk-3 selfish genetic element in Neurospora fungi is transmitted to offspring in a biased manner through spore killing. In crosses between an Sk-3 strain and a Spore killing-sensitive (Sk-S) strain, nearly all offspring that fail to inherit Sk-3 are killed. Although a mutation called rfk-2UV, which disrupts spore killing by Sk-3, was isolated in an earlier work, its precise location within the Sk-3 element was not identified. Here, we demonstrate that rfk-2UV is a T>G transversion mutation within a protein coding gene called rfk-2. The rfk-2 gene is located on Chromosome III, and it encodes a transcript that undergoes RNA splicing and at least two RNA editing events. One of these editing events changes a UAG stop codon to a UGG tryptophan codon and allows for production of a 102 amino acid protein. Additionally, while our results demonstrate that rfk-2 is required for spore killing, they also show that it is insufficient for the process. Sk-3 is thus more complex than previously anticipated. Rather than using a single gene to produce its killer, Sk-3 requires multiple genes to kill spores and break the rules of Mendelian Inheritance.

genetics↗

bioRxiv2021

RNA editing controls meiotic drive by a Neurospora Spore killer

Neurospora Sk-2 is a complex meiotic drive element that is transmitted to offspring through sexual reproduction in a biased manner. Sk-2s transmission mechanism involves spore killing, and recent evidence has demonstrated that its spore killing mechanism is mediated by a gene called rfk-1. The native rfk-1 sequence, referred to as rfk-1+, encodes an early UAG stop codon and a late UAA stop codon. When translation stops at the early stop codon, a 102 amino acid protein called RFK-1A is produced, and when translation stops at the late stop codon, a 130 amino acid protein called RFK-1B is produced. We show that expression of RFK-1B occurs when the early stop codon undergoes adenosine-to-inosine (A-to-I) mRNA editing (UAG is edited to UIG), and that this editing event is required for spore killing. We also show that RFK-1B, but not RFK-1A, acts as a poison when it is ectopically expressed within the vegetative tissue of a non-Sk-2 strain. Furthermore, we show that RFK-1B toxicity can be neutralized in vegetative tissue by co-expressing RSKSk-2, which is presumed to be Sk-2s antidote protein. Finally, we show that rfk-1+s first intron, or a truncated version of this intron, when present, improves phenotypic expression of RFK-1B in vegetative tissue. Overall, our results demonstrate that Sk-2 uses A-to-I mRNA editing to control when its killer protein (poison) is produced, and that the primary killing and resistance functions of Sk-2 can be established within a non-Sk-2 strain by the ectopic expression of only two genes.

genetics↗

bioRxiv2020

Investigating expression of a human optimized cas9 transgene in Neurospora crassa

The CRISPR-associated Cas9 enzyme is used in molecular biology to engineer the genomes of a wide range of organisms. While Cas9 can be injected or transfected into a target cell to achieve the desired goal, there are situations where stable expression of Cas9 within a target organism is preferable. Here, we show that the model filamentous fungus Neurospora crassa is recalcitrant to heterologous expression of a human-optimized version of Streptococcus pyogenes cas9. Furthermore, partial optimization of cas9 by synonymous codon exchange failed to improve its expression in the fungus. Finally, we show that transgene expression can be detected when cas9Hs sequences are placed in the 3 UTR regions of transgene-derived mRNAs, but not when the same sequences are in the translated part of the transgene-derived mRNA. This finding suggests that the primary obstacle to high cas9Hs expression levels in N. crassa is translational in nature.

molecular biology↗

bioRxiv2019

Comparative genomics guides elucidation of vitamin B12 biosynthesis in novel human associated Akkermansia

BackgroundAkkermansia muciniphila is a mucin-degrading bacterium found in the gut of most healthy humans and is considered a next-generation probiotic. However, knowledge of the genomic and physiological diversity of human associated Akkermansia is limited, as only one species has been formally described. ResultsTo begin to fill this knowledge gap, we reconstructed 35 high-quality metagenome assembled genomes from children and combined them with 40 other publicly available genomes from adults and mice for comparative genomic analysis. We identified at least four species-level phylogroups (AmI-AmIV) with distinct functional potentials. Most notably, we identified the presence of putative cobalamin (vitamin B12) biosynthesis genes within the AmII (n=26/28) and AmIII (n=2/2) phylogroups. To test these predictions, 10 novel strains of Akkermansia were isolated from adults and screened for essential vitamin B12 biosynthesis genes via PCR. Two strains of the AmII phylogroup were positive for the presence of vitamin B12 biosynthesis genes, while all AmI strains, including the type strain A. muciniphila MucT, were negative. To demonstrate vitamin B12 biosynthesis, we measured the production of acetate, succinate, and propionate in the presence and absence of vitamin supplementation in representative strains of the AmI and AmII phylogroups since cobalamin is a cofactor in propionate metabolism. Results show that the Akkermansia AmII strain produced acetate and propionate in the absence of supplementation, which is indicative of de novo vitamin B12 biosynthesis. In contrast, acetate and succinate were the main fermentation products for the AmI strains when vitamin B12 was not supplied in the culture medium. ConclusionsWe identified Akkermansia strains as potentially important vitamin B12 biosynthetic bacteria in the human gut. This novel physiological trait of human associated Akkermansia may impact how these bacteria interact with the human host and other members of the human gut microbiome.

genomics↗

bioRxiv2019

Recombination-independent recognition of DNA homology for meiotic silencing in Neurospora crassa

Pairing of homologous chromosomes represents a critical step of meiosis in nearly all sexually reproducing species. While in some organisms meiotic pairing requires programmed DNA breakage and recombination, in many others it engages homologous chromosomes that remain apparently intact. The mechanistic nature of such recombination-independent pairing represents a fundamental question in molecular genetics. Using meiotic silencing by unpaired DNA (MSUD) in Neurospora crassa as a model process, we demonstrate the existence of a cardinally different approach to DNA homology recognition in meiosis. The main advantage of MSUD over other experimental systems lies in its ability to identify any relatively short DNA fragment lacking a homologous allelic partner. Here we show that MSUD does not rely on the canonical mechanism of meiotic recombination, yet it is promoted by REC8, a conserved component of the meiotic cohesin complex. We also show that certain patterns of interspersed homology are recognized as pairable during MSUD. Such patterns need to be co-linear and must contain short tracts of sequence identity spaced apart with a periodicity of 21 or 22 base-pairs. By using these values as a guiding parameter in all-atom molecular modeling, we discover that homologous double-stranded DNA molecules can associate by forming quadruplex-based contacts with an interval of 2.5 helical turns, which requires right-handed plectonemic coiling and additional conformational changes in the intervening double-helical segments. These results (i) reconcile genetic and biophysical lines of evidence for the existence of direct homologous dsDNA-dsDNA pairing, (ii) identify a role for this process in initiating post-transcriptional silencing, and (iii) suggest that chromosomes are cross-matched in meiosis by a precise mechanism that operates on intact double-stranded DNA molecules.

molecular biology↗