Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa

Bartholomai, Bradley M.; Gladfelter, Amy S.; Loros, Jennifer J.; Dunlap, Jay C.

doi:10.1016/j.fgb.2021.103615

Cited by 4 publications

(11 citation statements)

References 44 publications

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“…smFISH was recently adapted for use in Neurospora ( 27 – 29 ). Forty-eight fluorescently labeled oligonucleotides designed to bind the protein-coding sequence of frequency mRNA enable resolution of individual transcripts ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To begin investigation of the delays between RNA and protein expression peaks for core clock components, and to understand how frq transcripts are localized in Neurospora hyphae, we adapted single-molecule RNA fluorescence in situ hybridization (smFISH) for use in Neurospora ( 27 ), revealing that frq transcripts are nonrandomly clustered with nuclei when expressed during the circadian cycle. Surprisingly, despite robust cycling of frq transcript levels, only a small subset of nuclei are actively transcribing frq at any given time as it cycles in abundance throughout the circadian day.…”

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confidence: 99%

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PRD-2 mediates clock-regulated perinuclear localization of clock gene RNAs within the circadian cycle of Neurospora

Bartholomai

Gladfelter

Loros

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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The transcription–translation negative feedback loops underlying animal and fungal circadian clocks are remarkably similar in their molecular regulatory architecture and, although much is understood about their central mechanism, little is known about the spatiotemporal dynamics of the gene products involved. A common feature of these circadian oscillators is a significant temporal delay between rhythmic accumulation of clock messenger RNAs (mRNAs) encoding negative arm proteins, for example, frq in Neurospora and Per1-3 in mammals, and the appearance of the clock protein complexes assembled from the proteins they encode. Here, we report use of single-molecule RNA fluorescence in situ hybridization (smFISH) to show that the fraction of nuclei actively transcribing the clock gene frq changes in a circadian manner, and that these mRNAs cycle in abundance with fewer than five transcripts per nucleus at any time. Spatial point patterning statistics reveal that frq is spatially clustered near nuclei in a time of day–dependent manner and that clustering requires an RNA-binding protein, PRD-2 (PERIOD-2), recently shown also to bind to mRNA encoding another core clock component, casein kinase 1. An intrinsically disordered protein, PRD-2 displays behavior in vivo and in vitro consistent with participation in biomolecular condensates. These data are consistent with a role for phase-separating RNA-binding proteins in spatiotemporally organizing clock mRNAs to facilitate local translation and assembly of clock protein complexes.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

PRD-2 mediates clock-regulated perinuclear localization of clock gene RNAs within the circadian cycle of Neurospora

Bartholomai

Gladfelter

Loros

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…It has previously been found that enzymes used in lysis of fungal cell walls are not universally effective ( Bartholomai et al, 2021 ). The primary reason for this inconsistent effectiveness is the variability in fungal cell wall composition.…”

Section: Resultsmentioning

confidence: 99%

“…SNR of bacterial features and 18S rRNA improved when the lysozyme, glucanase, and chitinase were combined. Treatment with ethanol has also been observed to aid in permeabilization by disrupting membranes, extracting lipids, and denaturing proteins ( Bartholomai et al, 2021 ). When the fungal isolate was treated with the addition of ethanol, the SNR of bacterial 16S rRNA was further improved and we observed an increase in 18S rRNA signal homogeneity.…”

Section: Resultsmentioning

confidence: 99%

“…Alternatively, optical imaging techniques, such as fluorescent in situ hybridization (FISH), afford improved spatial context of interactions by providing a wider field of view and enabling target specificity. As such, FISH remains a minimal requirement for in situ targeted-sequence analysis ( Higuchi and Takegawa, 2020 ; Bartholomai et al, 2021 ) and can be a valuable tool for assessing spatiotemporal aspects of bacteria with fungal hypha.…”

Section: Introductionmentioning

confidence: 99%

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Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria

et al. 2022

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Several bacteria have long been known to interact intimately with fungi, but molecular approaches have only recently uncovered how cosmopolitan these interactions are in nature. Currently, bacterial–fungal interactions (BFI) are inferred based on patterns of co-occurrence in amplicon sequencing investigations. However, determining the nature of these interactions, whether the bacteria are internally or externally associated, remains a grand challenge in BFI research. Fluorescence in situ hybridization (FISH) is a robust method that targets unique sequences of interest which can be employed for visualizing intra-hyphal targets, such as mitochondrial organelles or, as in this study, bacteria. We evaluate the challenges and employable strategies to resolve intra-hyphal BFI to address pertinent criteria in BFI research, such as culturing media, spatial distribution of bacteria, and abundance of bacterial 16S rRNA copies for fluorescent labeling. While these experimental factors influence labeling and detection of endobacteria, we demonstrate how to overcome these challenges thorough permeabilization, appropriate media choice, and targeted amplification using hybridization chain reaction FISH. Such microscopy imaging approaches can now be utilized by the broader research community to complement sequence-based investigations and provide more conclusive evidence on the nature of specific bacterial–fungal relationships.

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Single-Molecule Fluorescent In Situ Hybridization (smFISH) for RNA Detection in the Fungal Pathogen Candida albicans

van Otterdijk,

Motealleh,

Wang

et al. 2024

Methods in Molecular Biology

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Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa

Cited by 4 publications

References 44 publications

PRD-2 mediates clock-regulated perinuclear localization of clock gene RNAs within the circadian cycle of Neurospora

PRD-2 mediates clock-regulated perinuclear localization of clock gene RNAs within the circadian cycle of Neurospora

Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria

Single-Molecule Fluorescent In Situ Hybridization (smFISH) for RNA Detection in the Fungal Pathogen Candida albicans

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