Detecting RNA base methylations in single cells by in situ hybridization

Ranasinghe, Rohan T.; Challand, Martin R.; Ganzinger, Kristina A.; Lewis, Benjamin F.; Softley, Charlotte; Schmied, Wolfgang H.; Horrocks, Mathew H.; Shivji, Nadia; Chin, Jason W.; Spencer, James; Klenerman, David

doi:10.1038/s41467-017-02714-7

Cited by 30 publications

(21 citation statements)

References 42 publications

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“…Fluorescence images were analysed using custom software written in MATLAB (MATLAB R2014b, The MathWorks, Inc.) adapted from Ranasinghe et al [31]. Briefly, vesicles were detected (i.e.…”

Section: Discussionmentioning

confidence: 99%

Freeze-thaw cycles induce content exchange between cell-sized lipid vesicles

et al. 2018

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Early protocells are commonly assumed to consist of an amphiphilic membrane enclosing an RNAbased self-replicating genetic system and a primitive metabolism without protein enzymes. Thus, protocell evolution must have relied on simple physicochemical self-organization processes within and across such vesicular structures. We investigate freeze-thaw (FT) cycling as a potential environmental driver for the necessary content exchange between vesicles. To this end, we developed a conceptually simple yet statistically powerful high-throughput procedure based on nucleic acidcontaining giant unilamellar vesicles (GUVs) as model protocells. GUVs are formed by emulsion transfer in glass bottom microtiter plates and hence can be manipulated and monitored by fluorescence microscopy without additional pipetting and sample handling steps. This new protocol greatly minimizes artefacts, such as unintended GUV rupture or fusion by shear forces. Using DNAencapsulating phospholipid GUVs fabricated by this method, we quantified the extent of content mixing between GUVs under different FT conditions. We found evidence of nucleic acid exchange in all detected vesicles if fast freezing of GUVs at −80°C is followed by slow thawing at room temperature. In contrast, slow freezing and fast thawing both adversely affected content mixing. Surprisingly, and in contrast to previous reports for FT-induced content mixing, we found that the content is not exchanged through vesicle fusion and fission, but that vesicles largely maintain their membrane identity and even large molecules are exchanged via diffusion across the membranes. Our approach supports efficient screening of prebiotically plausible molecules and environmental conditions, to yield universal mechanistic insights into how cellular life may have emerged.

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“…Fluorescence images were analysed using custom software written in MATLAB (MATLAB R2014b, The MathWorks, Inc.) adapted from Ranasinghe et al [31]. Briefly, vesicles were detected (i.e.…”

Section: Discussionmentioning

confidence: 99%

Freeze-thaw cycles induce content exchange between cell-sized lipid vesicles

et al. 2018

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“…Alternative approaches use immunoprecipitation and next-generation sequencing in pooled samples to gain insights into the stoichiometry of modified nucleotides while preserving sequence information (Dominissini et al, 2013). Additionally, emerging technologies aim to detect RNA modifications at the single-cell level (Ranasinghe et al, 2018).…”

Section: Future Prospects: Emerging Technologies To Investigate Rrna mentioning

confidence: 99%

Methylation of Ribosomal RNA: A Mitochondrial Perspective

et al. 2020

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Ribosomal RNA (rRNA) from all organisms undergoes post-transcriptional modifications that increase the diversity of its composition and activity. In mitochondria, specialized mitochondrial ribosomes (mitoribosomes) are responsible for the synthesis of 13 oxidative phosphorylation proteins encoded by the mitochondrial genome. Mitoribosomal RNA is also modified, with 10 modifications thus far identified and all corresponding modifying enzymes described. This form of epigenetic regulation of mitochondrial gene expression affects mitoribosome biogenesis and function. Here, we provide an overview on rRNA methylation and highlight critical work that is beginning to elucidate its role in mitochondrial gene expression. Given the similarities between bacterial and mitochondrial ribosomes, we focus on studies involving Escherichia coli and human models. Furthermore, we highlight the use of state-of-the-art technologies, such as cryoEM in the study of rRNA methylation and its biological relevance. Understanding the mechanisms and functional relevance of this process represents an exciting frontier in the RNA biology and mitochondrial fields.

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“…Methylation types such as m 1 G and m 3 U destabilize the base-pairing, which prevents the probe from binding to the RNA. A methylation-insensitive probe carrying a different fluorophore and complementary to another region of the analysed RNA molecule serves as an internal control [ 40 ]. ( c ) m 6 A sites can be detected via the stalled RT of an m 6 A-containing RNA in case selenium-containing dTTP analogues are used [ 41 ].…”

Section: Approaches Based On Hybridization Propertiesmentioning

confidence: 99%

“…Another approach for detecting of RNA methylation at specific positions used DNA hybridization probes that are sensitive to methylation of their complementary RNA sequences [ 40 ]. This method—termed ‘methylation-sensitive RNA fluorescence in situ hybridization’ (MR-FISH)—allows to monitor RNA methylation at specific sites in single cells.…”

Section: Approaches Based On Hybridization Propertiesmentioning

confidence: 99%

Emerging approaches for detection of methylation sites in RNA

Ovcharenko¹,

Rentmeister²

2018

Open Biol.

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RNA methylations play a significant regulatory role in diverse biological processes. Although the transcriptome-wide discovery of unknown RNA methylation sites is essential to elucidate their function, the development of a bigger variety of detection approaches is desirable for multiple reasons. Many established detection methods for RNA modifications heavily rely on the specificity of the respective antibodies. Thus, the development of antibody-independent transcriptome-wide methods is beneficial. Even the antibody-independent high-throughput sequencing-based methods are liable to produce false-positive or false-negative results. The development of an independent method for each modification could help validate the detected modification sites. Apart from the transcriptome-wide methods for methylation detection de novo, methods for monitoring the presence of a single methylation at a determined site are also needed. In contrast to the transcriptome-wide detection methods, the techniques used for monitoring purposes need to be cheap, fast and easy to perform. This review considers modern approaches for site-specific detection of methylated nucleotides in RNA. We also discuss the potential of third-generation sequencing methods for direct detection of RNA methylations.

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Detecting RNA base methylations in single cells by in situ hybridization

Cited by 30 publications

References 42 publications

Freeze-thaw cycles induce content exchange between cell-sized lipid vesicles

Freeze-thaw cycles induce content exchange between cell-sized lipid vesicles

Methylation of Ribosomal RNA: A Mitochondrial Perspective

Emerging approaches for detection of methylation sites in RNA

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