Abstract
Methylated bases in tRNA, rRNA and mRNA control a variety of cellular processes, including protein synthesis, antimicrobial resistance and gene expression. Currently, bulk methods that report the average methylation state of ~104–107 cells are used to detect these modifications, obscuring potentially important biological information. Here, we use in situ hybridization of Molecular Beacons for single-cell detection of three methylations (m62A, m1G and m3U) that destabilize Watson–Crick base pairs. Our method—methylation-sensitive RNA fluorescence in situ hybridization—detects single methylations of rRNA, quantifies antibiotic-resistant bacteria in mixtures of cells and simultaneously detects multiple methylations using multicolor fluorescence imaging.
Highlights
Methylated bases in transfer RNA (tRNA), ribosomal RNA (rRNA) and messenger RNAs (mRNAs) control a variety of cellular processes, including protein synthesis, antimicrobial resistance and gene expression
We first tested whether a Molecular Beacon could detect the tetramethylation catalyzed by the methyltransferase (MTase) KsgA (Fig. 1a), one of the best-studied post-transcriptional modifications of rRNA
This technique verified that a Molecular Beacon designed to detect methylation by KsgA shows a transition for melting of an RNA/DNA duplex with complementary unmethylated RNA, but only a transition for melting of the hairpin loop in the presence of tetramethylated RNA (Fig. 1c)
Summary
Methylated bases in tRNA, rRNA and mRNA control a variety of cellular processes, including protein synthesis, antimicrobial resistance and gene expression. The observations that the fat mass and obesity-associated (FTO) protein demethylates N6-methyladenosine (m6A), 3methyluridine (m3U) and 3-methylthymidine (m3T) in singlestranded RNA7, 8 led to the realization that eukaryotic messenger RNAs (mRNAs) undergo dynamic and widespread methylations at N6 and N1 of adenine[9,10,11,12,13], which likely regulate expression This flurry of recent discoveries has refocused attention on posttranscriptional RNA modifications[14], and spawned a new field: epitranscriptomics. Cellular RNAs are digested to individual nucleosides that are separated by liquid chromatography to identify modified bases, which are put into sequence context by a separate reverse transcription assay, analyzed using gel electrophoresis[15] These bulk methods have detection limits in the femtomole to picomole range, which even for abundant species like rRNAs report the average methylation state of ~104–107 cells[16]. MR-FISH is sensitive to single methylations, and can characterize the composition of heterogeneous mixtures of cells that differ only in RNA methylation
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