Simultaneous Detection of mRNA and Protein in Single Cells Using Immunofluorescence-Combined Single-Molecule RNA FISH

The long non-coding RNA X-inactive specific transcript (XIST) mediates the transcriptional silencing of genes on the X chromosome. Here we show that in human cells XIST is highly methylated with at least 78 N6-methyladenosine (m6A) residues—a reversible base modification of unknown function in long non-coding RNAs. We show that m6A formation in XIST, as well as in cellular mRNAs, is mediated by RNA binding motif protein 15 (RBM15) and its paralogue RBM15B, which bind the m6A-methylation complex and recruit it to specific sites in RNA. This results in the methylation of adenosine nucleotides in adjacent m6A consensus motifs. Furthermore, we show that knockdown of RBM15 and RBM15B, or knockdown of methyltransferase like 3 (METTL3), an m6A methyltransferase, impairs XIST-mediated gene silencing. A systematic comparison of m6A-binding proteins shows that YTH domain containing 1 (YTHDC1) preferentially recognizes m6A residues on XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of m6A. These data reveal a pathway of m6A formation and recognition required for XIST-mediated transcriptional repression.

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“…43 with some modifications. In brief, siRNA-transfected and non-transfected HEK293T cells were seeded on poly-L-lysine-coated no.…”

Section: Methodsmentioning

confidence: 99%

m6A RNA methylation promotes XIST-mediated transcriptional repression

Patil

Chen

Pickering

et al. 2016

Nature

1,378

1,666

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“…RNA fluorescence in situ hybridization combined with immunofluorescence (FISH-IF) allows detection and localization of RNA–protein complexes at the cellular level in fixed cells or tissues (12,122,123). FISH-IF utilizes fluorescently labeled nucleic acid probes complementary to the desired RNA and antibodies detecting the protein of interest.…”

Section: Validation Of Ms Datamentioning

confidence: 99%

“…FISH-IF utilizes fluorescently labeled nucleic acid probes complementary to the desired RNA and antibodies detecting the protein of interest. Recent advances in RNA FISH, such as single-molecule FISH (smFISH), allow for the analysis of proteins co-localizing with individual RNA molecules in single cells (123). However, because of the limitations of optical resolution, the FISH-IF technique does not provide evidence for the direct interaction between proteins and RNA.…”

Section: Validation Of Ms Datamentioning

confidence: 99%

Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases

Jazurek¹,

Ciesiolka²,

Staręga-Rosłan³

et al. 2016

Nucleic Acids Res

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RNA–protein complexes play a central role in the regulation of fundamental cellular processes, such as mRNA splicing, localization, translation and degradation. The misregulation of these interactions can cause a variety of human diseases, including cancer and neurodegenerative disorders. Recently, many strategies have been developed to comprehensively analyze these complex and highly dynamic RNA–protein networks. Extensive efforts have been made to purify in vivo-assembled RNA–protein complexes. In this review, we focused on commonly used RNA-centric approaches that involve mass spectrometry, which are powerful tools for identifying proteins bound to a given RNA. We present various RNA capture strategies that primarily depend on whether the RNA of interest is modified. Moreover, we briefly discuss the advantages and limitations of in vitro and in vivo approaches. Furthermore, we describe recent advances in quantitative proteomics as well as the methods that are most commonly used to validate robust mass spectrometry data. Finally, we present approaches that have successfully identified expanded repeat-binding proteins, which present abnormal RNA–protein interactions that result in the development of many neurological diseases.

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“…Simultaneous detection of mRNA and protein quantity and their subcellular distribution in single cells by combining a RNase-free modification of the immunofluorescence (IF) technique and the smRNA FISH method observed direct interaction of RNase MCPIP1 with IL-6 mRNA (Kochan et al, 2015). Real-time live imaging using laser-scanning confocal microscope with photon-counting detectors for quantitative studies of transcription in culture cells and model animals have been achieved by smRNA-FISH and GFP-tagged reporter gene for RNA polymerase (Gregor et al, 2014).…”

Section: Single-cell Dna Structural and Rna Transcriptional Analysesmentioning

confidence: 99%

Fluorescence In situ Hybridization: Cell-Based Genetic Diagnostic and Research Applications

Cui

Shu

2016

Front. Cell Dev. Biol.

165

125

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Fluorescence in situ hybridization (FISH) is a macromolecule recognition technology based on the complementary nature of DNA or DNA/RNA double strands. Selected DNA strands incorporated with fluorophore-coupled nucleotides can be used as probes to hybridize onto the complementary sequences in tested cells and tissues and then visualized through a fluorescence microscope or an imaging system. This technology was initially developed as a physical mapping tool to delineate genes within chromosomes. Its high analytical resolution to a single gene level and high sensitivity and specificity enabled an immediate application for genetic diagnosis of constitutional common aneuploidies, microdeletion/microduplication syndromes, and subtelomeric rearrangements. FISH tests using panels of gene-specific probes for somatic recurrent losses, gains, and translocations have been routinely applied for hematologic and solid tumors and are one of the fastest-growing areas in cancer diagnosis. FISH has also been used to detect infectious microbias and parasites like malaria in human blood cells. Recent advances in FISH technology involve various methods for improving probe labeling efficiency and the use of super resolution imaging systems for direct visualization of intra-nuclear chromosomal organization and profiling of RNA transcription in single cells. Cas9-mediated FISH (CASFISH) allowed in situ labeling of repetitive sequences and single-copy sequences without the disruption of nuclear genomic organization in fixed or living cells. Using oligopaint-FISH and super-resolution imaging enabled in situ visualization of chromosome haplotypes from differentially specified single-nucleotide polymorphism loci. Single molecule RNA FISH (smRNA-FISH) using combinatorial labeling or sequential barcoding by multiple round of hybridization were applied to measure mRNA expression of multiple genes within single cells. Research applications of these single molecule single cells DNA and RNA FISH techniques have visualized intra-nuclear genomic structure and sub-cellular transcriptional dynamics of many genes and revealed their functions in various biological processes.

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Simultaneous Detection of mRNA and Protein in Single Cells Using Immunofluorescence-Combined Single-Molecule RNA FISH

Cited by 48 publications

References 19 publications

m6A RNA methylation promotes XIST-mediated transcriptional repression

m6A RNA methylation promotes XIST-mediated transcriptional repression

Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases

Fluorescence In situ Hybridization: Cell-Based Genetic Diagnostic and Research Applications

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