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Recognition of cellular RNAs by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids

ABSTRACT: Recognition of cellular RNAs by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids

PROVIDER: PRJNA594934 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10669423.fastq.gz	Fastqsanger.gz
	SRR10669424.fastq.gz	Fastqsanger.gz
	SRR10669425.fastq.gz	Fastqsanger.gz
	SRR10669426.fastq.gz	Fastqsanger.gz
	SRR10669427.fastq.gz	Fastqsanger.gz

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Recognition of cellular RNAs by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids

Project description:We showed that the majority of the signal observed in S9.6 immunofluorescence microscopy images of fixed human cells arises from RNA, not RNA:DNA hybrids. S9.6 staining is quantitatively unchanged by pre-treatment with the human RNA:DNA hybrid-specific nuclease, RNase H1, despite experimental verification that the enzyme was active in situ. S9.6 staining was, however, significantly sensitive to pre-treatments with RNase T1, and in some cases RNase III, two ribonucleases that specifically degrade single-stranded and double-stranded RNA, respectively. In contrast, genome-wide maps obtained by high-throughput DNA sequencing after S9.6-mediated DNA:RNA Immunoprecipitation (DRIP) are RNase H1-sensitive and RNase T1- and RNase III-insensitive. Altogether, these data demonstrate that the S9.6 antibody, though capable of recognizing RNA:DNA hybrids in situ and in vitro, suffers from a lack of specificity that precludes reliable imaging of RNA:DNA hybrids and renders associated imaging data inconclusive in the absence of controls for its promiscuous recognition of cellular RNAs.

2021-03-17 | GSE141833 | GEO

Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids.

Project description:The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid-specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.

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