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

Abstract

The contribution of RNA:DNA hybrid metabolism to cellular processes and disease states has become a prominent topic of study. The S9.6 antibody recognizes RNA:DNA hybrids with a subnanomolar affinity, making it a broadly used tool to detect and study RNA:DNA hybrids. However, S9.6 also binds double-stranded RNA in vitro with significant affinity. Though frequently used in immunofluorescence microscopy, the possible reactivity of S9.6 with non-RNA:DNA hybrid substrates in situ, particularly RNA, has not been comprehensively addressed. Furthermore, S9.6 immunofluorescence microscopy has been methodologically variable and generated discordant imaging datasets. In this study, we find that the majority of the S9.6 immunofluorescence signal observed in fixed human cells arises from RNA, not RNA:DNA hybrids. S9.6 staining was quantitatively unchanged by pre-treatment with the human RNA:DNA hybrid-specific nuclease, RNase H1, despite experimental verification in situ that S9.6 could recognize RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to pre-treatments with RNase T1, and in some cases RNase III, two ribonucleases that specifically degrade singlestranded 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 IIIinsensitive. 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.

Best practices for the visualization, mapping, and manipulation of R‐loops

“…While RNA:DNA hybrids are expected to show an exclusively nuclear and mitochondrial (as well as—in plants—chloroplastic) distribution, most studies report that the majority of the IF signals detected by S9.6 antibodies is cytoplasmic. Recent results (preprint: Smolka et al , 2020) show that, under normal conditions in human cells, the S9.6 signal does not overlap with mitochondria, in agreement with prior work (Koo et al , 2015; Silva et al , 2018). In addition, this cytoplasmic signal was found not to be sensitive to exogenous RNase H treatment (Silva et al , 2018; preprint: Smolka et al , 2020), even under conditions where RNase H was proven effective against transfected in vitro labeled RNA:DNA hybrids (preprint: Smolka et al , 2020).…”

“…In addition, this cytoplasmic signal was found not to be sensitive to exogenous RNase H treatment (Silva et al , 2018; preprint: Smolka et al , 2020), even under conditions where RNase H was proven effective against transfected in vitro labeled RNA:DNA hybrids (preprint: Smolka et al , 2020). Instead, the sensitivity of the cytoplasmic signal to RNA‐specific ribonucleases T1 and III implicated that the majority of the S9.6 signal in standard IF microscopy studies derives from RNA, but not from RNA:DNA hybrids (Silva et al , 2018; preprint: Smolka et al , 2020). These observations do raise substantive concerns about the results of past S9.6‐based imaging studies.…”

“…Approaches using sonication for genome fragmentation are now also available [qDRIP‐seq (Crossley et al , 2020) and sDRIP‐seq (preprint: Smolka et al , 2020)], allowing high‐resolution, strand‐specific R‐loop mapping. Owing to the fact that sonication breaks R‐loops down to two‐stranded RNA:DNA hybrids, close attention to library building strategies should be paid in this case: It is necessary to adopt techniques that can convert such two‐stranded RNA:DNA hybrids back into dsDNA, either through an ssDNA ligation step as in qDRIP‐seq (Crossley et al , 2020), or by introducing a second strand‐synthesis step prior to adapter ligation, as in sDRIP‐seq (preprint: Smolka et al , 2020). Results from both methods show strong agreement (Fig 2A).…”

SF3B1-targeted Splicing Inhibition Triggers Global Alterations in Transcriptional Dynamics and R-Loop Metabolism

“…These findings contrast with previous studies which reported that PladB and other splicing inhibitors caused broad R-loops gains (Nguyen et al, 2018;Nguyen et al, 2017;Wan et al, 2015). Such claims, however, relied on S9.6-based imaging approaches which are unreliable without proper controls due to the affinity of the S9.6 antibody for structured RNAs (Hartono et al, 2018;Phillips et al, 2013;Smolka et al, 2020). The observations that PladB treatment triggers a broad enlargement of RNA-rich nuclear speckles (Effenberger et al, 2014) suggests that RNA distribution is profoundly altered, further raising the possibility of significant imaging artefacts.…”

“…Mapping and all downstream analysis was performed on two independent biological replicates. R-loops were also mapped using sDRIP-seq, a variant of DRIP-seq that permits strand-specific R-loop mapping after genome fragmentation using sonication (Smolka et al, 2020).…”