Azuma, S. | bioRxiv Science

bioRxiv2025

EC-isHCR: a rapid method for in situ hybridization chain reaction in diverse animal samples

The in situ hybridization chain reaction (isHCR) visualizes RNA across multiple spatial scales, from organs to subcellular structures, in diverse samples. We previously proposed a rapid protocol, EC-isHCR, for Drosophila embryos and ovaries. Whether EC-isHCR retains the features of conventional isHCR, including wide-spatial-scale analyses in various samples, however, has remained unclear. Here, we show that EC-isHCR enables robust RNA detection in a broad range of samples, such as whole-mount fruit fly, parasitoid wasp, and aphid preparations; paraffin sections of trout; frozen mouse sections; and human cultured cells. Moreover, EC-isHCR enabled detection of subcellular RNA localization. EC-isHCR also visualized co-localization of RNA with phase-separated condensates in fruit fly embryos and detected the protrusion-enriched mRNA in HeLa cells. To broaden the applicability of EC-isHCR, we developed an automated probe design tool (https://github.com/ShuntaYorimoto/hcrkit). By combining this tool with EC-isHCR, we provide a fast and versatile framework to visualize mRNAs. This framework will help reduce the barrier to using fast isHCR and thereby facilitate research across diverse areas of the life sciences. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/696653v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1852584org.highwire.dtl.DTLVardef@621682org.highwire.dtl.DTLVardef@1b3bf5corg.highwire.dtl.DTLVardef@1d86841_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- EC-isHCR enables rapid acquisition of high-contrast images. - EC-isHCR preserves features of conventional isHCR, including versatile sample compatibility and high-resolution imaging. - An automated probe design tool was developed for EC-isHCR. - EC-isHCR/probe tool framework will help reduce the barrier to using fast isHCR.

bioengineering↗

bioRxiv2024

TRBP regulates poly(I:C)-mediated apoptotic pathway by modulating miRNA biogenesis

Interferon (IFN) response is usually triggered by the invasion of viral RNAs, and is known to suppress RNA silencing triggered by microRNAs (miRNAs). However, the detailed mechanism regulating RNA silencing during IFN response has not been elucidated. We previously reported that a virus sensor protein, LGP2, which is an upregulated protein during IFN response, interacts with an RNA-binding protein, TRBP. Since TRBP is an RNA-binding protein which enhances miRNA biogenesis in collaboration with Dicer, the interaction of TRBP with LGP2 inhibits the interaction with Dicer and suppresses the TRBP-mediated miRNA biogenesis. During Sendai virus infection, we found that the inhibition of miRNA maturation caused by TRBP depletion resulted in the induction of apoptosis. However, the global gene expression network regulated by miRNAs during viral infection is not understood. In this study, we analyzed comprehensive data from RNA-sequencing and small RNA-sequencing during the antiviral response induced by poly(I:C) transfection, and investigated how TRBP regulates expression profiles of miRNAs and the downstream mRNAs. A group of miRNAs, including miRNAs preferably bind to TRBP, were downregulated by poly(I:C) transfection. Although about 60 % of miRNAs were situated in the introns of their host genes, the expression levels of mature miRNAs were not correlated with those of host genes. Therefore, the downregulation of the expression levels of mature miRNAs was considered to be not caused by transcriptional inhibition of host genes but by the inhibition of TRBP-mediated miRNA processing. Target prediction analysis suggested that the downregulated miRNAs in the presence of TRBP but not in the absence of TRBP mainly targeted transcription factors. Furthermore, these transcription factors were included in the regulators of apoptosis- and cell cycle-related factors. Some of these regulators were also predicted to be the direct targets of downregulated miRNAs in the presence of TRBP. Thus, it was revealed that the downregulation of miRNAs in the presence of TRBP regulates the expression of apoptosis- and cell cycle-related genes either directly or indirectly through adjusting the expression levels of transcription factors. These results suggest that TRBP has a pivotal role in the determination of cell fates during viral infection regulating the miRNA-mRNA regulatory networks through modifying miRNA biogenesis.

molecular biology↗