Imaging and quantification of human and viral circular RNAs

Abstract

We present a robust approach for cellular detection, imaging, localization, and quantification of human and viral encoded circular RNAs (circRNA) using amplified fluorescence in situ hybridization (ampFISH). In this procedure, a pair of hairpin probes bind next to each other at contiguous stretches of sequence and then undergo a conformational reorganization which initiates a target-dependent hybridization chain reaction (HCR) resulting in deposition of an amplified fluorescent signal at the site. By harnessing the capabilities of both ampFISH and single-molecule FISH (smFISH), we selectively identified and imaged circular RNAs and their linear counterparts derived from the human genome, SARS-CoV-2 (an RNA virus), and human cytomegalovirus (HCMV, a DNA virus). Computational image processing facilitated accurate quantification of circular RNA molecules in individual cells. The specificity of ampFISH for circular RNA detection was confirmed through an in situ RNase R treatment that selectively degrades linear RNAs without impacting circular RNAs. The effectiveness of circular RNA detection was further validated by using ampFISH probes with mismatches and probe pairs that do not bind to the continuous sequence in their target RNAs but instead bind at segregated sites. An additional specificity test involved probes against the negative strands of the circular RNA sequence, absent in the cell. Importantly, our technique allows simultaneous detection of circular RNAs and their linear counterparts within the same cell with single molecule sensitivity, enabling explorations of circular RNA biogenesis, subcellular localization, and functions.

Graphical Abstract

Figure 1.

Imaging circular and linear RNAs encoded by human gene HIPK3 using ampFISH and smFISH. (A) A Schematic description of how ampFISH is used to detect contiguous sequences at back-splice junction (BSJ) in the circular RNAs. When bound to the circular RNA at BSJ the ampFISH probe pairs are juxtaposed and are able to interact with each other, causing a conformational reorganization which releases an HCR initiator sequence. The freed initiator creates amplified signals when HCR hairpins are provided. (B) Organization of human HIPK3 gene and the locations of the smFISH and ampFISH probes. The ampFISH probe pairs can bind to the terminal sequences on exon 2 in the linear RNA but cannot interact with each other and therefore do not generate any HCR signals from linear RNA. smFISH probes are directly labeled and 48 probes are tiled over exon 1. (C) Imaging of linear and circular HIPK3 RNAs with smFISH and ampFISH. The probes, the targeted RNA species, and the conditions are indicated on the left of each row of images, and the image components are shown on the top. DAPI represents the nucleus of the cells. The white dotted lines represent the enlarged areas. Two columns of images on the right show spot detection analysis where yellow circles are drawn around each detected spot and the blue lines represent cell boundaries. Each spot corresponds to single RNA molecule. smFISH signals are shown by green and ampFISH signals are shown by red color. The bottom panel represents the RNase A treatment of cells followed by ampFISH staining to detect positive strand circRNA of HIPK3. (D) The bar graphs show quantification of number of spots per cell from several fields of images. The size bars represent 20 μm. Data represent mean RNA copies/cell ± SE. At least 10 cells were analyzed in each category.

Figure 2.

Co-imaging HIPK3 circular and linear RNAs. The circular RNA was imaged using ampFISH probes (red signals) and the linear RNA was imaged using smFISH probes (green signals). The probe locations are depicted in Figure 1. Both kinds of probes were hybridized together and after removal of excess probes by washing, HCR was performed. The cell conditions are indicated on the left and the detected RNA species on the top. DAPI was used to stain nuclei. The enlarged images are the magnified from the selected area of the merged image (white dotted line). Number of spots detected in single cells in each category are shown on the right. The scale bars represent 20 μm. Data are mean number of RNAs per cell ± SE, n = 5 cells.

Figure 3.

Detection of HIPK3 linear transcripts using a pair of ampFISH probes targeted at the junction between exons 3 and 4 and the HIPK3 circular transcripts using a pair of ampFISH probes targeted at their back-splice junction (BSJ). The former is confirmed by using 48 smFISH probes against the linear transcripts in the same images. (A) Results of a triplex imaging experiment are shown where top panels correspond to the unprocessed z-stack merges from the indicated channels and the bottom panels are obtained by application of a Laplacian filter on the z-stack layers and merger of the layers followed by contrasting. Right most panels are composite images of the three channels created using color codes indicated by the labels above or over the images. In the bottom right panel, circles of different colors identify the spots that were detected by our image processing algorithm in each channel. The yellow and purple colored circles identify the smFISH linear spots that were localized with either ampFISH linear and ampFISH circular, respectively, the latter can arise only spuriously. (B) Percent of smFISH linear spots that were colocalized with spots created by the ampFISH linear or ampFISH circular probes. Number of cells analyzed 68. Error bars represent 95% confidence interval.

Figure 4.

Imaging a circular RNA encoded by SARS-CoV-2 together with its genomic RNA. (A) Graphical representation of organization of SARS-CoV-2 genome, locations of the smFISH and ampFISH probes. circular RNA29122/28295 is created from the N-gene sequence during the replication process. (B) In the multiplex images the genomic RNA was imaged by hybridizing a set of 48 smFISH probes labeled with Texas Red (green) and the circular RNA was imaged by hybridizing an ampFISH probe pair followed by HCR using hairpins labeled with Cy5 (red). DAPI was used to stain the nuclei. The conditions and the probes used are indicated on the left and the targets are indicated on top. All infected cells display strong signals for genomic RNA but the bystander cells don’t show any. The target complementary region of each ampFISH probe in the bottom panel contains three mutations which will prevent their binding; and the target complementary regions of the probes in the second panel from the bottom bind 26 nt away from each other which will not allow them to interact. (C) Integrated fluorescence intensities within infected cells for genomic RNA and circular RNA with and without RNase R treatment. Data represent mean ± SE, n = 6 cells. A white line in each image represents a scale bar of 20 μm.

Figure 5.

Imaging an HCMV circular RNA, circular RNA number 5, and an HCMV linear RNA transcript UL24, in HCMV-infected MRC-5 cells with ampFISH. (A) The probes employed are indicated in the left and the targets are indicated on the top. The smFISH RNA signals are represented by red color, HCMV-GFP infected cell by green, and DAPI stain by blue. Right two columns show the output of spot detection image processing, where yellow ovals are drawn around the detected spots and blue outlines are drawn around each cell. (B) The graphs show the average number of RNA molecules/cell ± SE, n = 10 cells. A scale bar of 20 μm is present in the images.

Figure 6.

Upregulation of expression of human circular RNA, circHIPK3(2) in HCMV infected cells. (A) MRC-5 cells in a field where some cells are infected with HCMV and some are not, were imaged using ampFISH probes against human circular RNA. The ampFISH probes are indicated on the left and the labels are shown on the top. The HCMV infected cells can be identified by GFP expression (green). The ampFISH signal is shown in red. (B) The ampFISH spots were quantified in each kind of cells and are presented as bar graphs for the average molecules per cell for each indicated categories. Error bars are ± SE, n = 10 cells and size bars represent 10 μm.