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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.

SUBMITTER: Jaijyan DK

PROVIDER: S-EPMC11347131 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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Imaging and quantification of human and viral circular RNAs.

Jaijyan Dabbu Kumar DK Yang Shaomin S Ramasamy Santhamani S Gu Alison A Zeng Mulan M Subbian Selvakumar S Tyagi Sanjay S Zhu Hua H

Nucleic acids research 20240801 15

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 sit ...[more]

PMID: 39051561

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Project description:Recent studies have identified circular RNAs (circRNAs) expressed from the Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) human DNA tumor viruses. To gain initial insights into the potential relevance of EBV circRNAs in virus biology and disease, we assessed the circRNAome of the interspecies homologue rhesus macaque lymphocryptovirus (rLCV) in a naturally occurring lymphoma from a simian immunodeficiency virus (SIV)-infected rhesus macaque. This analysis revealed rLCV orthologues of the latency-associated EBV circular RNAs circRPMS1_E4_E3a and circEBNA_U. Also identified in two samples displaying unusually high lytic gene expression was a novel rLCV circRNA that contains both conserved and rLCV-specific RPMS1 exons and whose backsplice junctions flank an rLCV lytic origin of replication (OriLyt). Analysis of a lytic infection model for the murid herpesvirus 68 (MHV68) rhadinovirus identified a cluster of circRNAs near an MHV68 lytic origin of replication, with the most abundant of these, circM11_ORF69, spanning the OriLyt. Lastly, analysis of KSHV latency and reactivation models revealed the latency associated circRNA originating from the vIRF4 gene as the predominant viral circRNA. Together, the results of this study broaden our appreciation for circRNA repertoires in the Lymphocryptovirus and Rhadinovirus genera of gammaherpesviruses and provide evolutionary support for viral circRNA functions in latency and viral replication.IMPORTANCE Infection with oncogenic gammaherpesviruses leads to long-term viral persistence through a dynamic interplay between the virus and the host immune system. Critical for remodeling of the host cell environment after the immune responses are viral noncoding RNAs that modulate host signaling pathways without attracting adaptive immune recognition. Despite the importance of noncoding RNAs in persistent infection, the circRNA class of noncoding RNAs has only recently been identified in gammaherpesviruses. Accordingly, their roles in virus infection and associated oncogenesis are unknown. Here we report evolutionary conservation of EBV-encoded circRNAs determined by assessing the circRNAome in rLCV-infected lymphomas from an SIV-infected rhesus macaque, and we report latent and lytic circRNAs from KSHV and MHV68. These experiments demonstrate utilization of the circular RNA class of RNAs across 4 members of the gammaherpesvirus subfamily, and they identify orthologues and potential homoplastic circRNAs, implying conserved circRNA functions in virus biology and associated malignancies.

Dataset Information

Imaging and quantification of human and viral circular RNAs.

Publications

Imaging and quantification of human and viral circular RNAs.

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