RNA sequencing has revolutionized our understanding of gene expression and regulation. However, traditional RNA sequencing methods do not provide information on the spatial organization of RNAs within cells. RNA in situ conformation sequencing (RIC-seq) is a powerful technique that allows for the global in situ profiling of RNA-RNA spatial interactions.
RIC-seq is a technique based on high-throughput sequencing that permits the profiling of RNA-RNA interactions in situ. This revolutionary method leverages the power of ligation, to fuse RNA molecules that are in the vicinity of each other in cells, which is followed by reverse transcription and sequencing of the resulting chimeric RNA molecules. Through a deep analysis of the obtained sequencing data, RIC-seq can effectively identify RNA-RNA interactions on a genome-wide scale, uncovering the intricate relationships between different RNA molecules.
The development of RIC-seq was spurred by the pressing need for a technique that can capture the spatial organization of RNA molecules within cells. Even though traditional RNA sequencing can offer information about the abundance of distinct RNA molecules, it fails to shed light on their spatial organization. RIC-seq surpasses this limitation by identifying RNA molecules that are in close proximity to each other within cells, providing valuable insights into the spatial organization of RNAs, which can have a significant impact on our understanding of cellular processes.
RIC-seq is a three-step process that involves sample preparation, library construction, and sequencing. The sample preparation step involves cross-linking of RNA molecules within cells using formaldehyde. This is followed by the isolation of RNA molecules and fragmentation using RNase enzymes.
The library construction step involves the ligation of RNA molecules that are near each other within cells. This is achieved using a biotinylated RNA linker that binds to the 5' phosphate of the fragmented RNA molecules. The linker is then ligated to the 3' end of the RNA molecules using T4 RNA ligase.
The resulting RNA molecules are reverse-transcribed using a primer that binds to the linker sequence. This generates chimeric cDNAs that contain sequences from both the ligated RNA molecules. The chimeric cDNAs are then sequenced using high-throughput sequencing technologies.
The sequencing data obtained from RIC-seq is analyzed to identify RNA-RNA interactions. This involves the alignment of the sequencing reads to the reference genome and the identification of chimeric reads that contain sequences from two different RNA molecules. The identified RNA-RNA interactions can be visualized using genome browsers such as the UCSC Genome Browser.
Schematic diagram of the RIC-seq procedures (Cao et al., 2021)
RIC-seq has many applications in the field of RNA biology. It can identify long-range RNA-RNA interactions that regulate gene expression. Long-range RNA-RNA interactions can occur between coding and non-coding RNAs and can play an important role in gene regulation. Ric-seq can also identify RNA-RNA interactions involved in RNA processing and splicing. In addition, this technique allows the study of RNA virus-host interactions. RNA viruses replicate within host cells and their replication is dependent on interactions between viral and host RNA. Ric-seq can be used to identify these interactions, providing insight into viral replication and pathogenesis.
RIC-seq faithfully captures RNA secondary structures and tertiary interactions (Cai et al., 2020).