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  • Exploring RNA Structure at the Single-Cell Level

    Click image for larger version  Name:	Low-Res_Picture1.jpg Views:	0 Size:	132.4 KB ID:	325408
    Different cells have different RNA structures and studying those structures offers insights into cell functions. Photo credit: A*STAR’s GIS




    Introduction to Sc-SPORT
    The Genome Institute of Singapore (GIS) under A*STAR has developed a novel method for sequencing single-cell RNA, offering a fresh perspective on the study of RNA structures in individual cells. This technique, named Sc-SPORT (Single Cell Structure Probing Of RNA Transcripts), stands out by focusing on RNA shape rather than its sequence to identify biomarkers critical for human development and disease.

    The Significance of RNA Structure in Cellular Function
    Unlike previous methods that generalized RNA structures across millions of cells, Sc-SPORT allows for the examination of RNA shapes at the level of individual cells. This is crucial as the shape of RNA in each cell can vary significantly, influencing different cellular functions. Prior approaches, which relied on mass RNA analysis, often missed these individual variabilities in RNA structure and function.

    The Technology Behind Sc-SPORT
    The recent study, “RNA structure profiling at single-cell resolution reveals new determinants of cell identity,” published in Nature Methods on January 4, 2024, highlights this innovative approach. The Sc-SPORT technique was developed by Dr. Wan Yue, Deputy Executive Director, and Principal Investigator at A*STAR’s GIS, in collaboration with Dr. Jiaxu Wang, A*STAR’s GIS fellow, and Dr. Roland Huber from A*STAR’s Bioinformatics Institute (BII). This method marks a first in exploring RNA structure heterogeneity and targeting rare cell types at the single-cell level.

    Advancements in Single-Cell RNA Analysis
    The traditional approach to RNA structure analysis required millions of cells, resulting in a generalized overview that blurred individual cellular differences. Sc-SPORT, however, enables a more detailed appreciation of the unique information within each cell and helps identify potential anomalies in cellular function.

    Dr. Wan Yue commented on the significance of this advancement: “Unlike traditional methods that study millions of cells at once, this breakthrough enables the examination of RNA structures in each cell individually, unveiling unique patterns and biomarkers, enriching our understanding of cellular fate. In the face of the COVID-19 pandemic, caused by an RNA virus, understanding how RNA folds within cells becomes paramount. This study not only sheds light on the fundamental functions of RNA but also paves the way for unprecedented insights into the potential of RNA as a game-changer in biomedical sciences.”

    Methods and Results
    Sc-SPORT involves several precise steps for RNA structure analysis in single cells. Initially, cells are dissociated into a suspension and treated with NAI-N3. After centrifugation and resuspension, individual cells are isolated using mouth pipetting and placed into tubes containing a special buffer. This is followed by a thermocycler-run fragmentation and primer annealing process. Next, a reverse transcription (RT) reaction mix is added and the RT program is run. The RT products undergo PCR amplification. The PCR products are purified, prepared for sequencing using the Illumina Nextra XT kit, and sequenced on the Illumina Hi-Seq 4000. Data analysis includes trimming and mapping of reads, identifying mutations, calculating reactivities, quantifying and normalizing expression levels, performing cell cycle assignments, alternative splicing quantification, and quality control.

    The study demonstrated the accuracy of sc-SPORT in analyzing RNA structures and applied this technique to examine RNA in human embryonic stem cells (hESCs) and during various stages of neuronal differentiation. Key findings include the observation that individual RNA transcripts can assume diverse structures in different cells. The study also revealed that RNA-binding proteins can regulate these structural differences, contributing to the distinct identities of individual cells. This research provides initial insights into the dynamics, regulation, and function of RNA structures within single cells during the process of neurogenesis.

    Potential Applications and Future Directions
    This advanced method of studying RNA structures in single cells opens new avenues for identifying structure biomarkers that could be dysregulated in diseases. It adds a novel layer to existing single-cell information and could serve as a unique biomarker or drug target for diseases where RNA levels remain constant.

    Professor Liu Jian Jun, Acting Executive Director at A*STAR’s GIS, emphasized “In the fast-evolving landscape of RNA research, the work conducted at GIS underlines our commitment to deciphering the complexities of RNA. The heightened awareness of RNA's role, especially in the context of the COVID-19 pandemic, contributes to better pandemic preparedness and offers new insights for potential treatments against RNA viruses.” The team is currently refining scale-up strategies for single-cell RNA structure sequencing and applying this technique to more complex cellular developmental processes and cancer studies.

    Original Publication
    Wang, J., Zhang, Y., Zhang, T. et al. RNA structure profiling at single-cell resolution reveals new determinants of cell identity. Nat Methods (2024). https://doi.org/10.1038/s41592-023-02128-y

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