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Researchers develop method for deciphering positional rules in splicing
![Figure 1. Diagram of the CRIC-seq method. Credit: Xue Yuanchao's group Researchers develop method for deciphering positional rules in splicing](https://scx1.b-cdn.net/csz/news/800a/2023/researchers-develop-me-1.jpg)
A research team led by Prof. Xue Yuanchao from the Institute of Biophysics of the Chinese Academy of Sciences has developed a new method for global profiling of in-situ RNA–RNA contacts associated with a specific RNA-binding protein (RBP) and revealed positional mechanisms by which PTBP1-associated RNA loops regulate cassette exon splicing.
This study was published online in Molecular Cell on March 22.
In eukaryotes, the same pre-mRNA can produce multiple protein isoforms to execute similar or different biological functions through alternative splicing. Several longstanding models proposed that RBPs may regulate alternative splicing by modulating long-range RNA–RNA interactions (RRI). However, direct experimental evidence was lacking.
To fill the knowledge gap, the researchers created a capture RIC-seq (CRIC-seq) method to map specific RBP-associated RRIs by leveraging the principle of RIC-seq technology and immunoprecipitation-mediated enrichment. RIC-seq technology had been developed by Prof. Xue's group in 2020 for global profiling of all expressing RBP-mediated RRIs rather than a specific one.
Using the CRIC-seq method and their self-developed data analysis pipeline, the researchers obtained high-confidence RRIs mediated by PTBP1, hnRNPA1, or SRSF1 and generated functional 3D RNA maps to investigate the positional mechanisms of these RBPs from the new angle of RNA spatial conformation.
![Figure 2. PTBP1-associated RNA loops in splicing regulation and tumor cell growth. Credit: Xue Yuanchao's group Researchers develop method for deciphering positional rules in splicing](https://scx1.b-cdn.net/csz/news/800a/2023/researchers-develop-me-2.jpg)
Similar to hnRNPA1, but unlike SRSF1, the researchers found that PTBP1-mediated RNA loops tended to be enriched on one side of the intron when enhancing the splicing of cassette exons. In contrast, when repressing splicing, PTBP1-mediated RNA loops tended to span the cassette exons. These findings demonstrate that PTBP1 can regulate cassette exon splicing via mediating specific RNA loops in a position-dependent manner.
According to minigene-based analysis, the researchers found that only wild-type (WT) PTBP1, but not C23S mutant PTBP1, can form a homodimer to rescue the corresponding splicing changes after PTBP1 knockdown.
Furthermore, the CRISPR-dCasRx-mediated tethering assay confirmed that RNA loops engineered by WT PTBP1 can modulate the usage of non-PTBP1-regulated cassette exons. These results demonstrate that PTBP1 dimerization can drive RNA looping to modulate splicing.
In addition, by integrating cancer-related splicing quantitative trait loci (sQTLs) with PTBP1-associated RRIs, the researchers identified 121 sQTLs that potentially affect splicing by altering RNA loops. Unexpectedly, one sQTL located in the intronic loops of HERPUD2 can induce exon 7 skipping, thus generating a short isoform to promote the proliferation of HeLa cells. These results demonstrate how cancer-related non-coding mutations modulate RNA spatial conformation to promote tumor cell growth.
More information: Rong Ye et al, Capture RIC-seq reveals positional rules of PTBP1-associated RNA loops in splicing regulation, Molecular Cell (2023). DOI: 10.1016/j.molcel.2023.03.001
Journal information: Molecular Cell
Provided by Chinese Academy of Sciences