Quantitative interactomics screen reveals the role of the lncRNA ANRIL during viral infection
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Abstract
Recent advances in deep sequencing technologies have revealed that while only 2-5% of the human genome encodes protein-coding genes, an estimated 80% of these genes are actively transcribed into a variety of non-coding RNAs. Among these, long non-coding RNAs represent a major subclass, with over 35,000 annotated genes. Despite their abundance, most of these genes remain functionally uncharacterized.
The antisense non-coding RNA in the INK4 locus (ANRIL) is transcribed from the disease-associated hotspot 9p21.3, which has been implicated in cancer development and cardiovascular disorders, yet its molecular functions remain poorly defined. To systematically characterize ANRIL, a quantitative mass spectrometry-based interactomics screen using 14 nonoverlapping RNA fragments covering the longest ANRIL isoforms was performed. This strategy identified 310 interacting proteins, each exhibiting distinct binding patterns across different fragments, revealing both previously described and yet unknown interactors.
Among the newly identified proteins, the uncharacterized protein C7orf50 bound to two ANRIL fragments. Functional characterization revealed an enrichment of C7orf50 interactors involved in ribosomal biogenesis, including known ANRIL-binding members of the PeBoW complex. These findings suggest a potential role of C7orf50 during ribosomal maturation and indicate an additional target of ANRIL-mediated regulation during ribosome biogenesis.
Furthermore, computational analysis of the ANRIL interactome recapitulated previously reported biological functions, such as chromatin remodeling, while also suggesting previously unrecognized roles in virus infection. Interestingly, ANRIL knockdown led to altered expression of genes associated with interferon signaling and viral entry. Infection studies with dsDNA and ssRNA viruses resulted in virus-specific modulation of ANRIL expression. Notably, ANRIL was upregulated in response to cedar virus (CedV) infection in a time- and dose-dependent manner. Transcriptomic analysis revealed a concurrent increase in immune response genes, particularly those involved in type I interferon signaling. Furthermore, ANRIL lockdown led to increased CedV RNA levels and titers, demonstrating a link between ANRIL and viral infection. Preliminary mechanistic insights indicated that the knockdown of the newly identified ANRIL-interacting protein INO80 also increased CedV RNA levels. However, further analyses are required to determine the precise molecular mechanisms involved.
Overall, the unbiased RNA-protein interaction screen provides insights into how ANRIL mediates various cellular functions through protein interactions and paves the way for future investigations into the role of ANRIL in health and disease.