Conserved architecture of a functional lncRNA-protein interaction in the DNA damage response pathway

Long non-coding RNA (lncRNAs) structures are emerging as key drivers in cellular functions. Unlike proteins, lncRNAs exhibit diverse patterns of conservation, reflecting the evolutionary genetic pressures they have undergone. The long intergenic non-coding RNA p21 (lincRNA-p21) is a low-conserved, mammal-specific lncRNA. It regulates the cis activation of its neighboring gene Cdkn1A, which encodes the p21 protein, an essential player in the DNA damage response. The syntenic mouse and human lincRNA-p21 genes display low sequence similarity, and their transcripts show distinct cellular localization and different additional cellular functions. Despite these evolutionarily divergent features, both transcripts interact with the same partner in the nucleus, the heterogeneous nuclear ribonucleoprotein K (hnRNP K), to regulate the expression of the Cdkn1a gene. Elucidating the molecular bases of the lincRNA-p21 interaction with hnRNP K is crucial for understanding the activation of the p21-dependent DNA damage response. However, it remains unknown which RNA and protein motifs are involved in this interaction, and whether those motifs are shared between mice and humans. To address this question, we employed in vitro, in cellulo, and ex cellulo SHAPE-MaP to determine the secondary structures of the full-length mouse (3 kb) and human (4 kb) lincRNA-p21 transcripts. We identified a conserved structural RNA motif that comprises a [~]20-nucleotide terminal stem-loop containing a conserved tetranucleotide UCAY. In silico analyses, molecular dynamics simulations, and in vitro protein-RNA binding assays support the interaction of lincRNA-p21 and this RNA motif with the KH3 domain of hnRNP K. Using eCLIP-qRT-PCR and fluorescence correlation spectroscopy (FCS), we demonstrate that a mutant lacking the conserved structural RNA motif is no longer able to bind the hnRNP K protein within cells. In conclusion, our study reveals a conserved lncRNA-protein interaction between two syntenic transcripts with divergent sequences and structures, highlighting evolutionary pressure to maintain a critical cellular stress-control element. Our data provide molecular details for targeting a specific RNA-protein interaction essential for regulating the cellular response to DNA damage.