Convergence in LINE-1 nucleotide variations can benefit redundantly forming triplexes with lncRNA in mammalian X-chromosome inactivation

Abstract

Abstract Background Associations between X-inactive transcript (Xist)–long noncoding RNA (lncRNA) and chromatin are critical intermolecular interactions in the X-chromosome inactivation (XCI) process. Despite high-resolution analyses of the Xist RNA-binding sites, specific interaction sequences are yet to be identified. Based on elusive features of the association between Xist RNA and chromatin and the possible existence of multiple low-affinity binding sites in Xist RNA, we defined short motifs (≥5 nucleotides), termed as redundant UC/TC (r-UC/TC) or AG (r-AG) motifs, which may help in the mediation of triplex formation between the lncRNAs and duplex DNA. Results The study showed that r-UC motifs are densely dispersed throughout mouse and human Xist/XIST RNAs, whereas r-AG motifs are even more densely dispersed along opossum RNA-on-the-silent X (Rsx) RNA, and also along both full-length and truncated long interspersed nuclear elements (LINE-1s, L1s) of the three species. Predicted secondary structures of the lncRNAs showed that the length range of these sequence motifs available for forming triplexes was even shorter, mainly 5- to 9-nucleotides long. Quartz crystal microbalance (QCM) measurements and Monte Carlo (MC) simulations indicated that minimum-length motifs can reinforce the binding state by increasing the copy number of the motifs in the same RNA or DNA molecule. Further, r-AG motifs in L1s had a similar length-distribution pattern, regardless of the similarities in the length or sequence of L1s across the three species; this also applies to high-frequency mutations in r-AG motifs, which suggests convergence in L1 sequence variations. Conclusions Multiple short motifs in both RNA and duplex DNA molecules could be brought together to form triplexes with either Hoogsteen or reverse Hoogsteen hydrogen bonding, by which their associations are cooperatively enhanced. This novel triplex interaction could be involved in associations between lncRNA and chromatin in XCI, particularly at the sites of L1s. Potential binding of Xist/XIST/Rsx RNAs specifically at L1s is most likely preserved through the r-AG motifs conserved in mammalian L1s through convergence in L1 nucleotide variations and by maintaining a particular r-UC/r-AG motif ratio in each of these lncRNAs, irrespective of their poorly conserved sequences.

Keywords

• LINE-1
• Long noncoding RNA
• X-chromosome inactivation
• Xist RNA
• XIST RNA
• Rsx RNA