ADAR RNA editing on antisense RNAs results in apparent U-to-C base changes on overlapping sense transcripts.

Despite hundreds of RNA modifications described to date, only RNA editing results in a change in the nucleotide sequence of RNA molecules compared to the genome. In mammals, two kinds of RNA editing have been described so far, adenosine to inosine (A-to-I) and cytidine to uridine (C-to-U) editing. Recent improvements in RNA sequencing technologies have led to the discovery of a continuously growing number of editing sites. These methods are powerful but not error-free, making routine validation of newly-described editing sites necessary. During one of these validations on DDX58 mRNA, along with A-to-I RNA editing sites, we encountered putative U-to-C editing. These U-to-C edits were present in several cell lines and appeared regulated in response to specific environmental stimuli. The same findings were also observed for the human long intergenic non-coding RNA p21 (hLincRNA-p21). A more in-depth analysis revealed that putative U-to-C edits result from A-to-I editing on overlapping antisense RNAs that are transcribed from the same loci. Such editing events, occurring on overlapping genes transcribed in opposite directions, have recently been demonstrated to be immunogenic and have been linked with autoimmune and immune-related diseases. Our findings, also confirmed by deep transcriptome data, demonstrate that such loci can be recognized simply through the presence of A-to-I and U-to-C mismatches within the same locus, reflective A-to-I editing both in the sense-oriented transcript and in the cis-natural antisense transcript (cis-NAT), implying that such clusters could be a mark of functionally relevant ADAR1 editing events.

Conserved Pseudoknots in lncRNA MEG3 Are Essential for Stimulation of the p53 Pathway.

Long non-coding RNAs (lncRNAs) are key regulatory molecules, but unlike with other RNAs, the direct link between their tertiary structure motifs and their function has proven elusive. Here we report structural and functional studies of human maternally expressed gene 3 (MEG3), a tumor suppressor lncRNA that modulates the p53 response. We found that, in an evolutionary conserved region of MEG3, two distal motifs interact by base complementarity to form alternative, mutually exclusive pseudoknot structures ("kissing loops"). Mutations that disrupt these interactions impair MEG3-dependent p53 stimulation in vivo and disrupt MEG3 folding in vitro. These findings provide mechanistic insights into regulation of the p53 pathway by MEG3 and reveal how conserved motifs of tertiary structure can regulate lncRNA biological function.

Inverted repeat Alu elements in the human lincRNA-p21 adopt a conserved secondary structure that regulates RNA function.

LincRNA-p21 is a long intergenic non-coding RNA (lincRNA) involved in the p53-mediated stress response. We sequenced the human lincRNA-p21 (hLincRNA-p21) and found that it has a single exon that includes inverted repeat Alu elements (IRAlus). Sense and antisense Alu elements fold independently of one another into a secondary structure that is conserved in lincRNA-p21 among primates. Moreover, the structures formed by IRAlus are involved in the localization of hLincRNA-p21 in the nucleus, where hLincRNA-p21 colocalizes with paraspeckles. Our results underscore the importance of IRAlus structures for the function of hLincRNA-p21 during the stress response.

HOTAIR forms an intricate and modular secondary structure.

Long noncoding RNAs (lncRNAs) have recently emerged as key players in fundamental cellular processes and diseases, but their functions are poorly understood. HOTAIR is a 2,148-nt-long lncRNA molecule involved in physiological epidermal development and in pathogenic cancer progression, where it has been demonstrated to repress tumor and metastasis suppressor genes. To gain insights into the molecular mechanisms of HOTAIR, we purified it in a stable and homogenous form in vitro, and we determined its functional secondary structure through chemical probing and phylogenetic analysis. The HOTAIR structure reveals a degree of structural organization comparable to well-folded RNAs, like the group II intron, rRNA, or lncRNA steroid receptor activator. It is composed of four independently folding modules, two of which correspond to predicted protein-binding domains. Secondary structure elements that surround protein-binding motifs are evolutionarily conserved. Our work serves as a guide for "navigating" through the lncRNA HOTAIR and ultimately for understanding its function.