Long noncoding RNAs (lncRNAs) influence cellular function through binding events that often depend on the lncRNA secondary structure. One such lncRNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), is upregulated in many cancer types and has a myriad of protein- and miRNA-binding sites. Recently, a secondary structural model of MALAT1 in noncancerous cells was proposed to form 194 hairpins and 13 pseudoknots. That study postulated that, in cancer cells, the MALAT1 structure likely varies, thereby influencing cancer progression. This work analyzes how that structural model is expected to change in K562 cells, which originated from a patient with chronic myeloid leukemia (CML), and in HeLa cells, which originated from a patient with cervical cancer. Dimethyl sulfate-sequencing (DMS-Seq) data from K562 cells and psoralen analysis of RNA interactions and structure (PARIS) data from HeLa cells were compared to the working structural model of MALAT1 in noncancerous cells to identify sites that likely undergo structural alterations. MALAT1 in K562 cells is predicted to become more unstructured, with almost 60% of examined hairpins in noncancerous cells losing at least half of their base pairings. Conversely, MALAT1 in HeLa cells is predicted to largely maintain its structure, undergoing 18 novel structural rearrangements. Moreover, 50 validated miRNA-binding sites are affected by putative secondary structural changes in both cancer types, such as miR-217 in K562 cells and miR-20a in HeLa cells. Structural changes unique to K562 cells and HeLa cells provide new mechanistic leads into how the structure of MALAT1 may mediate cancer in a cell-type specific manner.

Recent studies suggest noncoding RNAs interact with genomic DNA, forming an RNA·DNA-DNA triple helix. With at least one RNA·DNA-DNA triple helix predicted in the promoter regions of most human genes, RNA·DNA-DNA triple helices may be a common mechanism for regulating transcription. Additionally, cells could employ RNA modifications to regulate the formation of these triple helices. With over 143 naturally occurring RNA modifications, we hypothesize that some modifications stabilize RNA·DNA-DNA triple helices while others destabilize them. Here, we focus on a pyrimidine-motif triple helix composed of canonical U·A-T and C·G-C base triples. We employed electrophoretic mobility shift assays and microscale thermophoresis to examine how eleven different RNA modifications at a single position in an RNA·DNA-DNA triple helix affect stability: 5-methylcytidine (m5C), 5-methyluridine (m5U or rT), 3-methyluridine (m3U), pseudouridine (Ψ), 4-thiouridine (s4U), N6-methyladenosine (m6A), inosine (I), and each nucleobase with 2'-O-methylation (Nm). Compared to the unmodified U·A-T base triple, some modifications have no significant change in stability (Um·A-T), some have ~2.5-fold decreases in stability (m5U·A-T, Ψ·A-T, and s4U·A-T), and some completely disrupt triple helix formation (m3U•A-T). To identify potential biological examples of RNA·DNA-DNA triple helices controlled by an RNA modification, we searched RMVar, a database for RNA modifications mapped at single-nucleotide resolution, for lncRNAs containing an RNA modification within a pyrimidine-rich sequence. Using electrophoretic mobility shift assays, the binding of DNA-DNA to a 22-mer segment of human lncRNA Al157886.1 was destabilized by ~1.7-fold with the substitution of m5C at known m5C sites. Therefore, the formation and stability of cellular RNA·DNA-DNA triple helices could be influenced by RNA modifications.

The formation of pyrimidine‐motif RNA●DNA‐DNA (R●D‐D) triple helices (or triplexes), in which ‘●’ and ‘‐’ represent Hoogsteen and Watson‐Crick interactions, has been proposed for over 60 years, but the stability of these structures with RNA modifications has yet to be studied. Eleven RNA modifications were chosen for this study based on their presence in human transcripts and their effects on human health: 5‐methylcytidine (m5C), 5‐methyluridine (m5U), pseudouridine (Ψ), 2ʹ‐O‐methyladenosine (Am), 2ʹ‐O‐methylcytidine (Cm), 2ʹ‐O‐methylguanosine (Gm), 2ʹ‐O‐methyluridine (Um), 3‐methyluridine (m3U), 4‐thiouridine (s4U), inosine (I), and N6‐methyladenosine (m6A). Several of these had been previously found to stabilize or destabilize other nucleic acid duplex and triplex structures. Using both native gel‐shift assays and microscale thermophoresis, the relative stability of a single modified position in a pyrimidine‐motif R●D‐D triple helix was measured at neutral pH. All eleven modifications were found to either have no effect or to destabilize the R●D‐D triple helix, ranging from 2‐fold to complete disruption of binding. For each of the canonical R●D‐D base triples (U●A‐T and C●G‐C), the 2ʹ‐O‐methyl modifications were found to be the least destabilizing, whereas those that directly interfered with the Hoogsteen interactions, such as m3U, were the most destabilizing modifications. As the formation of R●D‐D triple helices in promoter regions of DNA leads to transcriptional enhancement and repression, this study reveals that some RNA modifications could potentially inhibit R●D‐D triplex formation as another level of transcriptional regulation.