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Superposition of MALAT1 th11 and MALAT1 Core RNA triple helices. (A) WC-helix of MALAT1 th11 triple helix (tricolor ribbon representation as described in Figure 1) superposed with WC-helix of MALAT1 Core triple helix (tan ribbon representation). RMSD is calculated over 424 atom pairs of WC-helix. (B) Shift of Hoogsteen strand in MALAT1 th11 triple helix (blue stick representation) relative to that in MALAT1 Core (tan stick representation) is observed in superposition analysis. The distances in red font between corresponding P atoms (blue and tan circles) are shown by dashed red lines. Nucleotide numbering is shown in schematics for each triple helix. The C72-G41 base pair in MALAT1 Core is marked by navy blue box and corresponding A75-U44 pair in MALAT th11 is marked by yellow box while U13 in Hoogsteen strand is orange.
Source publication
Three-dimensional structures have been solved for several naturally occurring RNA triple helices, although all are limited to six or fewer consecutive base triples, hindering accurate estimation of global and local structural parameters. We present an X-ray crystal structure of a right-handed, U•A-U-rich RNA triple helix with 11 continuous base tri...
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... G5-C54 and G6-C53 base pairs in stem I interact with A67 and A68, respectively, to form type II and I A-minor interactions, a common tertiary motif in RNA structures (Supplementary Figure S4A- B and Table S2) (32,33). These two consecutive A-minor interactions consequently form a ribose zipper motif (Supplementary Figure S4C), which is another structural element that stabilizes the tertiary fold of MALAT1 th11 (34,35). Both the A-minor interactions and ribose zipper are present in the MALAT1 Core (Supplementary Figure S1B) (12). ...
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... the A-minor interactions and ribose zipper are present in the MALAT1 Core (Supplementary Figure S1B) (12). Additional non-canonical nucleotide interactions present in MALAT1 th11 are as follows: (i) GTP1-C58 in stem I interact via their Hoogsteen edges in a trans orientation (tHH in Supplementary Figure S4D), (ii) G2-U57 in stem I (Supplementary Figure S4E) and G25-U32 in stem II (not shown) form cis Watson-Crick (cWC) wobble pairs and (iii) G27-A30 in stem II interact via their sugar/Hoogsteen edges in a trans conformation (tSH in Supplementary Figure S4F and Table S2) (36). Overall, most nucleotide interactions in MALAT1 th11 are similar to those observed previously in the MALAT1 Core structure (Supplementary Figure S1B and C) (12). ...
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... the A-minor interactions and ribose zipper are present in the MALAT1 Core (Supplementary Figure S1B) (12). Additional non-canonical nucleotide interactions present in MALAT1 th11 are as follows: (i) GTP1-C58 in stem I interact via their Hoogsteen edges in a trans orientation (tHH in Supplementary Figure S4D), (ii) G2-U57 in stem I (Supplementary Figure S4E) and G25-U32 in stem II (not shown) form cis Watson-Crick (cWC) wobble pairs and (iii) G27-A30 in stem II interact via their sugar/Hoogsteen edges in a trans conformation (tSH in Supplementary Figure S4F and Table S2) (36). Overall, most nucleotide interactions in MALAT1 th11 are similar to those observed previously in the MALAT1 Core structure (Supplementary Figure S1B and C) (12). ...
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... the A-minor interactions and ribose zipper are present in the MALAT1 Core (Supplementary Figure S1B) (12). Additional non-canonical nucleotide interactions present in MALAT1 th11 are as follows: (i) GTP1-C58 in stem I interact via their Hoogsteen edges in a trans orientation (tHH in Supplementary Figure S4D), (ii) G2-U57 in stem I (Supplementary Figure S4E) and G25-U32 in stem II (not shown) form cis Watson-Crick (cWC) wobble pairs and (iii) G27-A30 in stem II interact via their sugar/Hoogsteen edges in a trans conformation (tSH in Supplementary Figure S4F and Table S2) (36). Overall, most nucleotide interactions in MALAT1 th11 are similar to those observed previously in the MALAT1 Core structure (Supplementary Figure S1B and C) (12). ...
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... structural differences occur in the Hoogsteen strand (RMSD 1.6 ˚ A for all corresponding atom pairs) rather than the WC-helix portion (RMSD 1 ˚ A for all corresponding atom pairs in each strand) (Supplementary Figure S9). By superposing the WC-helix portions of MALAT1 th11 and MALAT1 Core, we determined an RMSD value of 1.1 ˚ A over 424 atom pairs ( Figure 4A). Importantly, MALAT1 th11 avoids the Nucleic Acids Research, 2020, Vol. ...
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... steric clash because the phosphate backbone near U13 of Hoogsteen strand, namely C12, U14 and U15, are shifted 2.5-4 ˚ A away from the corresponding positions, i.e. C12, U13 and U14, in the superposed MALAT1 Core triple helix ( Figure 4B). This expansion by the Hoogsteen strand accommodates all nucleotides, including U13, in the Hoogsteen strand to form a continuous RNA triple helix (Figure 4 and Supplementary Figure S9). ...
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... U13 and U14, in the superposed MALAT1 Core triple helix ( Figure 4B). This expansion by the Hoogsteen strand accommodates all nucleotides, including U13, in the Hoogsteen strand to form a continuous RNA triple helix (Figure 4 and Supplementary Figure S9). However, subtle irregularities do occur, such as the lack of 2 -OH . . . ...
Citations
... Additionally, LNAs can form tighter double-and triple-stranded structures than unmodified DNA and RNA [50][51][52][53][54][55][56][57]. We chose an all-LNA backbone for L15 and PS-L15 because triple helices prefer the C3′-endo conformation, theoretically creating more favorable binding conditions when an all-LNA oligonucleotide is the "middle" purinerich strand of a triple helix [58]. L15 and PS-L15 are 15 nucleotides in length and have an A9GCA4 sequence ( Figure 1E), an A-rich tract sequence that was previously shown to interact with the MALAT1 and MENβ SLs [16]. ...
... Additionally, LNAs can form tighter double-and triple-stranded structures than unmodified DNA and RNA [50][51][52][53][54][55][56][57]. We chose an all-LNA backbone for L15 and PS-L15 because triple helices prefer the C3 ′ -endo conformation, theoretically creating more favorable binding conditions when an all-LNA oligonucleotide is the "middle" purine-rich strand of a triple helix [58]. L15 and PS-L15 are 15 nucleotides in length and have an A 9 GCA 4 sequence ( Figure 1E), an A-rich tract sequence that was previously shown to interact with the MALAT1 and MENβ SLs [16]. ...
Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and multiple endocrine neoplasia-β (MENβ) are two long noncoding RNAs upregulated in multiple cancers, marking these RNAs as therapeutic targets. While traditional small-molecule and antisense-based approaches are effective, we report a locked nucleic acid (LNA)-based approach that targets the MALAT1 and MENβ triple helices, structures comprised of a U-rich internal stem-loop and an A-rich tract. Two LNA oligonucleotides resembling the A-rich tract (i.e., A9GCA4) were examined: an LNA (L15) and a phosphorothioate LNA (PS-L15). L15 binds tighter than PS-L15 to the MALAT1 and MENβ stem loops, although both L15 and PS-L15 enable RNA•LNA-RNA triple-helix formation. Based on UV thermal denaturation assays, both LNAs selectively stabilize the Hoogsteen interface by 5–13 °C more than the Watson–Crick interface. Furthermore, we show that L15 and PS-L15 displace the A-rich tract from the MALAT1 and MENβ stem loop and methyltransferase-like protein 16 (METTL16) from the METTL16-MALAT1 triple-helix complex. Human colorectal carcinoma (HCT116) cells transfected with LNAs have 2-fold less MALAT1 and MENβ. This LNA-based approach represents a potential therapeutic strategy for the dual targeting of MALAT1 and MENβ.
... R = purine, Y = pyrimidine, "⦁" = Hoogsteen H-bonding, ":" = Watson-Crick Hbonding. Structure adapted from PDB ID: 6SVS [18] riboswitches in bacteria and facilitate RNA protection from degradation [21][22][23][24][25][26]. Exogenous RNA triple helices have great potential for application in imaging of endogenous RNAs, targetspecific inhibition of translation, and inhibition of pre-miRNA processing. ...
Nucleic acid probes are valuable tools in biology and chemistry and are indispensable for PCR amplification of DNA, RNA quantification and visualization, and downregulation of gene expression. Recently, triplex-forming oligonucleotides (TFO) have received increased attention due to their improved selectivity and sensitivity in recognizing purine-rich double-stranded RNA regions at physiological pH by incorporating backbone and base modifications. For example, triplex-forming peptide nucleic acid (PNA) oligomers have been used for imaging a structured RNA in cells and inhibiting influenza A replication. Although a handful of programs are available to identify triplex target sites (TTS) in DNA, none are available that find such regions in structured RNAs. Here, we describe TFOFinder, a Python program that facilitates the identification of intramolecular purine-only RNA duplexes that are amenable to forming parallel triple helices (pyrimidine/purine/pyrimidine) and the design of the corresponding TFO(s). We performed genome- and transcriptome-wide analyses of TTS in Drosophila melanogaster and found that only 0.3% (123) of total unique transcripts (35,642) show the potential of forming 12-purine long triplex forming sites that contain at least one guanine. Using minimization algorithms, we predicted the secondary structure(s) of these transcripts, and using TFOFinder, we found that 97 (79%) of the identified 123 transcripts are predicted to fold to form at least one TTS for parallel triple helix formation. The number of transcripts with potential purine TTS increases when the strict search conditions are relaxed by decreasing the length of the probe or by allowing up to two pyrimidine inversions or 1-nucleotide bulge in the target site. These results are encouraging for the use of modified triplex forming probes for live imaging of endogenous structured RNA targets, such as pre-miRNAs, and inhibition of target-specific translation and viral replication.
... ; https://doi.org/10.1101/2023.04.26.538412 doi: bioRxiv preprint backbone modification has been employed to eliminate this unfavorable interaction, which resulted in high TFO binding specificity and sensitivity, and in a greater mismatch discrimination as compared to using DNA or RNA TFO [9][10][11]. Triplex formation can further be favored and stabilized by employing base modifications [11][12][13][14][15][16][17]. ...
Nucleic acid probes are valuable tools in biology and chemistry and are indispensable for PCR amplification of DNA, RNA quantification and visualization, and downregulation of gene expression. Recently, triplex forming oligonucleotides (TFO) have received increased attention due to their improved selectivity and sensitivity in recognizing purine-rich double-stranded RNA regions at physiological pH by incorporating backbone and base modifications. For example, triplex forming peptide nucleic acid (PNA) oligomers have been used for imaging structured RNA in cells and inhibiting influenza A replication. Although a handful of programs are available to identify triplex target sites (TTS) in DNA, none are available that find such regions in structured RNAs. Here, we describe TFOFinder , a Python program that facilitates the identification of intramolecular purine-only RNA duplexes that are amenable to forming parallel triple helices (pyrimidine/purine/pyrimidine). We performed genome- and transcriptome-wide analyses of TTS in Drosophila melanogaster and found that only 0.3% (123) of total unique transcripts (35,642) show the potential of forming 12-purine long triplex forming sites that contain at least one guanine. Using minimization algorithms, we predicted the secondary structure(s) of these transcripts, and using TFOFinder , we found that 97 (79%) of the identified 123 transcripts are predicted to fold to form at least one TTS for parallel triple helix formation. The number of transcripts with potential purine TTS increases when the strict search conditions are relaxed by decreasing the length of the probes or by allowing up to two pyrimidine inversions or 1-nucleotide bulge in the target site. These results are encouraging for the use of modified triplex forming probes for live imaging of endogenous structured RNA targets, such as pre-miRNAs, and inhibition of target-specific translation and viral replication.
... 22 Finally, some gene-regulatory RNA riboswitches [23][24][25] leverage triplexes as binding pockets of ligands regardless of nucleobase moiety. Despite their biological or technological importance, 26 the structures of only a few longer triplex structures have been solved (e.g., PDB: 6SVS 27 ). Although these structures possess well-defined contacts, the degree of flexibility displayed by the third, triplex-forming oligomer, is unknown, yet is likely important in understanding how these molecules interact with partners. ...
... Average helical parameters of RNA ensembles generated from WAXS-driven MD were compared with those of a realistic U,A-U rich triplex. 27 Only the regions designed to be triplex were taken into account. ...
RNA triple helices are commonly observed tertiary motifs that are associated with critical biological functions, including signal transduction. Because the recognition of their biological importance is relatively recent, their full range of structural properties has not yet been elucidated. The integration of solution wide-angle X-ray scattering (WAXS) with molecular dynamics (MD) simulations, described here, provides a new way to capture the structures of major-groove RNA triplexes that evade crystallographic characterization. This method yields excellent agreement between measured and computed WAXS profiles and allows for an atomically detailed visualization of these motifs. Using correlation maps, the relationship between well-defined features in the scattering profiles and real space characteristics of RNA molecules is defined, including the subtle conformational variations in the double-stranded RNA upon the incorporation of a third strand by base triples. This readily applicable approach has the potential to provide insight into interactions that stabilize RNA tertiary structure that enables function.
... 8,13 Structure-function studies by the Steitz lab demonstrated that the C+GC base triple punctuating the UAU triplex stem (Scheme 1) contributes significantly to triplex stability, 6,14 though Brown and co-workers successfully engineered a mutant that folds with an uninterrupted 10 UAU base-triple stem. 15 The unique ENE triplex architecture has been targeted by small molecule library screens, 16,17 a useful method to identify synthetic binders via the displacement of RNA binding fluorophores. 18,19 Binders were found that can both disrupt 17 and enhance 20 ENE triplex stability, resulting in increased and decreased sensitivity to exonuclease digestion, respectively. ...
... (5) Non-canonical interaction-based RNA modules are RNA structural modules that are formed by non-canonical interactions and can be predicted by module prediction methods (Cruz and Westhof, 2011;Zirbel et al., 2015). Some recent determined RNA structures have demonstrated the importance of these non-canonical interactions (Butcher and Pyle, 2011): the recently crystalized MALAT1_th11 RNA (Ruszkowska et al., 2020) shows a UA-U-rich RNA triple helix with 11 consecutive base triples ( Figure 3A); the two transsugar-Hoogsteen G:A base-pairs in the kink-turn module (Huang et al., 2019a) enables its folding in 3D ( Figure 3B); and the triple interactions in the pseudoknot structure of the glutamine-II riboswitch (Huang et al., 2019b) is known to be crucial for ligand binding (Figure 3C). A structural module in RNA is a set of ordered non-Watson-Crick basepairs embedded between Watson-Crick pairs, which are recurrent in the RNA structure ( Figure 4B). ...
RNA is a unique bio-macromolecule that can both record genetic information and perform biological functions in a variety of molecular processes, including transcription, splicing, translation, and even regulating protein function. RNAs adopt specific three-dimensional conformations to enable their functions. Experimental determination of high-resolution RNA structures using x-ray crystallography is both laborious and demands expertise, thus, hindering our comprehension of RNA structural biology. The computational modeling of RNA structure was a milestone in the birth of bioinformatics. Although computational modeling has been greatly improved over the last decade showing many successful cases, the accuracy of such computational modeling is not only length-dependent but also varies according to the complexity of the structure. To increase credibility, various experimental data were integrated into computational modeling. In this review, we summarize the experiments that can be integrated into RNA structure modeling as well as the computational methods based on these experimental data. We also demonstrate how computational modeling can help the experimental determination of RNA structure. We highlight the recent advances in computational modeling which can offer reliable structure models using high-throughput experimental data.
... Since then, it has been established that RNA triple helices can perform a variety of cellular functions: Metal-ion binding to facilitate F I G U R E 1 Composition and structural arrangement of major-groove RNA triple helices. Hydrogen-bonding interactions (gray dashed lines) are shown for the two canonical major-groove base triples: (a) U•A-U and C + •G-C (ball-and-stick representation of base triples from PDB 6SVS; Ruszkowska, Ruszkowski, Hulewicz, Dauter, & Brown, 2020). Interactions are shown along with the Hoogsteen and Watson-Crick faces. ...
... For RNA triple helices, the structural parameters of poly(U•A-U) structures were derived from models generated using a combination of X-ray fiber diffraction data and computational modeling (Arnott & Bond, 1973;Arnott et al., 1976;Chandrasekaran, Giacometti, & Arnott, 2000;Raghunathan, Miles, & Sasisekharan, 1995). More recently, an X-ray crystal structure was solved for a U•A-U-rich RNA triple helix spanning 11 consecutive base triples, thereby revealing the global and local structural parameters ( Figure 2 and Table 2; Ruszkowska et al., 2020). Overall, the right-handed RNA triple helix, which has a central C•G-C base triple flanked on both sides by five U•A-U base triples, is an A-family conformer and is quantitatively similar to A 0 -RNA, which is effectively an underwound form of A-RNA (Table 2; Arnott, Hukins, Dover, Fuller, & Hodgson, 1973;Ruszkowska et al., 2020;Tanaka et al., 1999). ...
... More recently, an X-ray crystal structure was solved for a U•A-U-rich RNA triple helix spanning 11 consecutive base triples, thereby revealing the global and local structural parameters ( Figure 2 and Table 2; Ruszkowska et al., 2020). Overall, the right-handed RNA triple helix, which has a central C•G-C base triple flanked on both sides by five U•A-U base triples, is an A-family conformer and is quantitatively similar to A 0 -RNA, which is effectively an underwound form of A-RNA (Table 2; Arnott, Hukins, Dover, Fuller, & Hodgson, 1973;Ruszkowska et al., 2020;Tanaka et al., 1999). Despite the presence of a Hoogsteen strand, the RNA triple helix has a diameter of 24 Å, which is nearly identical to those of double helices (Table 2). ...
It has been nearly 63 years since the first characterization of an RNA triple helix in vitro by Gary Felsenfeld, David Davies, and Alexander Rich. An RNA triple helix consists of three strands: A Watson–Crick RNA double helix whose major‐groove establishes hydrogen bonds with the so‐called “third strand”. In the past 15 years, it has been recognized that these major‐groove RNA triple helices, like single‐stranded and double‐stranded RNA, also mediate prominent biological roles inside cells. Thus far, these triple helices are known to mediate catalysis during telomere synthesis and RNA splicing, bind to ligands and ions so that metabolite‐sensing riboswitches can regulate gene expression, and provide a clever strategy to protect the 3′ end of RNA from degradation. Because RNA triple helices play important roles in biology, there is a renewed interest in better understanding the fundamental properties of RNA triple helices and developing methods for their high‐throughput discovery. This review provides an overview of the fundamental biochemical and structural properties of major‐groove RNA triple helices, summarizes the structure and function of naturally occurring RNA triple helices, and describes prospective strategies to isolate RNA triple helices as a means to establish the “triplexome”.
This article is categorized under:
• RNA Structure and Dynamics > RNA Structure and Dynamics
• RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry
• RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems
Abstract
The three‐dimensional structures of naturally occurring RNA triple helices are displayed with the three strands denoted in blue, purple, and green. Collectively, these RNA triple helices play important biological roles in catalysis, binding to ligands (orange), and stabilizing RNA.
By combining in silico, biophysical, and in vitro experiments, we decipher the topology, physical, and potential biological properties of hybrid-parallel nucleic acids triplexes, an elusive structure at the basis of life. We found that hybrid triplex topology follows a stability order: r(Py)-d(Pu)·r(Py) > r(Py)-d(Pu)·d(Py) > d(Py)-d(Pu)·d(Py) > d(Py)-d(Pu)·r(Py). The r(Py)-d(Pu)·d(Py) triplex is expected to be preferred in the cell as it avoids the need to open the duplex reducing the torsional stress required for triplex formation in the r(Py)-d(Pu)·r(Py) topology. Upon a massive collection of melting data, we have created the first predictor for hybrid triplex stability. Leveraging this predictor, we conducted a comprehensive scan to assess the likelihood of the human genome and transcriptome to engage in triplex formation. Our findings unveil a remarkable inclination—of both the human genome and transcriptome—to generate hybrid triplex formation, particularly within untranslated (UTRs) and regulatory regions, thereby corroborating the existence of a triplex-mediated regulatory mechanism. Furthermore, we found a correlation between nucleosome linkers and Triplex-forming sequence (TFS) which agree with a putative role of triplexes in arranging chromatin structure.
The accumulation of the 8-kb oncogenic long noncoding MALAT1 RNA in cells is dependent on the presence of a protective triple helix structure at the 3′ terminus. While recent studies have examined the functional importance of numerous base triples within the triplex and its immediately adjacent base pairs, the functional importance of peripheral duplex elements has not been thoroughly investigated. To investigate the functional importance of a peripheral linker region that was previously described as unstructured, we employed a variety of assays including thermal melting, protection from exonucleolytic degradation by RNase R, small-angle X-ray scattering, biochemical ligation and binding assays, and computational modeling. Our results demonstrate the presence of a duplex within this linker that enhances the functional stability of the triplex in vitro, despite its location more than 40 Å from the 3′ terminus. We present a full-length model of the MALAT1 triple helix-containing RNA having an extended rod-like structure and comprising 33 layers of coaxial stacking interactions. Taken together with recent research on a homologous triplex, our results demonstrate that peripheral elements anchor and stabilize triplexes in vitro. Such peripheral elements may also contribute to the formation and stability of some triple helices in vivo.
