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Structural insights into the stabilization of MALAT1 noncoding RNA by a bipartite triple helix

Nature Structural & Molecular Biology volume 21, pages 633–640 (2014)Cite this article

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Abstract

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a highly abundant nuclear long noncoding RNA that promotes malignancy. A 3′-stem-loop structure is predicted to confer stability by engaging a downstream A-rich tract in a triple helix, similar to the expression and nuclear retention element (ENE) from the KSHV polyadenylated nuclear RNA. The 3.1-Å-resolution crystal structure of the human MALAT1 ENE and A-rich tract reveals a bipartite triple helix containing stacks of five and four U•A-U triples separated by a C+•G-C triplet and C-G doublet, extended by two A-minor interactions. In vivo decay assays indicate that this blunt-ended triple helix, with the 3′ nucleotide in a U•A-U triple, inhibits rapid nuclear RNA decay. Interruption of the triple helix by the C-G doublet induces a 'helical reset' that explains why triple-helical stacks longer than six do not occur in nature.

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Figure 1: Overview of ENE+A structures and their importance for RNA accumulation.
Figure 2: Hydrogen-bonding interactions in the triple helix of the MALAT1 ENE+A core RNA.
Figure 3: Structural features of the C+•G-C and C-G in the MALAT1 ENE+A core.
Figure 4: Destabilizing structural features predicted between extended Hoogsteen and Watson strands of RNA triplexes.
Figure 5: The MALAT1 ENE+A exhibits a single phase of RNA decay in vivo.
Figure 6: Accumulation levels of βΔ1,2-MALAT1 ENE+A+mascRNA containing mutations in the C+•G-C and C-G nucleotides.

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Acknowledgements

We are grateful for staff assistance at Advanced Photon Source beamline 24-ID and National Synchrotron Light Source beamline X-25, plasmids from K. Prasanth (University of Illinois, Urbana-Champaign, pSV40-mMALAT1) and A. Alexandrov (Yale University, AVA2136) and iridium (III) hexamine trichloride from S. Strobel (Yale University). We thank P. Moore, K. Tycowski and J. Withers for critical review of the manuscript, A. Miccinello for editorial work and all Steitz-laboratory members for thoughtful discussions. This work was supported by US National Institutes of Health grants GM026154 (J.A.S.) and GM022778 (T.A.S.), a Postdoctoral Fellowship (grant 122267-PF-12-077-01-RMC) from the American Cancer Society (J.A.B.) and the Steitz Center for Structural Biology, Gwangju Institute of Science and Technology, Republic of Korea (J.W.). J.A.S. and T.A.S. are supported as investigators of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA

    Jessica A Brown, Jimin Wang, Max L Valenstein, Thomas A Steitz & Joan A Steitz

  2. Department of Chemistry, Yale University, New Haven, Connecticut, USA

    David Bulkley & Thomas A Steitz

  3. Howard Hughes Medical Institute, Yale University, New Haven, Connecticut, USA

    Therese A Yario, Thomas A Steitz & Joan A Steitz

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Contributions

J.A.B. and J.A.S. designed research; J.A.B., D.B., M.L.V. and T.A.Y. performed research; J.A.B., D.B. and J.W. analyzed data; T.A.S. and J.A.S. oversaw research; J.A.B. and J.A.S. wrote the paper; and all authors discussed the results and commented on the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Overview of crystal structures for the oligo(A)-bound ENE cores from KSHV PAN and MALAT1 RNAs.

(a) A schematic diagram (left) is shown for the KSHV PAN ENE core in complex with oligo A9 next to the cartoon representation of the corresponding crystal structure (right)1. ENE nucleotides and oligo A9 are colored green and purple, respectively. Non-native sequence is colored gray. Specific types of hydrogen-bonding interactions are as defined in Figure 1a. (b) Identical to panel (a) except the schematic and crystal structure are for the MALAT1 ENE+A core.

Supplementary Figure 2 Composition and arrangement of three RNA molecules in the asymmetric unit.

(a) The asymmetric unit contains three molecules of the MALAT1 ENE+A core RNA, which are labeled molecule A (green), B (orange) and C (blue) with the A-rich tract (purple for A, teal for B and red for C). Crystal-packing interactions are denoted by arrows and briefly described. The visible 5′ and 3′ ends are labeled. (b) Superposition of the three NCS-related molecules shows that the greatest structural deviations occur in stem I (R.m.s. deviation values relative to molecule A were 5.25 and 2.90 Å for B and C, respectively) and the linker region, while the triple helix (R.m.s. deviation values relative to molecule A were 0.76 and 0.96 Å for B and C, respectively) and stem II (R.m.s. deviation values relative to molecule A were 0.61 and 0.92 Å for B and C, respectively) are more similar.

Supplementary Figure 3 Experimental electron density maps and final refined maps.

(a) Experimental maps generated by MAD heavy-atom phasing using ShelX (1.5 σ) superimposed on the final models (sticks) for molecules A (left), B (middle) and C (right). The ENE (green for A, orange for B and blue for C) and A-rich tract (purple for A, teal for B and red for C) are colored sticks. Nucleotides are labeled according to the positions in the MALAT1 ENE+A core schematic in Figure 1a. This view is a closeup of nucleotides in the three different strands of the triple helix: Hoogsteen (U11, C12, U13, U14), Watson (A70, G71, C72, A73) and Crick (C42, U43). (b) Improved experimental maps using density modification and heavy-atom refinement methods (2.0 σ) for the same region in (a). (c) Final 2Fobs-Fcalc electron density maps using calculated phases (1.5 σ), where Fobs and Fcalc denote the observed and calculated amplitudes, respectively. Region is same as described in (a).

Supplementary Figure 4 A-minor interactions observed in MALAT1 ENE+A core crystal structure.

(a) Schematic diagram of the MALAT1 ENE+A core RNA with the boxed region indicating the A-minor interactions. Hydrogen-bonding interactions are as defined in Figure 1a. (b) Cartoon representation of the two A-minor interactions involving A65 (purple) with G6-C50 (green) and A64 (purple) with G5-C51 (green). Hydrogen bonds for the A-minor interactions are dark blue dashed lines while Watson-Crick hydrogen bonds are light blue dashed lines.

Supplementary Figure 5 Plots of major- and minor-groove widths observed in the MALAT1 ENE+A core crystal structure.

Major-groove (a) and minor-groove (b) widths between the Watson and Crick strands (W-C, black line) and between the Hoogsteen and Crick strands (red line, H-C) are shown. Phosphate-to-phosphate distances are in gray dashed lines for ideal A-form dsRNA and ideal B-form dsDNA created by Coot2. Distances were obtained by measuring direct phosphate-to-phosphate distances by base-pair step. The steps for the major and minor grooves are Pn to Pn+4 and Pn to Pn+3, respectively, starting with the terminal G-U base pair of stem I. A complete list of distances for each phosphate pair is in Supplementary Table 4.

Supplementary Figure 6 ENE+A triplexes are structurally different from other RNA triplexes.

Various RNA triplexes (Hoogsteen and Crick strands in orange and Watson strand in blue) were superimposed onto the phosphate-ribose atoms of the MALAT1 ENE+A core using U•A-U(2-4) (Hoogsteen and Crick strands in green and Watson strand in purple). RNA triplexes include (a) U•A-U(2-4) from the KSHV PAN ENE core+A9 structure (PDB ID 3P22), (b) C•PreQ1-U/U•A-U(1-2) from the PreQ1-II riboswitch structure (PDB ID 4JF2), (c) U•U•SAM/U•A-U(1-2) from the SAM-II riboswitch structure (PDB ID 2QWY), (d) U•A-U(1-3) from the human telomerase structure (PDB ID 1YMO) and (e) U•A-U(2-4) from the K. lactis telomerase structure (PDB ID 2M8K). Nucleotides are labeled in the Watson, Crick and Hoogsteen strands. These overlays correspond to Supplementary Table 6.

Supplementary Figure 7 Uncropped images of northern blots.

Uncropped images are shown for each of the northern blots in (a) Figure 1d, (b) Figure 1e, (c-f) Figure 5c, and (g) Figure 6c. RNA sizes are indicated to the left of the blot and the large arrowhead on the right points to the desired RNA target for each probe.

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Supplementary Figures 1–7 and Supplementary Tables 1–6. (PDF 2415 kb)

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Brown, J., Bulkley, D., Wang, J. et al. Structural insights into the stabilization of MALAT1 noncoding RNA by a bipartite triple helix. Nat Struct Mol Biol 21, 633–640 (2014). https://doi.org/10.1038/nsmb.2844

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