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Structural basis of MALAT1 RNA maturation and mascRNA biogenesis

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Abstract

The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) long noncoding RNA (lncRNA) has key roles in regulating transcription, splicing, tumorigenesis, etc. Its maturation and stabilization require precise processing by RNase P, which simultaneously initiates the biogenesis of a 3′ cytoplasmic MALAT1-associated small cytoplasmic RNA (mascRNA). mascRNA was proposed to fold into a transfer RNA (tRNA)-like secondary structure but lacks eight conserved linking residues required by the canonical tRNA fold. Here we report crystal structures of human mascRNA before and after processing, which reveal an ultracompact, quasi-tRNA-like structure. Despite lacking all linker residues, mascRNA faithfully recreates the characteristic ‘elbow’ feature of tRNAs to recruit RNase P and ElaC homolog protein 2 (ELAC2) for processing, which exhibit distinct substrate specificities. Rotation and repositioning of the D-stem and anticodon regions preclude mascRNA from aminoacylation, avoiding interference with translation. Therefore, a class of metazoan lncRNA loci uses a previously unrecognized, unusually streamlined quasi-tRNA architecture to recruit select tRNA-processing enzymes while excluding others to drive bespoke RNA biogenesis, processing and maturation.

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Fig. 1: Overall structures of human mascRNA before and after processing.
Fig. 2: Structural comparison of mascRNA with tRNAPhe.
Fig. 3: Comparison of mascRNA and tRNAPhe thermostabilities and mutational effects.
Fig. 4: Molecular basis of pre-mascRNA recruitment of human RNase P.
Fig. 5: Molecular basis of pre-mascRNA recruitment of human ELAC2.
Fig. 6: mascRNA escapes aminoacylation despite binding GlnRS.
Fig. 7: Comparison of diverse RNA topologies that drive tRNA mimicry.

Data availability

Atomic coordinates and structure factor amplitudes for the human premature and mature mascRNA were deposited to the PDB under accession codes 8V1H and 8V1I, respectively. The raw sequencing data were deposited to the Gene Expression Omnibus (GEO) under accession number GSE248290. Source data are provided with this paper.

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Acknowledgements

We thank I. Botos for computational support, G. Piszczek and D. Wu for support in biophysical analyses, Y. He and N. Tjandra for cell culture support, the NIAMS Genomics and Technology Section for sequencing support and C. Stathopoulos, K. Grafanaki, A. Umuhire Juru, K. Suddala and A. Brasington for discussions. This work was supported by the Intramural Research Program of the National Institutes of Health (NIH), the National Institute of Diabetes and Digestive and Kidney Diseases (ZIADK075136 to J.Z.), the National Institute for Arthritis and Musculoskeletal and Skin Disease, the National Cancer Institute and an NIH Deputy Director for Intramural Research Challenge Award to J.Z. and Y.-X.W. This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science user facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357.

Author information

Authors and Affiliations

  1. Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, USA

    Ilias Skeparnias, Charles Bou-Nader & **wei Zhang

  2. RNA Molecular Biology Laboratory, National Institute for Arthritis and Musculoskeletal and Skin Disease, Bethesda, MD, USA

    Dimitrios G. Anastasakis & Markus Hafner

  3. Basic Science Program, Frederick National Laboratory for Cancer Research, Small-Angle X-Ray Scattering Core Facility of National Cancer Institute, Frederick, MD, USA

    Lixin Fan & Yun-**ng Wang

  4. Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA

    Yun-**ng Wang

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  1. Ilias Skeparnias
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Contributions

I.S. and J.Z. conceptualized and designed the work. I.S. prepared all RNA and protein samples and crystals and performed all biochemical and biophysical assays. I.S. and C.B.-N. collected diffraction data and determined and refined the structures. I.S. and J.Z. analyzed the structures. D.G.A. and M.H. contributed to cell culture, enzyme preparations, RT–qPCR and RNA-seq analyses. L.F. and Y.-X.W. contributed to biophysical analyses. All authors contributed to data interpretation and paper preparation.

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Correspondence to **wei Zhang.

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Extended data

Extended Data Fig. 1 Secondary structures of human pre-tRNAGln, pre-mascRNA and mascRNA and crystal structures of human mascRNA and pre-mascRNA.

a, b, Secondary structures of pre-tRNAGln (a) and pre-mascRNA (b). Both RNAs contain 30-nucleotides (nts) of natural 5′-leader and 3′-trailer sequences (red). RNase P and ELAC2 cleavage sites are indicated by blue arrowheads. c, Secondary structure of the crystallization construct corresponding to the post-cleavage mascRNA prior to 3′ CCA addition. d, Front, back, and side views of mascRNA structure as in c, colored as Fig. 1. e, Secondary structure of the crystallization construct that corresponds to the pre-mascRNA. The 9-base pair (bp) acceptor stem extension is shown in grey. To facilitate crystallization, the original 8-bp AU-rich extended acceptor stem was extended by 1-bp, its GC content increased, and the bulged A removed. f, Front, back, and side views of the pre-mascRNA structure as in e, colored as Fig. 1.

Extended Data Fig. 2 Representative X-ray crystallographic electron density maps and crystal-packing arrangements and interactions in the pre-mascRNA and mascRNA crystals.

a, b, Composite simulated anneal-omit 2|Fo|-|Fc| electron density calculated using the final model (1.0 s.d.) superimposed with the final refined model of the pre-mascRNA (a) and portion of the map showing the elbow region (b). c, d, Composite simulated anneal-omit 2|Fo|-|Fc| electron density calculated using the final model (1.0 s.d.) superimposed with the final refined model of mascRNA (c) and portions of the map showing the elbow region (d). e, Overall crystal-packing arrangements in the pre-mascRNA crystals. A reference molecule is shown in blue, while its symmetry-related molecules are in salmon. f, g, Detailed packing interfaces between pre-mascRNA molecules at the elbow (f) and termini (g) regions. h, Overall crystal-packing arrangements in the mascRNA crystals, colored as in e. i-k, Detailed packing interfaces between mascRNA molecules at the termini (i) and ASL (j, k) regions. Different interactions were observed between the chain A (j) and B (k).

Extended Data Fig. 3 Conservation of the secondary structures of mascRNA and menRNA and secondary structures of human pre-mascRNAs used in in-cell RNase P or ELAC2 processing assays.

a, Nucleotides with ~100%, 90%, and 75% sequence conservation are colored in red, black, and orange, respectively, based on Rfam1. R and Y represent purines (A or G) and pyrimidines (C or U), respectively. b, Secondary structures of pre-mascRNA including either a 5′-leader (left) or a 3′-trailer (right). The gray residues (5′-GG or 3′-UU) were appended during cloning into destination vectors. c, Representative IGV plots for WT and mutant pre-mascRNAs mapped to an artificial genome of the pre-mascRNA sequence (in b).

Extended Data Fig. 4 CD analyses of mascRNA variants.

a, Temperature-dependent CD spectra (upper) and unfolding transitions (lower) of mascRNA mutants in the presence of 10 mM Mg2+. The measured melting temperature (Tm) for each RNA is indicated. b, Temperature-dependent CD spectra (upper) and unfolding transitions (lower) of mascRNA ASL (anticodon stem loop) mutants in the absence of Mg2+. The measured melting temperature (Tm) for each RNA is indicated. Secondary structures are shown in Fig. 3a.

Source data

Extended Data Fig. 5 CD analyses of tRNAGln mascRNA variants in the absence of Mg2+.

Temperature-dependent CD spectra (upper) and unfolding transitions (lower) of tRNAGln and mascRNA mutants without Mg2+. The measured melting temperature (Tm) for each RNA is indicated. All RNAs are the mascRNA unless explicitly labelled as tRNAGln.

Source data

Extended Data Fig. 6 tRNA interfaces with processing and transport enzymes inform their mascRNA interactions.

a, Recognition of the tRNA elbow by the human RNase P (PDB: 6AHU, left)2 and its modelled interaction with pre-mascRNA elbow (right), based on tRNA-mascRNA structural alignment. b, Detailed stacking interfaces between the tRNA elbow and the interdigitated double T-loop motif (IDTM) of RNase Ps from bacteria (PDB: 3Q1Q)3, archaea (PDB: 6K0B)4, yeast (PDB: 6AH3)5 and metazoans (PDB: 6AHU)2. The elbow base pair is generally weakened or opened. c-e, Recognition of the tRNA elbow by RNase Z (c, PDB: 2FK6)6, CCA-adding enzyme (d, PDB: 1SZ1)7 and Exportin-t (e, PDB: 3ICQ)8 (left) and their modelled interactions with the mascRNA elbow (right).

Extended Data Fig. 7 Kinetic analyses of RNase P cleavage of WT and select mutant pre-mascRNAs.

a, Plots of remaining, uncleaved RNAs as functions of time for WT, G15C + C41A, G8U + C18U, U40G, ΔA44U45, ASLGGG, and ASLGAAA mascRNAs, whose gels are shown in Fig. 4d. b, Representative in vitro RNase P cleavage analyses of G8U, G11U, G14C, G14U, G15C, C18U, U39G, C41G, C41A and C41U mascRNAs. n = 3 for WT, G11U, G14C, C18U, U40G, U39G, C41G; n = 2 for G8U, G14U, G15C, G8U + C18U, C41A, C41U, G15C + C41A, ΔA44U45, ASLGGG and ASLGAAA. Biologically independent replicates are indicated as individual data points. All rates are shown in Fig. 4g.

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Extended Data Fig. 8 Kinetic analyses of RNase P cleavage of additional mutant pre-mascRNAs.

Representative in vitro RNase P cleavage analyses of G15C + C41G, G14C + C41A, A42U, A43U, A43G, A44U, A44G, A44C, U45C, U45G, U45A, and Δ3′-UCCA pre-mascRNAs, and C41G and C41A pre-mascRNA minihelix variants. Secondary structures of the C41G and C41A mascRNA minihelices are shown at the bottom. The 30-nt 5′ leader that precedes the minihelices (Extended Data Fig. 1) is omitted for clarity. n = 3 for A42U, A43G, A44U, A44G, U45C, U45G, U45A; n = 2 for G15C + C41G, G14C + C41A, A43U, A44C, Δ3′-UCCA, and pre-mascRNA minihelices C41G and C41A. Biologically independent replicates indicated as individual data points. All rates are shown in Fig. 4g.

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Extended Data Fig. 9 Kinetic analyses of human ELAC2 cleavage of WT and select mutant pre-mascRNAs and tRNAs.

Representative in vitro ELAC2 cleavage analyses of G18U + G19U tRNAGln, WT, G8U, G11U, G14C, G15C, G14C + G15C, C18U, G8U + C18U, U39G, U40G, C41G, A42U, A44G, and 3′-UCCA pre-mascRNAs, and pre-mascRNA minihelix. The 30-nt 3′ trailer (Extended Data Fig. 1) following the minihelix is omitted for clarity.). All rates are summarized in Fig. 5c (n = 2 biologically independent replicates, indicated as individual data points).

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Extended Data Fig. 10 Secondary structures of tRNAGln and mascRNA variants used for fluorescence polarization titration analysis with GlnRS.

a, Secondary structures of tRNAGln and mascRNA variants. b,c, Fluorescence polarization titration analysis of the tRNAGln and mascRNA variants binding to GlnRSΔNTD (b) or the NTD alone (c). Data are presented as mean ± s.d. (n = 3 biologically independent replicates).

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Supplementary information

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Source Data Figs. 3–6 and Extended Data Figs. 4, 5 and 7–10

Statistical source data for all main figures and extended data figures, where applicable.

Source Data Figs. 4–6 and Extended Data Figs. 7–9

Unprocessed gels for all main figures and extended data figures, where applicable.

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Skeparnias, I., Bou-Nader, C., Anastasakis, D.G. et al. Structural basis of MALAT1 RNA maturation and mascRNA biogenesis. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01340-4

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