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Crystal structures of the Lsm complex bound to the 3′ end sequence of U6 small nuclear RNA

Abstract

Splicing of precursor messenger RNA (pre-mRNA) in eukaryotic cells is carried out by the spliceosome1, which consists of five small nuclear ribonucleoproteins (snRNPs) and a number of accessory factors and enzymes2. Each snRNP contains a ring-shaped subcomplex of seven proteins and a specific RNA molecule2,3,4. The U6 snRNP contains a unique heptameric Lsm protein complex, which specifically recognizes the U6 small nuclear RNA at its 3′ end. Here we report the crystal structures of the heptameric Lsm complex, both by itself and in complex with a 3′ fragment of U6 snRNA, at 2.8 Å resolution. Each of the seven Lsm proteins interacts with two neighbouring Lsm components to form a doughnut-shaped assembly, with the order Lsm3–2–8–4–7–5–6. The four uridine nucleotides at the 3′ end of U6 snRNA are modularly recognized by Lsm3, Lsm2, Lsm8 and Lsm4, with the uracil base specificity conferred by a highly conserved asparagine residue. The uracil base at the extreme 3′ end is sandwiched by His 36 and Arg 69 from Lsm3, through π–π and cation–π interactions, respectively. The distinctive end-recognition of U6 snRNA by the Lsm complex contrasts with RNA binding by the Sm complex in the other snRNPs. The structural features and associated biochemical analyses deepen mechanistic understanding of the U6 snRNP function in pre-mRNA splicing.

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Figure 1: Structure of the Lsm2–8 heptameric complex.
Figure 2: Recognition of U6 snRNA by the Lsm2–8 heptameric complex.
Figure 3: Structural comparison with the Sm complex.
Figure 4: Lsm3 anchors the 3′ end of RNA elements.

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Acknowledgements

We thank S. Huang and J. He at SSRF beamline BL17U and N. Shimizu, T. Kumasaka, and S. Baba at the Spring-8 beamline BL41XU for on-site assistance. This work was supported by funds from National Natural Science Foundation of China projects 31130002 and 31021002.

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Authors and Affiliations

Authors

Contributions

L.Z., J.H., and Y.S. designed all experiments. L.Z., J.H., Y.Z., R.W., G.L., P.Y., and C.Y. performed the experiments. All authors contributed to data analysis. L.Z., J.H., C.Y., and Y.S. contributed to manuscript preparation. Y.S. wrote the manuscript.

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Correspondence to Yigong Shi.

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The authors declare no competing financial interests.

Additional information

Atomic coordinates and structure factors have been deposited in the Protein Data Bank. The PDB codes of RNA-free Lsm2–8 complex are 4M77 and 4M78 for space groups I212121 and P21, respectively. The PDB codes of Lsm2–8 bound to the RNA elements 5′-UUCGUUUU-3′ and 5′-UUUCGUUU-3′ are 4M7A and 4M7D, respectively. The PDB code of Lsm1–7 is 4M75.

Extended data figures and tables

Extended Data Figure 1 Purification and characterization of the recombinant Lsm2–8 complex.

a, Purification of the Lsm2–8 complex. A representative chromatogram of anion exchange chromatography (Source-15Q) for the recombinant Lsm2–8 complex is shown in the left panel. A representative SDS–PAGE gel is shown in the right panel. The protein bands were excised, trypsinized and analysed by mass spectrometry, which confirmed the presence of all seven Lsm proteins. b, Alignment of U6 snRNA 3′ end sequences from seven eukaryotic species. Invariant bases are highlighted in red. The U6 snRNA sequences from multiple species share four consecutive uridine nucleotides at their 3′ ends. The predicted secondary structure of U6 snRNA (shown in the right panel) has been reported37.

Extended Data Figure 2 Measurement of dissociation constants between the Lsm2–8 heptameric complex and various RNA oligonucleotides derived from the 3′ end of U6 snRNA by isothermal titration calorimetry (ITC).

ag, The representative raw ITC data and the fitted binding curves are shown for the RNA oligonucleotides 5′-UUUU-3′ (a), 5′-GUUUU-3′ (b), 5′-CGUUUU-3′ (c), 5′-UCGUUUU-3′ (d), 5′-UUCGUUUU-3′ (e), 5′-AUUUCGUUUU-3′ (f), 5′-AUUUAUUUCGUUUU-3′ (g).

Extended Data Figure 3 Measurement of dissociation constants between the Lsm2–8 complex and various RNA oligonucleotides, each containing a single base replacement of the sequence 5′-UUCGUUUU-3′.

ag, The representative raw ITC data and the fitted binding curves are shown for the RNA oligonucleotides 5′-UUCGUUUC-3′ (a), 5′-UUCGUUCU-3′ (b), 5′-UUCGUCUU-3′ (c), 5′-UUCGCUUU-3′ (d), 5′-UUCAUUUU-3′ (e), 5′-UUUGUUUU-3′ (f), and as negative control for non-specific RNA 5′-CCCCCCCC-3′ (g).

Extended Data Figure 4 Protein engineering of Lsm2–8 complex.

a, Sequence alignment of Lsm1 through Lsm8 from Saccharomyces cerevisiae. The Lsm4 protein has an extended sequence highly enriched by Asn. The two amino acids that are invariant among Lsm1 through Lsm7, but not in Lsm8, are marked by red arrows. b, Truncation of the C-terminal flexible sequences in Lsm4 (residues 94–188) and Lsm8 (residues 97–109) and seven engineered mutations (Cys45Ser in Lsm2, Cys37Ser/Cys63Ser in Lsm3, and Cys22Ser/Cys51Ser/Lys17Leu/Ile38Leu in Lsm8) have no significant effect on the binding affinity between U6 snRNA and the Lsm2–8 heptameric complex. Shown here are results of isothermal titration calorimetry (ITC).

Extended Data Figure 5 Electron density maps and structure details of the Lsm2–8 heptameric complex.

a, 2Fo − Fc electron density of an intact Lsm2–8 heptameric complex, contoured at 1σ. b, 2Fo − Fc electron density of the Lsm8 component, contoured at 1σ. c, Representative 2Fo − Fc electron density around Lsm8 residues 25–40 (backbone trace in yellow) and 50–60 (magenta), contoured at 1σ. d, Each Lsm component interacts with two neighbouring Lsm proteins primarily through intermolecular hydrogen bonds mediated by main chain groups. Hydrogen bonds are represented by red, dashed lines. e, Specific association between neighbouring Lsm proteins involve side-chain-mediated interactions. The left panel shows a hydrogen bond between Tyr 8 of Lsm8 and Asn 59 of Lsm2 and a salt bridge between Asp 7 of Lsm8 and Lys 39 of Lsm2. The right panel displays a hydrogen bond between Thr 12 of Lsm6 and Asp 38 of Lsm5 and van der Waals contact between Val 11 of Lsm6 and Ile 44 of Lsm5. The distance is labelled in Å.

Extended Data Figure 6 Electron density maps of an RNA segment bound to the Lsm2–8 heptameric complex.

a, The 2Fo − Fc electron density of the bound RNA, contoured at 1σ, is coloured magenta (left panel). A close-up, stereo view of the 2Fo − Fc electron density of the bound RNA is shown in the right panel. b, Confirmation of correct RNA sequence assignment. Shown here is the anomalous density for bromine (Br), shown in magenta mesh and contoured at 5σ. U110 in the octanucleotide is substituted by 5-Br-U. The resulting RNA oligonucleotide was co-crystallized with the Lsm2–8 complex. X-ray diffraction data were collected and anomalous signals calculated. c, The octanucleotide remained intact in the crystals. RNA was extracted from crystals, end-labelled by 32P, and visualized on denaturing gel (lane 3). Three synthetic RNAs of various lengths were similarly end-labelled by 32P and visualized for comparison (lanes 1, 2, and 4). This result shows that the octanucleotide in the crystals remained intact during crystallization. Shown here is a representative gel out of three independent experiments.

Extended Data Figure 7 Structural alignment between RNA-free and RNA-bound states of the Lsm2–8 complex.

a, Overall structural alignment between RNA-free and RNA-bound states of the Lsm2–8 complex. RNA-free structure is presented in light yellow whereas RNA-bound state is shown in grey. b, The side chains of Phe 35 and Arg 63 of Lsm2 reorient to sandwich the base U111. c, The side chain of Arg 72 in re-positioned to form cation–π interaction with the base U109. d, A representative local conformational change. The side chain of Gln 57 in Lsm7 undergoes local changes upon binding to U6 snRNA, resulting in formation of a hydrogen bond with the base G108.

Extended Data Figure 8 Specific recognition of the three bases U109, U110, and U111 by the Lsm proteins.

a, The base U111 is recognized by Lsm2. Hydrogen bonds are represented in red dashed lines. The uracil base is sandwiched by π–π and cation–π interactions. The base-specific contacts are conferred by hydrogen bonds from a highly conserved Asn residue and from main chain amide nitrogen atoms of the residues Gly 64-Ser 65. b, The base U110 is recognized by Lsm8. Compared to U111 and U112, the π–π interaction is absent for U110 and there are only three base-specific hydrogen bonds. c, The base U109 is recognized by Lsm4. Compared to U111 and U112, there are only three base-specific hydrogen bonds for U109.

Extended Data Figure 9 2′,3′-cyclic phosphorylation of U6 snRNA had a relatively minor effect on the binding affinity for the Lsm2–8 complex.

a, Analysis of in vitro transcribed and processed 28-nt RNA fragment derived from the 3′ end of U6 snRNA by denaturing gel electrophoresis. The 2′,3′-cyclophosphate at the 3′ end of U6 snRNA fragment (U6>P) was generated by 3′ end Hammerhead ribozyme cleavage. Lane 1 shows the RNA sample with 2′,3′-cyclophosphate. A portion of this sample was treated by T4 polynucleotide kinase to remove the 2′,3′-cyclophosphate group, which resulted in slight up-shift of the RNA band on the gel (lane 2). As control, another portion of RNA was treated by T4 polynucleotide kinase 3′-phosphatase-minus (NEB) (lane 3). b, The U6 snRNA fragments, with or without 2′,3′-cyclophosphate, were incubated with increasing concentrations of the Lsm2–8 complex as indicated and applied to native agarose gel. Following SYBRgold staining, the RNA bands were quantified using Progel software. This experiment was independently repeated three times. The dissociation constants were approximately 83.6 ± 9.0 nM and 113.3 ± 7.6 nM for the U6 snRNA fragment with and without 2′,3′-cyclophosphate, respectively.

Extended Data Table 1 Statistics of data collection and refinement

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Zhou, L., Hang, J., Zhou, Y. et al. Crystal structures of the Lsm complex bound to the 3′ end sequence of U6 small nuclear RNA. Nature 506, 116–120 (2014). https://doi.org/10.1038/nature12803

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