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Core structure of the U6 small nuclear ribonucleoprotein at 1.7-Å resolution

Nature Structural & Molecular Biology volume 21, pages 544–551 (2014)Cite this article

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Abstract

The spliceosome is a dynamic assembly of five small nuclear ribonucleoproteins (snRNPs) that removes introns from eukaryotic pre-mRNA. U6, the most conserved of the spliceosomal small nuclear RNAs (snRNAs), participates directly in catalysis. Here, we report the crystal structure of the Saccharomyces cerevisiae U6 snRNP core containing most of the U6 snRNA and all four RRM domains of the Prp24 protein. It reveals a unique interlocked RNP architecture that sequesters the 5′ splice site–binding bases of U6 snRNA. RRMs 1, 2 and 4 of Prp24 form an electropositive groove that binds double-stranded RNA and may nucleate annealing of U4 and U6 snRNAs. Substitutions in Prp24 that suppress a mutation in U6 localize to direct RNA-protein contacts. Our results provide the most comprehensive view to date of a multi-RRM protein bound to RNA and reveal striking coevolution of protein and RNA structure.

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Figure 1: Conformational changes in U6 snRNA during the splicing cycle.
Figure 2: Structure of the yeast U6–Prp24 complex.
Figure 3: New structural motifs in the U6–Prp24 complex.
Figure 4: Prp24 interactions with the U6 asymmetric bulge.
Figure 5: Suppressors of U6-A62G cold sensitivity map to the RNA-protein interface.
Figure 6: U6-A62G–suppressor mutations are expected to destabilize the interaction between Prp24 and U6 snRNA.
Figure 7: An electropositive groove in Prp24 binds double-stranded RNA.
Figure 8: Proposed mechanism for Prp24-mediated annealing of U4 and U6 snRNAs.

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Acknowledgements

We are grateful to J. Doudna, C. Guthrie, A. Hoskins, H. Noller, A.M. Pyle and members of the Brow and Butcher laboratories for helpful discussions and critical reading of the manuscript, C. Bingman for advice and technical assistance in performing crystallization screening and B. Bhattacharyya and J. Keck for acquisition of diffraction data. Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under contract no. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by grant 085P1000817. This work was supported by a grant from the US National Institutes of Health (no. GM065166 to S.E.B. and D.A.B.).

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  1. Elizabeth C Curran & Kristie L Andrews

    Present address: Present addresses: Department of Pharmacology, University of Washington, Seattle, Washington, USA (E.C.C.), and Illinois College of Optometry, Chicago, Illinois, USA (K.L.A.).,

Authors and Affiliations

  1. Department of Biomolecular Chemistry, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Eric J Montemayor, Elizabeth C Curran, Kristie L Andrews, Christine N Treba & David A Brow

  2. Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Eric J Montemayor, Hong Hong Liao & Samuel E Butcher

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Contributions

E.J.M. and H.L. prepared crystallization samples; E.J.M. determined the structure; E.C.C., K.L.A., C.N.T. and D.A.B. performed the yeast genetic analysis; S.E.B. and D.A.B. supervised the work and wrote the manuscript with assistance from E.J.M.

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Correspondence to Samuel E Butcher or David A Brow.

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Integrated supplementary information

Supplementary Figure 1 Wall-eyed stereo image of the U6–Prp24 crystal structure.

(a) Cartoon representation of the interlocked RNP topology, colored as in Figure 2. (b) Electron density from the final 2mFo-DFc map contoured at 2.0 rmsd. The RNA (nucleotides 48-53 of the U6 asymmetric bulge) is colored yellow to enhance clarity, while the protein is colored as in Figure 2. Red spheres depict associated water molecules.

Supplementary Figure 2 RNA structural features of interest.

(a) U80, which plays an important role in pre-mRNA splicing63,64, is base-paired with C67 in the U6-A62G-Prp24 crystal structure. The red sphere depicts associated solvent. (b) The observed structure of the ISL is within experimental error (RMSD < 1 Å) of the solution structure of the isolated ISL65. (c) Similar to an unusual backbone conformation observed previously in duplex RNA66, there are two inverted ribose moieties in U6; however, this ribose inversion is dictated through extensive contacts between RNA and protein. The directionality of the backbone is depicted by highlighted O4' atoms (green). Inverted nucleotide A40 makes a non-Watson-Crick base pair with A91 at the end of the U6 telestem. The inversion in A42 is part of the aspartate bridge motif described in Figures 4a and 6a. (RRM3, which forms extensive contacts with RNA in this region of the structure, has been omitted for clarity.) RNA and protein are colored as in Figure 2.

Supplementary Figure 3 Mutations in PRP24 isolated as genomic suppressors of U4-G14C6 also suppress U6-A62G and U6-UA cold sensitivity.

Strains containing the indicated U6 and PRP24 alleles were serially diluted and plated at 16 °C and 37 °C. The Prp24-L217P substitution confers heat-sensitivity at 37 °C in addition to suppression at 16 °C6.

Supplementary Figure 4 Agreement between chemical-probing data and the U6–Prp24 crystal structure.

Observed contacts between Prp24 and U6 along the single-stranded asymmetric bulge help to explain previous chemical probing studies28, where blue nucleotides were protected from chemical modification, pink nucleotides were moderately reactive, and red nucleotides were strongly reactive. (Gray indicates no information.) Reactive base-paired regions in the ISL and telestem are consistent with helical breathing during the long incubation times used for chemical modification (40-80 minutes at 4°C)28. The RRM domains are colored as in Figure 2 and are shown with a partially transparent solvent accessible surface (calculated using a 1.4 Å probe radius).

Supplementary Figure 5 Proposed assembly mechanism of the interlocked U6–Prp24 structure.

The telestem, which is relatively unstable due to three non-canonical A-A or A-G pairs (Fig. 2a), likely exists only transiently in free U6 RNA. Binding of RRM2 to the ACAGA-box region induces binding of the RRM2 loop spanning residues 149-160 to oRRM4 (middle panel). Binding of RRM3 to U6 nucleotides 39-44 and 91-92 then stabilizes the telestem.

Supplementary Figure 6 Proposed architecture of the U6 snRNP from yeast.

(a) The Lsm ring46 (gray) is placed near the 3' tail of U6 RNA to allow association of the 3' nucleotide with the torus of the ring. (b) The register of the ring is made to be consistent with observed cross-linking9 between G30 (red surface) and Lsm2 (yellow) and (c) yeast two-hybrid13 data suggesting the C-terminal tail of Prp24 interacts with Lsm5, Lsm7 and Lsm8 (light green). The positioning of the SNFFL box in close proximity to the U6 telestem could explain previously reported cross-links between U6 nucleotides 28 and 29 with unidentified residues in Prp24 (ref. 28).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1 and 2, and Supplementary Note (PDF 8289 kb)

Architecture of the U6–Prp24 crystal structure

Structure of the U6 RNA–Prp24 complex highlighting the interlocked RNA-protein topology, aspartate bridged base pair, and extensive interactions of RRM2 with the conserved U6 ACAGA sequence. (MP4 19362 kb)

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Montemayor, E., Curran, E., Liao, H. et al. Core structure of the U6 small nuclear ribonucleoprotein at 1.7-Å resolution. Nat Struct Mol Biol 21, 544–551 (2014). https://doi.org/10.1038/nsmb.2832

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