Crystal structure of human spliceosomal U1 snRNP at 5.5 A resolution

Abstract

Human spliceosomal U1 small nuclear ribonucleoprotein particles (snRNPs), which consist of U1 small nuclear RNA and ten proteins, recognize the 5' splice site within precursor messenger RNAs and initiate the assembly of the spliceosome for intron excision. An electron density map of the functional core of U1 snRNP at 5.5 A resolution has enabled us to build the RNA and, in conjunction with site-specific labelling of individual proteins, to place the seven Sm proteins, U1-C and U1-70K into the map. Here we present the detailed structure of a spliceosomal snRNP, revealing a hierarchical network of intricate interactions between subunits. A striking feature is the amino (N)-terminal polypeptide of U1-70K, which extends over a distance of 180 A from its RNA binding domain, wraps around the core domain consisting of the seven Sm proteins and finally contacts U1-C, which is crucial for 5'-splice-site recognition. The structure of U1 snRNP provides insights into U1 snRNP assembly and suggests a possible mechanism of 5'-splice-site recognition.

Figure 1. Overall fold of U1 snRNA and the U1 snRNP core domain containing the seven Sm proteins

a, Human U1 snRNA forms four stem-loop (SL) structures: SL1 (red), SL2 (blue), SL3 (green) and SL4 (cyan). A four-helix junction with two co-axially stacked helices (SL1-SL2; SL3-helix H (magenta)) is separated from SL4 by the single-stranded Sm site (lime green). b, Anomalous difference peaks used to place the seven Sm proteins and U1C into the map. Se atoms of Se-methionine-labelled Sm proteins (red); a Zn atom of the Zn-finger of U1C (green); Hg atoms attached to residues C24 and mutant Q39C of U1C (purple). The 5′-end of U1 snRNA (orange) base-pairs with its counterpart from a neighboring complex (grey). c, Seven Sm proteins and U1C fitted into the map.

Figure 2. Structure of the U1 snRNP core domain

a, The Sm proteins and the experimental electron density map (contoured at 1σ). Cartwheel-shaped density in the central hole of the U1 snRNP core domain is attributed to the seven Sm site nucleotides. b, The N-terminus of D2 supporting helix H. The extended polypeptide of U1-70k interacts with D2. Seleno-methionine anomalous peaks from natural (M67; red) and engineered Met residues (I41M, E49M and E61M; orange) of U1-70k and of Sm protein D2 (Met11; cyan). The asterisk indicates a “bump” due to Phe-24 of D2. c, The N-terminus of Sm B protein interacting with the phosphate backbone of SL2. Navy blue spheres, anomalous peaks from seleno-methionine-labelled B protein.

Figure 3. A mechanism of 5′-splice site recognition

a, The 5′-end of U1 snRNA (blue) base-pairs with the same region of a neighbouring complex (orange), which mimics the 5′-splice site of pre-mRNA. C8 and A7 of U1 snRNA are highlighted (magenta). The experimental map is contoured at 1σ. Spheres represent anomalous peaks of Zn (green) and Hg (red). The asterisk indicates a “bump” due to Trp-41. b, The Zn-finger of yeast dimethylallyltransferase and the anti-codon stem of a tRNA substrate. c, Base-pairing between the 5′-ends of two U1 snRNAs within the U1 snRNP crystal. C8 and A7 are highlighted (magenta). d, Base-pairing between the 5′-end of U1 snRNA and a canonical 5′-splice site. The invariant first and second nucleotides of the intron (GU) are highlighted (red).

Figure 4. Interaction of U1-70k with SL1 of U1 snRNA, the core domain, and U1C

a, The RNA binding domain (RBD) of U1-70k and the α-helix extending along SL1 towards the core domain. b, The long α-helix B of U1C (red) held between the N-terminus of U1-70k (orange) and the C-terminal end of D3 (purple). c, Stereo view of U1 snRNP showing the entire path of U1-70k revealed by Se-methionine landmarks (orange spheres). d, Path of the N-terminal region of U1-70k crossing the Sm ring viewed in stereo from SL4 (omitted for clarity). Spheres indicate anomalous peaks from Se-methionines in U1-70k (natural, red; engineered, orange) and D3 (purple). Experimental electron density map is contoured at 1σ.

Figure 5. Model of the complete human U1 snRNP

a, Overview of a model of the complete U1 snRNP. Truncated SL2 was extended with A-form RNA and the crystal structure of the U1A-RNA complex was appended to the extended helix. The internal loop of SL2, consisting of four consecutive non-Watson-Crick base-pairs (red), is in a position to interact with B and D1. b, Two views of the complete U1 snRNP model approximately 45° apart with surface representation superimposed. Closely matching images are found in the gallery of negative stain images of U1 snRNP reported previously,.