pubmed.ncbi.nlm.nih.gov

Structure of the spliceosomal U4 snRNP core domain and its implication for snRNP biogenesis - PubMed

  • ️Sat Jan 01 2011

. 2011 May 26;473(7348):536-9.

doi: 10.1038/nature09956. Epub 2011 Apr 24.

Affiliations

Structure of the spliceosomal U4 snRNP core domain and its implication for snRNP biogenesis

Adelaine K W Leung et al. Nature. 2011.

Abstract

The spliceosome is a dynamic macromolecular machine that assembles on pre-messenger RNA substrates and catalyses the excision of non-coding intervening sequences (introns). Four of the five major components of the spliceosome, U1, U2, U4 and U5 small nuclear ribonucleoproteins (snRNPs), contain seven Sm proteins (SmB/B', SmD1, SmD2, SmD3, SmE, SmF and SmG) in common. Following export of the U1, U2, U4 and U5 snRNAs to the cytoplasm, the seven Sm proteins, chaperoned by the survival of motor neurons (SMN) complex, assemble around a single-stranded, U-rich sequence called the Sm site in each small nuclear RNA (snRNA), to form the core domain of the respective snRNP particle. Core domain formation is a prerequisite for re-import into the nucleus, where these snRNPs mature via addition of their particle-specific proteins. Here we present a crystal structure of the U4 snRNP core domain at 3.6 Å resolution, detailing how the Sm site heptad (AUUUUUG) binds inside the central hole of the heptameric ring of Sm proteins, interacting one-to-one with SmE-SmG-SmD3-SmB-SmD1-SmD2-SmF. An irregular backbone conformation of the Sm site sequence combined with the asymmetric structure of the heteromeric protein ring allows each base to interact in a distinct manner with four key residues at equivalent positions in the L3 and L5 loops of the Sm fold. A comparison of this structure with the U1 snRNP at 5.5 Å resolution reveals snRNA-dependent structural changes outside the Sm fold, which may facilitate the binding of particle-specific proteins that are crucial to biogenesis of spliceosomal snRNPs.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Overall structure of the U4 snRNP core domain

a, Side view of the core domain showing the ring with its flat face up and tapered face down. b, View from the flat face of the ring. The N- and C-terminal extensions of the Sm fold interact between SmD3 and SmB, and between SmD1 and SmD2. c, the heptameric ring is cleaved along a plane (dark blue patches) through SmG and SmD2, leaving the five subunits SmG-SmD3-SmB-SmD1-SmD2 that bind the penta-uridylate to form the protein envelope in the background. Loops L3, L5 and L2 of the Sm fold line the walls of the funnel shaped hole, whereas L4 is exposed on the tapered face. The bases of the Sm site nucleotides, such as U120 and U124, are bound between L3 and L5 near the rim on the flat face.

Figure 2
Figure 2. The Sm site RNA binds asymmetrically in the central hole of the heptamer ring

The Sm site heptad sequence is shown together with Sm proteins in the central hole of the core domain. Carbon atoms are colour-coded by chain: SmE (yellow), SmG (green), SmD3 (salmon), SmB (light blue), SmD1 (tan), SmD2 (cyan), SmF (purple), RNA (magenta). Nitrogen, oxygen, sulphur and phosphorus atoms are in blue, red, green and orange, respectively. A sharpened (B=−15 Å2) ncs-averaged electron density map is contoured at 8σ.

Figure 3
Figure 3. Interactions between the U4 Sm site heptad nucleotides and the Sm proteins

a, A119. b, U120. c, U121. d,U122. e, U123. f, U124. g, G125. Shown in dashed lines are the hydrogen-bonding interactions inferred from the residue configurations. Similarities with the RNA-free hetero-dimers and the Lsm-1 complex are present: the invariant Asn(L3.5) is buttressed by hydrogen bonds with the side chain of Asp(L3.1) and peptide amide of Gly(L5.2) in six cases (a-b, d-f), and with Glu-36D3(L3.1) via Tyr-62D3 and with the peptide amide of Gly-65D3(L5.2) in SmD3 (c); Met(L3.4) contributes van der Waals contacts to the base bound by a neighbouring Sm protein in six cases (a-f), and Gly(L5.2) is conserved in six cases (a-f) where a side chain would clash with the base contacting residues. The 2Fo-Fc map is shown sharpened with B=−15 Å2 and contoured at ~1.5σ. Atom colours are as in Figure 2.

Figure 4
Figure 4. snRNA-dependent structural changes of the U1 and U4 core domains

a, U1 snRNP and b, U4 core domain in the same orientation; c, the U4 core domain in tapered-face view. The N-terminal fragment of U1-70K (green), is overlaid onto the U4 core domain (b and c), and the Sm folds common to both structures are masked with a white envelope. The red circle indicates where U1 and U4 snRNAs come out from the central hole. In U4, the L4 loops of SmB and SmD2 contact the backbone of the 3′ stem (b). In U1,, SmD2 helix h0 points into the minor groove of RNA helix H (hH) (a), but in U4, SmD2 h0 is orientated almost orthogonal to this, with its N-terminus and the C-terminus of the masked SmD2 h1 both pointing at SmD1 helix h3 (b). The latter is positioned to obstruct the path of U1-70K (arrow). Moreover the first seven base pairs of the 3′-stem of U4 snRNA (orange) would clash on its 3′-strand with U1-70K (arrow) (c).

Similar articles

Cited by

References

    1. Burge CB, Tuschl T, Sharp PA. In: The RNA World II. Gesteland RR, Cech TR, Atkins JF, editors. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY: 1999. pp. 525–560.
    1. Will CL, Lührmann R. In: The RNA World. Gesteland RF, Cech TR, Atkins JF, editors. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY: 2006. pp. 369–400.
    1. Yu Y-T, Scharl EC, Smith CM, Steitz JA. In: The RNA World II. Gesteland RR, Cech TR, Atkins JF, editors. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY: 1999. pp. 487–524.
    1. Hinterberger M, Pettersson I, Steitz JA. Isolation of small nuclear ribonucleoproteins containing U1, U2, U4, U5, and U6 RNAs. J. Biol. Chem. 1983;258:2604–2613. - PubMed
    1. Bringmann P, Lührmann R. Purification of the individual snRNPs U1, U2, U5 and U4/U6 from HeLa cells and characterization of their protein constituents. EMBO J. 1986;5:3509–3516. - PMC - PubMed

Publication types

MeSH terms

Substances