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. 2002 Oct 15;21(20):5527-38.
doi: 10.1093/emboj/cdf544.

Hierarchical, clustered protein interactions with U4/U6 snRNA: a biochemical role for U4/U6 proteins

Stephanie Nottrott  1 , Henning Urlaub, Reinhard Lührmann
Affiliations

Hierarchical, clustered protein interactions with U4/U6 snRNA: a biochemical role for U4/U6 proteins

Stephanie Nottrott et al. EMBO J. .

Abstract

During activation of the spliceosome, the U4/U6 snRNA duplex is dissociated, releasing U6 for subsequent base pairing with U2 snRNA. Proteins that directly bind the U4/U6 interaction domain potentially could mediate these structural changes. We thus investigated binding of the human U4/U6-specific proteins, 15.5K, 61K and the 20/60/90K protein complex, to U4/U6 snRNA in vitro. We demonstrate that protein 15.5K is a nucleation factor for U4/U6 snRNP assembly, mediating the interaction of 61K and 20/60/90K with U4/U6 snRNA. A similar hierarchical assembly pathway is observed for the U4atac/U6atac snRNP. In addition, we show that protein 61K directly contacts the 5' portion of U4 snRNA via a novel RNA-binding domain. Furthermore, the 20/60/90K heteromer requires stem II but not stem I of the U4/U6 duplex for binding, and this interaction involves a direct contact between protein 90K and U6. This uneven clustering of the U4/U6 snRNP-specific proteins on U4/U6 snRNA is consistent with a sequential dissociation of the U4/U6 duplex prior to spliceosome catalysis.

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Figures

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Fig. 1. Binding of protein 61K and the 20/60/90K protein complex to U4/U6 snRNA duplex requires protein 15.5K. (A) SDS–PAGE of recombinant proteins 15.5K (left panel), GST–61K (middle panel) and purified native 20/60/90K protein complex (right panel). 20/60/90K complex was visualized by silver staining, 15.5K and GST–61K were stained with Coomassie Blue. (B) Secondary structure of human U4/U6 snRNA according to Brow and Guthrie (1988). (C) U4/U6 duplex formation as analyzed by native PAGE on a 6% gel. Lanes 1 and 2, 32P-labeled human U4 and U6 snRNAs; lanes 3 and 4, 32P-labeled U4/U6 duplexes. Labeled RNA is marked by an asterisk. (D) Co-immunoprecipitation of 32P-labeled U4/U6 duplexes and U4 and U6 snRNAs with anti-61K antibody. GST–61K was incubated with each of the four RNA substrates shown in (C) in the presence (lanes 5, 6, 9 and 10) or absence (lanes 3, 4, 7 and 8) of protein 15.5K. RNA co-precipitated with anti-61K antibody was analyzed on a denaturing 10% polyacrylamide gel and visualized by autoradiography. Lanes 1 and 2: 20% of total 32P-labeled U4 and U6 snRNA input. (E) Co-immunoprecipitation of 32P-labeled U4/U6 duplexes and U4 and U6 snRNAs with anti-60K antibody. 20/60/90K protein complex was incubated with each of the four RNA substrates shown in (C) in the presence (lanes 3, 4, 7 and 8) or absence (lanes 5 and 6) of protein 15.5K. RNA co-precipitated with anti-60K antibody was analyzed as above. Lanes 1 and 2: 15% of total 32P-labeled U4 and U6 snRNA input.
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Fig. 2. The 20/60/90K protein complex binds U4atac/U6atac snRNA duplex only in the presence of protein 15.5K. (A) Secondary structure of human U4atac/U6atac snRNA according to Padgett and Shukla (2002). (B) U4atac/U6atac duplex formation as analyzed by native PAGE on a 6% gel. Lanes 1 and 2, 32P-labeled human U4atac and U6atac snRNAs; lanes 3 and 4, 32P-labeled U4atac/U6atac duplexes. Labeled RNA is marked by an asterisk. (C) Co-immunoprecipitation of 32P-labeled U4atac and U6atac snRNAs and U4atac/U6atac duplexes with anti-60K antibody. The 20/60/90K complex was incubated with each of the four RNA substrates shown in (B) in the presence (lanes 3, 4, 7 and 8) or absence (lanes 5 and 6) of protein 15.5K. RNA co-precipitated with anti-60K antibody was analyzed as in Figure 1. Lanes 1 and 2: 15% of total 32P-labeled U4atac and U6atac snRNA input. (D) GST pull-down assay of [U4atac·15.5K·GST–61K] complexes. GST–61K was coupled to glutathione–Sepharose and incubated with 32P- labeled U4atac snRNA in either the absence (lane 3) or presence of protein 15.5K (lane 4). As a control, glutathione–Sepharose-coupled GST was incubated with U4atac snRNA in the presence of 15.5K (lane 2). Bound RNA was analyzed as above. Lane 1: 20% of total 32P-labeled U4atac RNA input.
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Fig. 3. The 20/60/90K complex binds a truncated U4/U6 snRNA duplex. (A) Secondary structure of a ‘minimal’ U4/U6 duplex comprising nucleotides 1–52 of U4 (U4 nts 1–52) and nucleotides 58–87 of U6 snRNA (U6 nts 58–87). (B) 32P-Labeled U6 nts 58–87, alone (lane 1) and base-paired with non-labeled U4 nts 1–52 (lane 2), analyzed on a 10% native gel. (C) Co-immunoprecipitation of the ‘minimal’ U4/U6 duplex bound to the 20/60/90K complex in the presence of protein 15.5K. U6 nts 58–87 alone (lane 2) or complexed with non-labeled U4 nts 1–52 (lane 3) were incubated with protein 15.5K and the 20/60/90K complex. RNA co-immunoprecipitated with the anti-60K antibody was analyzed as in Figure 1. Lane 1: 20% of total 32P-labeled U6 RNA nts 58–87 input.
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Fig. 4. Protein 61K binds the 5′ portion of U4 snRNA in the presence of protein 15.5K. (A) Secondary structure of U4 snRNA comprising nucleotide positions 1–52 and 20–52. (B) GST–61K was coupled to glutathione–Sepharose and incubated with 32P-labeled full-length U4 snRNA and the U4 snRNA fragments shown in (A), in either the presence (lanes 3, 6 and 9) or absence (lanes 2, 5 and 8) of protein 15.5K. Bound RNA was analyzed as in Figure 1. Lanes 1, 4 and 7: 20% of total input of the respective 32P-labeled RNA substrate.
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Fig. 5. Protein 61K can be cross-linked to U4 and U4atac snRNA. (A) Protein 61K–U4 snRNA cross-linking sites in purified native 25S tri-snRNPs. Left panel: primer extension analysis of U4 snRNA derived from UV-cross-linked tri-snRNPs (lane 1) or UV-irradiated, naked U4 snRNA (lane 4). Lanes 2 and 3: controls without UV irradiation. C, U, A and G are dideoxy sequence markers. Nucleotide positions of the reverse transcriptase stops are indicated on the right; a black bar indicates nucleotides of the U4 5′ stem–loop, and an open arrowhead marks the position of full-length U4 snRNA. Right panel: primer extension analysis of U4 snRNA after immunoprecipitation of UV-irradiated, denatured tri-snRNPs with antibodies against proteins 61K (lanes 5 and 6) or 60K (lanes 7 and 8), either with (lanes 6 and 8) or without UV irradiation (lanes 5 and 7). Nucleotide positions of the reverse transcriptase stops are indicated. (B) The cross-linked amino acid His270 is found in the central Nop56/Nop58 homology domain of protein 61K (Nop HD; residues 93–328; Makarova et al., 2002). Residues 263–283 of protein 61K were aligned with the homologous sequences of human Nop56 (amino acids 346–366; Y12065) and Nop58 (amino acids 330–350; AF123534) using the Clustal method. Identical residues are boxed in black, and conserved residues are highlighted in gray. His270 is marked by an arrow. (C) Protein 61K–U4atac snRNA cross-linking sites in reconstituted [15.5K·61K·U4atac/U6atac] complexes. MALDI-MS analysis identified the 61K peptide (260SGFSSTSVLPHTGY273) cross-linked to an RNase T1 oligonucleotide (42CAUAG46) in the U4atac 5′ stem–loop. The nucleotide composition (C1U1A2G1; [M] = 1632.227) was determined from the difference between the mono-isotopic mass of the cross-linked peptide–oligonucleotide complex ([M + H]+ = 3071.907) and that of the peptide alone ([M + H]+ = 1439.680). The cross-linked peptide moiety was determined by N-terminal sequence analysis.
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Fig. 6. Protein 90K of the 20/60/90K complex contacts U6 snRNA. (A) The 20/60/90K complex was incubated with U4/U6 duplex composed of 32P-labeled, full-length U6 snRNA and non-labeled U4 nucleotides 1–52. After UV irradiation, the products were digested with RNases A and T1, and separated by SDS–PAGE. In lane 4, U4/U6 duplex, protein 15.5K and the 20/60/90K complex were present during cross-linking. Lane 1, no UV irradiation; lane 2, all proteins omitted; lane 3, protein 15.5K omitted; lane 6, RNA duplex replaced by 32P-labeled U6 snRNA. In lane 5, the cross-linking product was digested with proteinase K. The 90K protein cross-link is marked on the right. (B) The 20/60/90K complex was incubated with the minimal U4/U6 duplex consisting of 5′ end-labeled U6 nucleotides 58–87 and non-labeled U4 nucleotides 1–52 (see Figure 3). Lane designations are as in (A) and indicated above each lane.
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Fig. 7. Proposed mechanism for disruption of U4/U6 snRNA during activation of the spliceosome. See text for details.
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