Spliceosomal U snRNP core assembly: Sm proteins assemble onto an Sm site RNA nonanucleotide in a specific and thermodynamically stable manner - PubMed
Spliceosomal U snRNP core assembly: Sm proteins assemble onto an Sm site RNA nonanucleotide in a specific and thermodynamically stable manner
V A Raker et al. Mol Cell Biol. 1999 Oct.
Abstract
The association of Sm proteins with U small nuclear RNA (snRNA) requires the single-stranded Sm site (PuAU(4-6)GPu) but also is influenced by nonconserved flanking RNA structural elements. Here we demonstrate that a nonameric Sm site RNA oligonucleotide sufficed for sequence-specific assembly of a minimal core ribonucleoprotein (RNP), which contained all seven Sm proteins. The minimal core RNP displayed several conserved biochemical features of native U snRNP core particles, including a similar morphology in electron micrographs. This minimal system allowed us to study in detail the RNA requirements for Sm protein-Sm site interactions as well as the kinetics of core RNP assembly. In addition to the uridine bases, the 2' hydroxyl moieties were important for stable RNP formation, indicating that both the sugar backbone and the bases are intimately involved in RNA-protein interactions. Moreover, our data imply that an initial phase of core RNP assembly is mediated by a high affinity of the Sm proteins for the single-stranded uridine tract but that the presence of the conserved adenosine (PuAU.) is essential to commit the RNP particle to thermodynamic stability. Comparison of intact U4 and U5 snRNAs with the Sm site oligonucleotide in core RNP assembly revealed that the regions flanking the Sm site within the U snRNAs facilitate the kinetics of core RNP assembly by increasing the rate of Sm protein association and by decreasing the activation energy.
Figures

All of the Sm proteins associate in a specific and stable manner with a biotinylated Sm site oligonucleotide. (A) Reconstitution with TPs was carried out with biotinylated Sm site oligonucleotide (AAUUUUUGA [lane 2]) or, as negative controls, in the absence of biotinylated RNA (lane 3) or with biotinylated SmC3-C7 (AACCCCCGA) oligonucleotide (lane 4). Following streptavidin-agarose precipitation, samples were washed extensively with buffer containing 150 mM KCl. Bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue. (B) Reconstitution with TPs was performed in either the presence or absence of biotinylated Sm site oligonucleotide (b-Sm site), as indicated above each lane. The salt stability of the coprecipitation of U1-specific and Sm proteins was analyzed by extensive washing with buffers containing various concentrations of salt (0.15 to 2 M KCl, as indicated above the lanes). Following SDS-polyacrylamide gel electrophoresis, proteins were visualized by staining first with Coomassie blue and then with silver.

Analysis of minimal core RNP particle sedimentation in sucrose gradients. (A) Sedimentation behavior of TPs incubated under reconstitution conditions in the absence of Sm site oligonucleotide. The reconstitution assays were fractionated on a 10 to 30% sucrose gradient containing 400 mM KCl. Fractionation was from top to bottom (corresponding to left to right). Proteins were analyzed by SDS-polyacrylamide gel electrophoresis and visualized by silver staining. (B) Sedimentation behavior of TPs after reconstitution with the Sm site oligonucleotide performed as described for panel A. Note that the apparent overabundance of nonshifted B and B′ protein following reconstitution can be attributed to the U2-specific A′ and B" proteins, which cosedimented with a peak in lanes 9 and 10 of both TP-containing gradients (A). Some B, B′, and D3 cosedimented as a free complex following reconstitution, most probably due to their slight overabundance. (C) Sedimentation behavior of the Sm site oligonucleotide. The amount of radioactivity in each fraction was determined by scintillation counting and expressed as the percentage of the input cpm. The dashed line indicates Sm site oligonucleotide in a mock reconstitution, and the solid line indicates Sm site oligonucleotide reconstituted with TPs. The protein sedimentation profile of the latter is shown in panel B.

Electron micrographs of negatively stained minimal core RNP particles purified by sucrose gradient sedimentation. (A) Overview micrograph of the minimal core RNP sample from fraction 15 of the RNP gradient (Fig. 2B). (B) Overview micrograph of native 10S core U5 snRNP particles, which contains only U5 snRNA and Sm proteins, isolated by an independent procedure and shown for comparison. (C and D) Typical views of the minimal core RNP (C) and the U5 core snRNP (D). Particles with similar structural details are arranged in rows (C) and each row corresponds to those described previously for U5 core snRNP particles (22) (D). Briefly, images in the first and second rows show forms with a line of stain that roughly bisects the core RNP domain, with additional short extensions in the second row; images in the third row show forms with a wedge-shaped structure; and images in the fourth and fifth rows show forms with a light or dark central dot. The bar at the bottom of each picture represents 10 nm with respect to the images depicted above it.

Formation of the minimal core RNP imparts N7 methylation sensitivity to the conserved A2 adenosine. The Sm site oligonucleotide was 3′-end labelled with [32P]pCp and incubated in the presence of TPs (lanes 3 and 4) or, as mock reconstitution assays, in the absence of TPs (lanes 1 and 2). RNA in each assay was then treated with NaBH4-aniline following incubation with DMS (lanes 2 and 4) or, as controls for nonspecific RNA cleavage by NaBH4-aniline, without DMS (lanes 1 and 3). The position of the N7-methylated adenosine is indicated by an arrow on the left. The weaker band approximately halfway between those marked A1 and A2 is probably due to N3 methylation of the cytidine of the 3′-pCp label. To verify the positions of the adenosines, 32P-labelled Sm site oligonucleotide was treated with DEPC (lane 5). A base ladder marker generated by hydroxyl radical cleavage of the 32P-labelled Sm site oligonucleotide was performed in parallel and is depicted schematically at the right, with the 5′-most nucleotide (Nt) of the cleaved RNA oligonucleotide indicated.

Comparison of core RNP assembly kinetics for the Sm site oligonucleotide and U4 and U5 snRNAs, as measured by EMSA. (A) Radiolabelled Sm site oligonucleotide (∼5 nM) was incubated with TP concentrations between 0 and 1,000 nM, as indicated (lanes 1 to 7). Competition of the Sm site oligonucleotide shift was performed by adding an excess of unlabelled Sm site oligonucleotide at the onset of the reconstitution in a 25-, 50-, 100-, or 500-molar excess over the labelled Sm site oligonucleotide; the TP concentration for these assays was 100 nM (lanes 8 to 12). Native gels contained either 8% (left) or 6% (right) acrylamide. (B) Radiolabelled, native HeLa U4 (lanes 1 to 7) or U5 (lanes 8 to 12) snRNA (∼5 nM) was incubated with various TP concentrations, as indicated above each lane. Complexes were analyzed as above, on native gels containing 6% acrylamide.

Rate of association of Sm proteins with U5 snRNA (A), U4 snRNA (B), or the Sm site oligonucleotide (C), as measured by EMSA and represented graphically. Radiolabelled RNA (∼5 nM) was incubated at 30°C (solid line) or 0°C (dashed line) in the presence of a TP concentration of approximately 100 nM for the Sm site oligonucleotide and 50 nM for the U4 and U5 snRNAs. Aliquots from each assay were withdrawn at time intervals ranging from 10 s to 5 min for the U4 and U5 snRNAs and 10 s to 40 min for the Sm site oligonucleotide, mixed with loading buffer, and applied to a running native polyacrylamide gel. The shifted complex for each RNA was measured densitometrically. The percentage of total complex, relative to the maximum concentration of complex formed at 30°C, is plotted on the y axis, and the time intervals are plotted on the x axis. The standard error was less than 7%.

Effects of substitutions of the 2′-hydroxyl groups of the Sm site oligonucleotide on the binding specificity of the Sm proteins. EMSA analysis of reconstitution mixtures containing the indicated radiolabelled oligonucleotide (5 nM) with no TPs (first lane of each group) or increasing concentrations (∼100, 300, and 600 nM) of TPs is shown. The oligonucleotides used are indicated above the gel. Sm site, wild-type Sm site oligonucleotide; deoxy-Sm; 2′-H at all positions; OMe-U7, 2′-OMe at the uridine at position 7; deoxy-U7, 2′-H at the uridine at position 7.

Effects of base modifications or deletions on oligonucleotide binding specificity of the Sm proteins. (A and B) EMSA analysis of reconstitution mixtures containing the indicated radiolabelled oligonucleotide (5 nM) with no TPs (first lane of each group) or increasing concentrations (∼100, 300, and 600 nM) of TPs is shown. The RNA oligonucleotides used are shown above the gel. Note that the dot at the top of lane 17 of panel B is due to a nonspecific contamination on the dried gel. Panels A and B represent results from two experiments with different RNAs. (C) Sequences of RNA oligonucleotides used in the assays in panels A and B.

Comparison of the thermodynamic stability of the RNP containing either the U9 or the Sm site oligonucleotide. Radiolabelled oligonucleotide (5 nM) was incubated for 45 min at 30°C with approximately 100 nM TPs. Following reconstitution, a 1,000-fold excess of cold Sm site oligonucleotide (5 μM) was added to each assay mixture. Incubation at 30°C was continued for 0 to 120 min, as indicated. Reconstitution assays were started at different times so that all sample incubations were completed simultaneously.
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