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. 2006 Jul;26(13):5146-54.
doi: 10.1128/MCB.02374-05.

Protein-protein and protein-RNA contacts both contribute to the 15.5K-mediated assembly of the U4/U6 snRNP and the box C/D snoRNPs

Annemarie Schultz  1 , Stephanie Nottrott, Nicholas James Watkins, Reinhard Lührmann
Affiliations

Protein-protein and protein-RNA contacts both contribute to the 15.5K-mediated assembly of the U4/U6 snRNP and the box C/D snoRNPs

Annemarie Schultz et al. Mol Cell Biol. 2006 Jul.

Abstract

The k-turn-binding protein 15.5K is unique in that it is essential for the hierarchical assembly of three RNP complexes distinct in both composition and function, namely, the U4/U6 snRNP, the box C/D snoRNP, and the RNP complex assembled on the U3 box B/C motif. 15.5K interacts with the cognate RNAs via an induced fit mechanism, which results in the folding of the surrounding RNA to create a binding site(s) for the RNP-specific proteins. However, it is possible that 15.5K also mediates RNP formation via protein-protein interactions with the complex-specific proteins. To investigate this possibility, we created a series of 15.5K mutations in which the surface properties of the protein had been changed. We assessed their ability to support the formation of the three distinct RNP complexes and found that the formation of each RNP requires a distinct set of regions on the surface of 15.5K. This implies that protein-protein contacts are essential for RNP formation in each complex. Further supporting this idea, direct protein-protein interaction could be observed between hU3-55K and 15.5K. In conclusion, our data suggest that the formation of each RNP involves the direct recognition of specific elements in both 15.5K protein and the specific RNA.

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Figures

FIG. 1.
FIG. 1.
Schematic representation of the three RNA motifs bound by 15.5K. The secondary structures of the U4/U6 snRNA (reference and references therein), U14 box C/D snoRNA (30, 31), and U3 snoRNA-specific box B/C motif (6, 31) are shown. The highly conserved internal loop elements recognized by 15.5K are depicted in white type on a black background. The two flanking stem structures (stems I and II) that along with the loop nucleotides are part of the k-turn structure are indicated. The conserved sequence of stem II in the box B/C and box C/D is indicated by black type on a gray background. Base pairing interactions between the U14 snoRNA and the 18S rRNA essential for modification and processing are shown. The Sm-binding site in the U4 snRNA is underlined. In addition, the minimal U6 sequence, which together with the U4 snRNA, is required for the binding of the CYPH/hPRP4/hPRP3 proteins is indicated by gray type (20).
FIG. 2.
FIG. 2.
Identification and mutation of conserved amino acids on the surface of 15.5K. A) Amino acid alignment of 15.5K from human (Homo sapiens), Saccharomyces cerevisiae (Snu13p), Caenorhabditis elegans (accession no. Q21568), Drosophila melanogaster (accession no. GH03082), Arabidopsis thaliana (accession no. A71421), and Xenopus laevis (accession no. AAH46579) with the human ribosomal protein L7a sequence using the ClustalV program. Conserved residues are shown on a gray background and are grouped as described by Schulz and Schirmer (24). Identical and conserved residues are indicated by white type on black background and by black type on gray background, respectively. Residues specifically conserved in 15.5K are depicted in white type on blue background. Amino acid positions are indicated on the left. The secondary structure of human 15.5K (29) is indicated above the alignment, and amino acids involved in RNA binding are indicated by dark blue circles. B) The five mutations introduced into 15.5K. C) The positions of the residues targeted for mutagenesis on the surface of 15.5K are depicted using the crystal structure of 15.5K bound to the 5′ stem-loop of the U4 snRNA (29). The surface plot of 15.5K is shown in white, and the U4 snRNA is shaded gray. The positions of the amino acids targeted for mutagenesis are indicated in either red (mutant 15.5K-1), green (15.5K-2), turquoise (15.5K-3), blue (15.5K-4), or yellow (15.5K-5). D) Gel mobility shift analysis of the interaction of recombinant 15.5K and 15.5K mutants with U4, U14 and U14 mutants. The mutant proteins outlined in panel B were incubated with 32P-labeled in vitro-transcribed snRNA or snoRNA transcripts, and the resulting RNA-protein complexes were resolved on a 6% native polyacrylamide gel and visualized by autoradiography. The identity of the protein used is indicated above each lane (−, no 15.5K; 15.5K-Wt, wild-type 15.5K). The positions of the protein-RNA complex (RNP) and the free RNA are indicated on the right. The RNA used is indicated on the left.
FIG. 3.
FIG. 3.
Binding of hPRP31 to the U4 snRNA requires residues in α-helix 3 of 15.5K. GST-hPRP31 was incubated with 32P-labeled U4 snRNA in the presence (lanes 2, 3, 4, 5, 6 and 7) or absence (lane 1) of either wild-type or mutant 15.5K. GST-hPRP31 was isolated using glutathione resin, and bound RNA was extracted and ethanol precipitated. The bound (GST-hPRP31) and unbound RNA (10% supernatant [10% Sup.]) was analyzed on a denaturing 8% polyacrylamide-7 M urea gel and visualized by autoradiography. The identity of the 15.5K protein used is indicated above each lane (15.5K-Wt, wild-type 15.5K).
FIG. 4.
FIG. 4.
The association of CYPH/hPRP4/hPRP3 to the U4/U6 snRNA requires multiple regions on the surface of 15.5K. Purified CYPH/hPRP4/hPRP3 complex was incubated with U4/U6 snRNA duplex (U4 snRNA base paired with 32P-labeled U6 nucleotides 58 to 87 [Fig. 1]) in the presence (lanes 2 to 7) or absence (lane 1) of wild-type or mutant 15.5K. The CYPH/hPRP4/hPRP3 complex was then immunoprecipitated using anti-hPRP4 antibody (α-hPRP4) and the coprecipitating RNA and the RNA present in the supernatant (5% Sup.) were analyzed on a denaturing 8% polyacrylamide-7 M urea gel and visualized by autoradiography. The identity of the 15.5K protein used is indicated above each lane (−, no 15.5K; 15.5K-Wt, wild-type 15.5K).
FIG. 5.
FIG. 5.
Multiple regions on the surface of 15.5K are required for box C/D snoRNP assembly. HeLa nuclear extract was preincubated with either 800 pmol of U4-SL1 (SL1) RNA oligonucleotide (+) or buffer (−). Radiolabeled U14 snoRNA was subsequently added in the presence (lanes 2 to 8) or absence (−) (lanes 1 and 2) of wild-type or mutant 15.5K. The binding of individual snoRNP proteins was then assayed by immunoprecipitation (see Materials and Methods). Bound RNAs were recovered and then separated on an 8% polyacrylamide-7 M urea gel. Antibodies used for immunoprecipitation are indicated on the right. Ten percent of the RNA after incubation in nuclear extract was used as input in the bottom gel. The 15.5K protein used is indicated above each lane (15.5K-Wt, wild-type 15.5K).
FIG. 6.
FIG. 6.
hU3-55K binding to the U3 box B/C motif requires a distinct region on the surface of 15.5K. HeLa nuclear extract was preincubated with either 800 pmol of U4-SL1 RNA oligonucleotide (+) or buffer (−). Radiolabeled U3 box B/C motif RNA was subsequently added in the presence (lanes 3 to 8) or absence (lanes 1 and 2) of recombinant 15.5K or mutant 15.5K. The binding of hU3-55K was then assayed by immunoprecipitation (see Materials and Methods). Bound RNAs were recovered and then separated on an 8% polyacrylamide-7 M urea gel. Five percent of the RNA after incubation in nuclear extract was used as input in the bottom gel. The 15.5K protein used is indicated above each lane (15.5K-Wt, wild-type 15.5K). α-hU3-55K, anti-hU3-55K antibody.
FIG. 7.
FIG. 7.
hU3-55K directly interacts with 15.5K. A) 35S-labeled hU3-55K was incubated with either GST-15.5K (0.25 or 25 pmol) in the absence (−) or presence (+) of U3 B/C motif RNA (U3BC) (lanes 3 to 6) or 25 pmol of GST (lane 2). Bound, radiolabeled proteins were purified using glutathione resin, resolved on a 12% polyacrylamide-SDS gel, and revealed by autoradiography. The amount of recombinant 15.5K used is indicated above each lane. Ten percent of the input material was used in the Input lane. GST 15.5K-WT, GST fused to wild-type 15.5K. B) 35S-labeled hU3-55K was incubated with 25 pmol of either wild-type (WT) or mutant GST-15.5K (lanes 3 to 8) or GST (lane 2). Bound, radiolabeled proteins were purified using glutathione resin and resolved on a 12% polyacrylamide-SDS gel. Ten percent of the input material was used in the Input lane. The identity of the GST protein used is indicated above each lane.
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