Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Jul;9(7):821-38.
doi: 10.1261/rna.2130503.

A structural, phylogenetic, and functional study of 15.5-kD/Snu13 protein binding on U3 small nucleolar RNA

Nathalie Marmier-Gourrier  1 , Antoine Cléry, Veronique Senty-Ségault, Bruno Charpentier, Florence Schlotter, Fabrice Leclerc, Régis Fournier, Christaine Branlant
Affiliations

A structural, phylogenetic, and functional study of 15.5-kD/Snu13 protein binding on U3 small nucleolar RNA

Nathalie Marmier-Gourrier et al. RNA. 2003 Jul.

Abstract

The 15.5-kD protein and its yeast homolog Snu13p bind U4 snRNA, U3 snoRNA, and the C/D box snoRNAs. In U4 snRNA, they associate with a helix-bulge-helix (K-turn) structure. U3 snoRNA contains two conserved pairs of boxes, C'/D and B/C, which were both expected to bind the 15.5-kD/Snu13 protein. Only binding to the B/C motif was experimentally demonstrated. Here, by chemical probing of in vitro reconstituted RNA/protein complexes, we demonstrate the independent binding of the 15.5-kD/Snu13 protein to each of the two motifs. Due to a highly reduced stem I (1 bp), the K-turn structure is not formed in the naked B/C motif. However, gel-shift experiments revealed a higher affinity of Snu13p for the B/C motif, compared to the C'/D motif. A phylogenetic analysis of U3 snoRNA, coupled with an analysis of Snu13p affinity for variant yeast C'/D and B/C motifs, and a study of the functionality of a truncated yeast U3 snoRNA carrying base substitutions in the C'/D and B/C motifs, revealed that conservation of the identities of residues 2 and 3 in the B/C K-turn is more important for Snu13p binding and U3 snoRNA function, than conservation of the identities of corresponding residues in the C'/D K-turn. This suggests that binding of Snu13p to K-turns with a very short helix I imposes sequence constraints in the bulge. Altogether, the data demonstrate the strong importance of the binding of the 15.5-kD/Snu13 protein to the C'/D and B/C motifs for both U3 snoRNP assembly and activity.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Structural analysis of the 15.5 kD/hU3 complex. (A) Primer extension analysis of the chemically modified hU3 RNA and hU3 RNA/15.5 kD protein complex. Fifty picomoles of recombinant 15.5 kD protein were incubated with about 15 fmoles of in vitro transcribed hU3 RNA, using the conditions described in Materials and Methods. The free RNA (−15.5 kD) and the RNA/protein complex (+15.5 kD) were subjected to the chemical reagents indicated at the top of the autoradiogram (DMS, CMCT, and kethoxal), using the conditions described in Materials and Methods. Lanes marked by – are control experiments performed on the RNA or RNA/protein complex in the absence of any chemical reagent. The 5′-end labeled oligonucleotide RT-hU3 was the primer for reverse transcription. The synthesized cDNAs were fractionated by electrophoresis on a 7% sequencing gel. Lanes U, G, C, and A correspond to a sequence ladder made with the same oligonucleotide. Nucleotide positions in hU3 RNA are indicated on the left of the panel. Boxes C′, B, C, and D are shown on the right of the autoradiogram. In (B) and (C), the chemical modifications observed for the naked hU3 RNA (B) and the protections observed in the hU3 RNA/15.5-kD complex (C) are represented on a revised version of the secondary structure previously proposed for the human U3 snoRNA (Parker and Steitz 1987). The conserved B, C, C′, and D boxes are indicated. The helices Ic′/d, IIc′/d, Ib/c, and IIb/c of the K-turn motifs are also indicated. Helix numbering from 1 to 5 is as for yeast U3 snoRNA (Segault et al. 1992). In (B), chemically modified residues are in colored circles: red, orange, and green circles represent strong, medium, and low levels of modification, respectively. The protected sequence CUU (169 to 171) and its possible partner sequence at the 5′ extremity of the RNA are shown by squared residues. In (C), nucleotides protected in the complex compared to naked RNA are in blue circles: the darkness of the blue reflects the level of protection. Residues with a reinforced reactivity in the complex are shown in red circles.
FIGURE 1.
FIGURE 1.
Structural analysis of the 15.5 kD/hU3 complex. (A) Primer extension analysis of the chemically modified hU3 RNA and hU3 RNA/15.5 kD protein complex. Fifty picomoles of recombinant 15.5 kD protein were incubated with about 15 fmoles of in vitro transcribed hU3 RNA, using the conditions described in Materials and Methods. The free RNA (−15.5 kD) and the RNA/protein complex (+15.5 kD) were subjected to the chemical reagents indicated at the top of the autoradiogram (DMS, CMCT, and kethoxal), using the conditions described in Materials and Methods. Lanes marked by – are control experiments performed on the RNA or RNA/protein complex in the absence of any chemical reagent. The 5′-end labeled oligonucleotide RT-hU3 was the primer for reverse transcription. The synthesized cDNAs were fractionated by electrophoresis on a 7% sequencing gel. Lanes U, G, C, and A correspond to a sequence ladder made with the same oligonucleotide. Nucleotide positions in hU3 RNA are indicated on the left of the panel. Boxes C′, B, C, and D are shown on the right of the autoradiogram. In (B) and (C), the chemical modifications observed for the naked hU3 RNA (B) and the protections observed in the hU3 RNA/15.5-kD complex (C) are represented on a revised version of the secondary structure previously proposed for the human U3 snoRNA (Parker and Steitz 1987). The conserved B, C, C′, and D boxes are indicated. The helices Ic′/d, IIc′/d, Ib/c, and IIb/c of the K-turn motifs are also indicated. Helix numbering from 1 to 5 is as for yeast U3 snoRNA (Segault et al. 1992). In (B), chemically modified residues are in colored circles: red, orange, and green circles represent strong, medium, and low levels of modification, respectively. The protected sequence CUU (169 to 171) and its possible partner sequence at the 5′ extremity of the RNA are shown by squared residues. In (C), nucleotides protected in the complex compared to naked RNA are in blue circles: the darkness of the blue reflects the level of protection. Residues with a reinforced reactivity in the complex are shown in red circles.
FIGURE 2.
FIGURE 2.
Structural analysis of the Snu13p/yU3AΔ2,3,4 complex. (A) Primer extension analysis of the chemically modified yU3AΔ2,3,4 RNA and yU3AΔ2,3,4 RNA/Snu13p complex. Sixty picomoles of recombinant Snu13p were incubated with about 20 fmoles of in vitro transcribed yU3AΔ2,3,4 RNA, in the conditions described in Materials and Methods. The free RNA (−Snu13p) and the RNA/protein complex (+Snu13p) were subjected to the chemical reagents indicated at the top of the autoradiogram (same legend as in Fig. 1A). The 5′-end labeled oligonucleotide RT-yU3 was used for primer extension analyses and sequence analysis (lanes U, G, C, and A). The synthesized cDNAs were fractionated by electrophoresis on a 7% sequencing gel. Nucleotide positions in yU3AΔ2,3,4 RNA are indicated on the left of the panel. Boxes C′, B, and C are shown on the right of the autoradiogram. In (B) and (C), the chemical modifications observed on the naked yU3AΔ2,3,4 RNA (B) and the protection observed in the yU3AΔ2,3,4/Snu13p complex (C) are represented on the deduced secondary structure of yU3AΔ2,3,4 RNA. Positions of residues in the entire S. cerevisiae U3A snoRNA are indicated in black squares. The broken arrows delimit the part of the RNA yU3AΔ2,3,4 (positions 72 to 130), whose analysis is shown in (A). The helices Ic′/d, IIc′/d, Ib/c, and IIb/c of the K-turn structures are indicated when they are expected to be present. In (B), nucleotides circled in red, orange, and green were modified at high, medium, and low levels, respectively. In (C), the residues protected in the RNA/protein complex are in blue circles. The darkness of the blue circles reflects the level of protection. Nucleotides with an increased reactivity in the complex are in red circles. In (D), 60 pmoles of the recombinant Snu13p were incubated with about 20 fmoles of yU3A RNA in the presence 2 μg of yeast tRNAs, using the conditions described in Materials and Methods. The protein complex (+Snu13p) and the free RNA (−Snu13p) were treated with the chemical reagents (same legend as in Fig. 1A). Primer extension analyses and sequence analysis (lanes U, G, C, and A) were performed with oligonucleotide RT-yU3. Positions of residues in the yeast U3A snoRNA are indicated on the right of the autoradiogram, box C is indicated on the left. In (E), the results obtained in (D) are schematically represented on the portion of the yeast U3A snoRNA secondary structure, corresponding to boxes B and C. Nucleotides modified in free RNA are circled and the color of the circles reflects the level of modification (same color code as in [B] and [C]). Protections in the complex are indicated by blue dots. The darkness of the blue reflects the level of protection. The C–G pair 115–252, expected to be formed upon protein binding, is indicated in a rectangle.
FIGURE 3.
FIGURE 3.
Phylogenic analysis of the K-turn structures formed by the C′/D and B/C boxes of U3 snoRNA. The sequence from human (Homo sapiens), the mouse (Mus musculus), Xenopus laevis, tomato (Lycopersicon esculentum), wheat (Triticum aestivum), Chlamydomonas reinhardtii, Euglena gracilis, Crithidia fasciculata, Tetrahymena thermophila, Schyzosaccharomyces pombe, Saccharomyces cerevisiae, Hansenula wingei, Kluyveromyces bulgaricus, Kluyveromyces delphensis, Kluyveromyces marxianus var. fragilis, and Kluyveromyces marxianus var. lactis, are numbered AF020531, X04258, Z12613, X14411, X63065, AJ001179, AF277396, X71349, U27297, X56982, X91037, X91005, Y14752, Z78432, Y14751, X87402 in GenBank: http://www.ncbi.nlm.nih.gov/. The sequence from Trypanosoma brucei U3 snoRNA was taken from Hartshorne and Toyofuku (1999). The sequence from Pichia anomala strain CBS 247, Pichia guilliermondii strain CBS 2030, Pichia pastoris strain CBS 704, and Pichia salictaria strain CBS 5456 are from this study (AJ507111, AJ507112, AJ507113, AJ507109, respectively numbered in GenBank). The K-turn structures, which can be formed by the C′/D motif of all the compared U3 snoRNAs, are shown in (A). These structures were divided into three blocks, according to the sequence of their internal loop. The K-turn structures, which can be formed by B/C motifs of all compared U3 snoRNAs, are shown in (B). In (C), the structures that can be formed at the junction between helices 5, 4, 2, and 3 in the S. cerevisiae, K. marxianus var. lactis, and P. salictaria U3 snoRNAs are compared. Numbering of helices 2, 3, 4, and 5 is done according to Segault et al. (1992). Nucleotide numbering in the S. cerevisiae U3 snoRNA, as well as the B and C boxes, and the helix IIb/c are indicated. The yields and positions of V1 RNase cleavages previously found in U3 snoRNA phenol-extracted from the S. cerevisiae U3 snoRNP (Mereau et al. 1997) are also represented on the S. cerevisiae U3 snoRNA structure (arrows associated with one gray square, two darker gray squares, or three very dark squares correspond to low, medium, or strong cleavage, respectively). In (D), the consensus motifs established for the C′/D and B/C motifs of the U3 snoRNAs compared in this study are shown, as well as the consensus motif established for the C/D motif of guide snoRNAs (Watkins et al. 2000) and the K-turn structure recognized by the 15.5-kD protein in human U4 snRNA (Nottrott et al. 1999).
FIGURE 4.
FIGURE 4.
Snu13p binds the C′/D and B/C motifs of U3 snoRNA independently. The base substitutions performed in the mutD and mutB variant yU3AΔ2,3,4 RNAs are shown in (A) (base substitutions are shown in gray characters). In (B), RNP complexes were formed by incubation of 5 fmoles of uniformly labeled WT or yU3AΔ2,3,4mutB RNA in the presence of Snu13p at a 125, 250, 500, or 1000 nM concentration or of 5 fmoles of yU3AΔ2,3,4mutD RNA in the presence of Snu13p at a 50, 75, 125, or 250 nM concentration. All complexes were formed in the presence of 10 μg of yeast tRNAs in the conditions described in Materials and Methods. Lanes (0) are control experiments in the absence of protein. Complexes were fractionated by electrophoresis in a nondenaturing 6% polyacrylamide gel. The positions of the RNA–protein complexes (RNP1 and RNP2 for yU3AΔ2,3,4 WT RNA and RNP for yU3AΔ2,3,4mutD and yU3AΔ2,3,4mutB RNAs) and of the free RNA (RNA) are indicated on the right of the autoradiograms.
FIGURE 5.
FIGURE 5.
Effects of point mutations in yU3C′/D and yU3B/C RNAs on Snu13p binding. The proposed secondary structure of the yU3C′/D RNA containing the C′/D motif of yeast U3 snoRNA is shown in (A). Positions in yeast U3 snoRNA of the extremities of the two yeast U3 snoRNA segments present in yU3C′/D RNA are indicated in black squares. The base substitutions that were generated at position 2 of the internal loop are indicated. (B) Autoradiograms of gel-shift assays performed with 5 fmoles of uniformely labeled WT and variant yU3C′/D RNAs and the recombinant Snu13 protein at various concentrations indicated below the lanes. (C) The proposed secondary structure of the yU3B/C RNA containing the B/C motif of U3 snoRNA. Positions in yeast U3 snoRNA of the extremities of the three yeast U3 snoRNA segments present in yU3B/C RNA are indicated in black rectangles. The base substitutions that were generated at positions 2 and 3 of the internal loop are indicated. (D) Autoradiograms of gel-shift assays performed with 5 fmoles of uniformly labeled WT and variant yU3B/C RNAs and the recombinant Snu13 protein at various concentrations indicated below the lanes.
FIGURE 6.
FIGURE 6.
Effects on cell growth of point mutations in the internal loops of the C′/D and B/C motifs of yU3AΔ2,3,4 RNA. In (A), the point mutations that were generated in the yU3AΔ2,3,4 RNA are represented on the secondary structure established for this RNA. The relative stabilities of the yU3AΔ2,3,4 WT and variant RNAs in the JH84 S. cerevisiae strain grown at 30°C in YPD medium (see Materials and Methods), were compared to that of the entire U3 snoRNA (yU3A) by Northern blot analysis (B). The 5′-end labeled oligonucleotide RT-yU3 was used as the probe and for an internal control, a simultaneous hybridization was made with a 5′-end labeled oligonucleotide complementary to U6 snRNA (RT-yU6, see Table 1). The values obtained by dividing the radioactivity measured in the band corresponding to U3 snoRNA or yU3AΔ2,3,4 RNA by that measured in the band corresponding to U6 snRNA are given below the lanes (U3A/U6 or yU3AΔ2,3,4/U6 for cells expressing the yU3AΔ2,3,4 WT or variant RNAs, and (yU3A+U3A)/U6 for cells expressing the yU3A RNA). Growth of the S. cerevisiae JH84 strain transformed with plasmid pASZ11 (−), pASZ11::yU3A, WT, or variant pASZ11::yU3AΔ2,3,4 on YPD plates at 20°C, 30°C, and 37°C is shown in (C). In these experiments, the S. cerevisiae JH84 cells were grown in liquid YPG medium for 48 h, and then 24 h in liquid YPD medium before spreading on YPD plates. Growth at 30°C and 37°C was for 48 h, growth at 20°C was for 72 h.
FIGURE 7.
FIGURE 7.
The nonconventional C/D box snoRNAs can form K-turn structures with one base pair instead of helix I, as the yU3AΔ2,3,4 B/C motif. The K-turn structures that can be formed by the C/D motifs of conventional (A) and nonconventional (B) C/D box snoRNAs, that were previously studied by Darzacq and Kiss (2000) are compared to the K-turn structures formed by the yU3AΔ2,3,4 B/C motif (C) and B/C motif of T. brucei U3 snoRNA (D).
See this image and copyright information in PMC

References

    1. Beltrame, M. and Tollervey, D. 1992. Identification and functional analysis of two U3 binding sites on yeast pre-ribosomal RNA. EMBO J. 11: 1531–1542. - PMC - PubMed
    1. ———. 1995. Base pairing between U3 and the pre-ribosomal RNA is required for 18S rRNA synthesis. EMBO J. 14: 4350–4356. - PMC - PubMed
    1. Beltrame, M., Henry, Y., and Tollervey, D. 1994. Mutational analysis of an essential binding site for the U3 snoRNA in the 5′ external transcribed spacer of yeast pre-rRNA. Nucleic Acids Res. 22: 5139–5147. - PMC - PubMed
    1. Borovjagin, A.V. and Gerbi, S.A. 1999. U3 small nucleolar RNA is essential for cleavage at sites 1, 2 and 3 in pre-rRNA and determines which rRNA processing pathway is taken in Xenopus oocytes. J. Mol. Biol. 286: 1347–1363. - PubMed
    1. ———. 2000. The spacing between functional cis-elements of U3 snoRNA is critical for rRNA processing. J. Mol. Biol. 300: 57–74. - PubMed

Publication types

MeSH terms