The hU3-55K Protein Requires 15.5K Binding to the Box B/C Motif as Well as Flanking RNA Elements for Its Association with the U3 Small Nucleolar RNA in Vitro

Granneman, Sander; Pruijn, Ger J. M.; Horstman, Wendy; Venrooij, Walther J. van; Lührmann, Reinhard; Watkins, Nicholas J.

doi:10.1074/jbc.m206631200

Cited by 41 publications

(73 citation statements)

References 42 publications

(112 reference statements)

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“…2F), presumably reflecting errors in reverse transcription at the nucleotide that is the site of protein-RNA cross-linking (6, 7). These results are in good agreement with previous predictions of an Rrp9 binding site near the box B/C motif (8,9,11).…”

Section: Resultssupporting

confidence: 83%

“…Two Rrp9 binding sites were mapped in U3, adjacent to the box B/C motif in helices 2 and 4. This location is consistent with previous studies (8,9,11), and the major Rrp9 cross-linking site was confirmed by primer extension. The archaeal dual guide box C/D snoRNP architecture is symmetric with 2 copies each of the orthologues of Nop1, Nop56/58, and Snu13 (15).…”

Section: Discussionsupporting

confidence: 81%

“…The archaeal dual guide box C/D snoRNP architecture is symmetric with 2 copies each of the orthologues of Nop1, Nop56/58, and Snu13 (15). In U3, 2 binding sites had been identified for Snu13 (8,16), suggesting that these might associate with 2 copies of Nop1 and single copies of Nop56 and Nop58. However, the stoichiometry of the snoRNP proteins and their exact binding sites were unclear.…”

Section: Discussionmentioning

confidence: 99%

“…Of these, Nop1, Nop56, Nop58, and Snu13 are common to all box C/D class snoRNAs, whereas Rrp9 is U3-specific. Rrp9 was selected to initially test the method, because the human Rrp9 orthologue (hU3-55K) was efficiently UV-cross-linked to U3 in vitro (8), and previous analyses had identified a potential binding site on U3 (8,9).…”

Section: Resultsmentioning

confidence: 99%

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Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs

Granneman

Kudla

Petfalski

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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328

446

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The U3 small nucleolar ribonucleoprotein (snoRNP) plays an essential role in ribosome biogenesis but, like many RNA-protein complexes, its architecture is poorly understood. To address this problem, binding sites for the snoRNP proteins Nop1, Nop56, Nop58, and Rrp9 were mapped by UV cross-linking and analysis of cDNAs. Cross-linked protein-RNA complexes were purified under highly-denaturing conditions, ensuring that only direct interactions were detected. Recovered RNA fragments were amplified after linker ligation and cDNA synthesis. Cross-linking was successfully performed either in vitro on purified complexes or in vivo in living cells. Cross-linking sites were precisely mapped either by Sanger sequencing of multiple cloned fragments or direct, high-throughput Solexa sequencing. Analysis of RNAs associated with the snoRNP proteins revealed remarkably high signal-to-noise ratios and identified specific binding sites for each of these proteins on the U3 RNA. The results were consistent with previous data, demonstrating the reliability of the method, but also provided insights into the architecture of the U3 snoRNP. The snoRNP proteins were also cross-linked to pre-rRNA fragments, with preferential association at known sites of box C/D snoRNA function. This finding demonstrates that the snoRNP proteins directly contact the pre-rRNA substrate, suggesting roles in snoRNA recruitment. The techniques reported here should be widely applicable to analyses of RNA-protein interactions.ribosome synthesis ͉ RNA modification ͉ RNA processing ͉ RNP structure ͉ yeast P roteomic approaches have identified many factors involved in ribosome synthesis in yeast, but we still lack detailed understanding of the architecture of the preribosomes and small nucleolar ribonucleoprotein (snoRNP) complexes that are required for their maturation. Several methods have been described that allow identification of protein-RNA interaction sites in native particles. RNA immunoprecipitation uses formaldehyde to cross-link RNA to proteins (1, 2). Caveats of this method are that formaldehyde also cross-links proteins to proteins and the immunoprecipitation step is performed under semidenaturing conditions, so a positive result does not demonstrate direct RNA-protein interaction, and the spatial resolution of the technique is low. Moreover, formaldehyde and other chemical cross-linkers may not enter the cores of large complexes. This problem can be avoided by cross-linking proteins and RNA with UV light, and several techniques have been reported (3-7).UV-induced protein-RNA cross-links can be detected by primer extension analysis on the RNA and by MALDI-MS on the protein (4). These approaches can detect cross-links on both protein and RNA but primer extension mapping on long RNAs is not practical without prior knowledge of the approximate cross-linking site and MS analyses require up to 50 pmol of RNP (5). The cross-linking and immunoprecipitation (CLIP) method identified protein-RNA interaction sites in mammalian cells by cloning of the covalentlya...

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Section: Resultssupporting

confidence: 83%

Section: Discussionsupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs

Granneman

Kudla

Petfalski

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

328

446

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show abstract

“…Mutagenesis suggests it is the helix-bulge structure, rather than sequence of base-pairing regions, that is crucial for 15.5K binding (Nottrott et al 1999;. Assembly of 15.5K with a radiolabeled fragment of the human U3 snoRNA, containing only the B/C box, was observed directly in HeLa cell nuclear extracts (Granneman et al 2002). Nucleotide analog interference mapping (NAIM) of a box C/D snoRNP reconstituted in Xenopus oocytes suggests that 15.5K interacts only with the box C/D motif in vivo and not with the box CЈ/DЈ (Weinstein Szewczak et al 2002).…”

Section: Introductionmentioning

confidence: 98%

Analysis of Snu13p mutations reveals differential interactions with the U4 snRNA and U3 snoRNA

Dobbyn

O’Keefe²

2004

RNA

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Pre-mRNA splicing is executed by the spliceosome, a complex of small nuclear RNAs (snRNAs) and numerous proteins. One such protein, 15.5K/Snu13p, is associated with the spliceosomal U4/U6.U5 tri-snRNP and box C/D small nucleolar ribonucleoprotein particles (snoRNPs), which act during preribosomal RNA (rRNA) processing. As such, it is the first splicing factor to be identified in two functionally distinct particles. 15.5K binds to an internal helix-bulge-helix (K-turn) structure in the U4 snRNA and two such structures in the U3 snoRNA. Previous work has concentrated on the structural basis of the interaction of 15.5K with the RNAs and has been carried out in vitro. Here we present a functional analysis of Snu13p in vivo, using a galactose inducible SNU13 strain to investigate the basis of three lethal mutations in Saccharomyces cerevisiae. Two are point mutations that map to the RNA-binding domain, and the third is a C-terminal deletion. These mutations result in accumulation of unspliced pre-mRNA, confirming a role for Snu13p in pre-mRNA splicing. In addition, these mutants also display rRNA processing defects that are variable in nature. Analysis of one mutant in the RNA-binding domain reveals a reduction in the levels of the U4 snRNA, U6 snRNA, and box C/D snoRNAs, but not H/ACA snoRNAs, supporting a role for Snu13p in accumulation and/or maintenance of specific RNAs. The mutations in the RNA-binding domain exhibit differential binding to the U4 snRNA and U3 snoRNA in vitro, suggesting that there are differences in the mode of interaction of Snu13p with these two RNAs.

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Ribosome Assembly

Nierhaus¹,

Lafontaine²

2004

Protein Synthesis and Ribosome Structure

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The hU3-55K Protein Requires 15.5K Binding to the Box B/C Motif as Well as Flanking RNA Elements for Its Association with the U3 Small Nucleolar RNA in Vitro

Cited by 41 publications

References 42 publications

Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs

Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs

Analysis of Snu13p mutations reveals differential interactions with the U4 snRNA and U3 snoRNA

Ribosome Assembly

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