An unexpected, conserved element of the U3 snoRNA is required for Mpp10p association

Wormsley, Steven; Samarsky, Dmitry; Fournier, Maurille J.; Baserga, Susan J.

doi:10.1017/s1355838201010238

Cited by 22 publications

(34 citation statements)

References 65 publications

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“…We did not observe fibrillarin binding to the U3BC RNA in our HeLa nuclear extract; however, under the same conditions we could observe fibrillarin binding to the box C/D motif (30). Furthermore, it has since been shown that the box B/C motif is not required for fibrillarin association with either the yeast or human U3 snoRNAs in vivo (40). 2 We therefore believe that the hU3-55K interacts directly with the 15.5K-bound U3BC RNA complex.…”

Section: Discussionsupporting

confidence: 40%

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

Granneman¹,

Pruijn²,

Horstman³

et al. 2002

Journal of Biological Chemistry

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The 15.5K protein directly binds to the 5 stem-loop of the U4 small nuclear RNA, the small nucleolar (sno) RNA box C/D motif, and the U3 snoRNA-specific box B/C motif. The box B/C motif has also been shown to be essential for the association of the U3 small nucleolar ribonucleoprotein-specific protein hU3-55K. We therefore set out to determine how 15.5K and hU3-55K recognize the box B/C motif. By using an in vitro assembly assay, we show that hU3-55K effectively binds a sub-fragment of the U3 snoRNA surrounding the B/C motif that we have named the U3BC RNA. The association of hU3-55K with the U3BC RNA is dependent on the binding of 15.5K to the box B/C motif. The association of hU3-55K with the U3BC RNA was found to be also dependent on a conserved RNA structure that flanks the box B/C motif. Furthermore, we show that hU3-55K, a WD 40 repeat containing protein, directly cross-links to the U3BC RNA. Our data support a new structural model of the box B/C region of the U3 snoRNA in which the box B/C motif is base-paired to form a structure highly similar to that of both the U4 5 stem-loop and the box C/D motif.The U3 small nucleolar RNA (snoRNA) 1 is essential for the processing of the small subunit ribosomal RNA (rRNA) in both vertebrates and fungi (1-3). This box C/D-type snoRNA functions by base pairing with the 5Ј-external transcribed spacer (ETS) of the pre-rRNA and the 5Ј-terminal region of the 18 S rRNA sequence. The latter interaction blocks the formation of a universally conserved central pseudoknot; a long range interaction essential for the formation and function of the small subunit rRNA (1, 4). Based on these observations, the U3 snoRNP has been proposed to function as an RNA chaperone in the assembly of the small subunit of the ribosome.The U3 snoRNP contains the core proteins common to all box C/D snoRNPs, namely fibrillarin (Nop1p in yeast), NOP56, NOP58 (Nop5p), and 15.5K (Snu13p in yeast) (5-12). These proteins are essential for the biogenesis, stability, and nucleolar localization of the box C/D snoRNPs. In addition the U3 snoRNA has been shown to be associated with several U3-specific proteins in yeast, namely Rrp9p (hU3-55K in human), Imp3p, Imp4p, Mpp10p, Dhr1p, Rcl1p, Sof1p,22). However, the purified yeast U3 snoRNP mono-particle was shown to contain only one U3-specific protein, namely Rrp9p (11), suggesting that many of the other U3 snoRNAassociated proteins may be part of a larger processing complex (13,15,20,21). Genetic studies in yeast have shown that all of the U3 snoRNP-specific proteins are essential for U3 snoRNP function but not for its biogenesis (13)(14)(15)(16)(17)(18)(19)22).The 3Ј domain of the U3 snoRNA can be folded into a stemrich structure that contains four conserved RNA sequence elements, namely boxes CЈ, B, C, and D. Boxes CЈ and D are found opposite each other at the base of the 3Ј domain and form the common core box C/D motif (previously termed the box CЈ/D motif but referred to here as the box C/D motif for clarity) (23-25). The box C/D motif is required for the biog...

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Section: Discussionsupporting

confidence: 40%

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

Granneman¹,

Pruijn²,

Horstman³

et al. 2002

Journal of Biological Chemistry

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“…In each mutant the complete element was replaced by an unrelated sequence. A similar approach has been used in the analysis of U3 snoRNA in other organisms (30,32,36,37,42,48).…”

Section: Resultsmentioning

confidence: 99%

“…However, mutations mutC, mutB, mutA, mutAЈ, and mut 5Ј hinge were all expressed at lower levels than the wild-type RNA, suggesting that other regions within the human U3 snoRNA may also function in the accumulation of the transcript. Interestingly, mutations in the 5Ј end of the yeast U3 snoRNA also result in reduced expression levels (48), suggesting that this effect is not specific to the human U3 snoRNA.…”

Section: Resultsmentioning

confidence: 99%

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Role of Pre-rRNA Base Pairing and 80S Complex Formation in Subnucleolar Localization of the U3 snoRNP

Granneman

Vogelzangs

Lührmann

et al. 2004

Molecular and Cellular Biology

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In the nucleolus the U3 snoRNA is recruited to the 80S pre-rRNA processing complex in the dense fibrillar component (DFC). The U3 snoRNA is found throughout the nucleolus and has been proposed to move with the preribosomes to the granular component (GC). In contrast, the localization of other RNAs, such as the U8 snoRNA, is restricted to the DFC. Here we show that the incorporation of the U3 snoRNA into the 80S processing complex is not dependent on pre-rRNA base pairing sequences but requires the B/C motif, a U3-specific protein-binding element. We also show that the binding of Mpp10 to the 80S U3 complex is dependent on sequences within the U3 snoRNA that base pair with the pre-rRNA adjacent to the initial cleavage site. Furthermore, mutations that inhibit 80S complex formation and/or the association of Mpp10 result in retention of the U3 snoRNA in the DFC. From this we propose that the GC localization of the U3 snoRNA is a direct result of its active involvement in the initial steps of ribosome biogenesis.The processing of eukaryotic pre-rRNA involves a series of endo-and exonucleolytic cleavages as well as a significant number of covalent, posttranscriptional modifications. Both the cleavage and modification events require small nucleolar RNAs (snoRNAs). The two major classes of snoRNA function as guide RNAs by base pairing with specific sites of modification in the substrate. The H/ACA snoRNAs function in the site-specific formation of pseudouridine, while the box C/D snoRNAs direct the 2Ј-O methylation of rRNA and certain snRNAs (reviewed in references 1 and 20). A subset of the box C/D snoRNAs that includes U3, U8, and U14 is essential for pre-rRNA cleavage events (17,19,26,31,33). These snoRNAs are proposed to function as molecular chaperones that use extensive rRNA complementary regions to orchestrate the folding and cleavage of the precursor transcript.The U3 snoRNA has two distinct functional domains (Fig. 1A). The 5Ј domain contains the sequence elements that are important for base pairing with the pre-rRNA (GAC box, box A, box AЈ, 5Ј hinge, and 3Ј hinge) (reviewed in reference 39). Box A base pairs with a region near the 5Ј terminus of the 18S rRNA and in doing so regulates the formation of an evolutionarily conserved pseudoknot structure (16,35). The 5Ј hinge and 3Ј hinge sequences are complementary to regions of the 5Ј external transcribed spacer (5Ј ETS) (6). The 3Ј hinge sequence has the potential to base pair with the pre-rRNA adjacent to the primary processing site. Interestingly, the 3Ј hinge region is more important for pre-rRNA processing in Xenopus laevis oocytes (6), while rRNA processing in Saccharomyces cerevisiae is more dependent on the 5Ј hinge sequence (3). The 3Ј domain of the U3 snoRNA contains the evolutionarily conserved and structurally related box CЈ/D motif (the C/D motif in other box C/D snoRNAs) and the U3-specific box B/C motif. The box C/D motif is essential for nucleolar localization, RNA stability, and 5Ј cap hypermethylation. In contrast, the U3-specific B/C motif is ...

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“…In both humans and yeast, Imp4p forms a complex with Mpp10p and Imp3p [24,25]. The protein complex then interacts with both U3 snoRNA and 35S rRNA to initiate the maturation of rRNAs [26][27][28]. Imp4p belongs to a protein superfamily that contains a conserved Brix (biogenesis of ribosomes in Xenopus) domain [29].…”

Section: Introductionmentioning

confidence: 99%

The U3 small nucleolar ribonucleoprotein component Imp4p is a telomeric DNA-binding protein

Hsieh¹,

Tu²,

Lee³

et al. 2007

Biochemical Journal

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Imp4p is a component of U3 snoRNP (small nucleolar ribonucleoprotein) involved in the maturation of 18S rRNA. We have shown that Imp4p interacts with Cdc13p, a single-stranded telomere-binding protein involved in telomere maintenance. To understand the role of Imp4p in telomeres, we purified recombinant Imp4p protein and tested its binding activity towards telomeric DNA using electrophoretic mobility-shift assays. Our results showed that Imp4p bound specifically to single-stranded telomeric DNA in vitro. The interaction of Imp4p to telomeres in vivo was also demonstrated by chromatin immunoprecipitation experiments. Significantly, the binding of Imp4p to telomeres was not limited to yeast proteins, since the hImp4 (human Imp4) also bound to vertebrate single-stranded telomeric DNA. Thus we conclude that Imp4p is a novel telomeric DNA-binding protein that, in addition to its role in rRNA processing, might participate in telomere function.

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An unexpected, conserved element of the U3 snoRNA is required for Mpp10p association

Cited by 22 publications

References 65 publications

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

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

Role of Pre-rRNA Base Pairing and 80S Complex Formation in Subnucleolar Localization of the U3 snoRNP

The U3 small nucleolar ribonucleoprotein component Imp4p is a telomeric DNA-binding protein

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