The box C/D small nucleolar RNPs (snoRNPs) are essential for the processing and modification of rRNA. The core box C/D proteins are restructured during human U3 box C/D snoRNP biogenesis; however, the molecular basis of this is unclear. Here we show that the U8 snoRNP is also restructured, suggesting that this may occur with all box C/D snoRNPs. We have characterized four novel human biogenesis factors (BCD1, NOP17, NUFIP, and TAF9) which, along with the ATPases TIP48 and TIP49, are likely to be involved in the formation of the pre-snoRNP. We have analyzed the in vitro protein-protein interactions between the assembly factors and core box C/D proteins. Surprisingly, this revealed few interactions between the individual core box C/D proteins. However, the novel biogenesis factors and TIP48 and TIP49 interacted with one or more of the core box C/D proteins, implying that they mediate the assembly of the pre-snoRNP. Consistent with this, we show that NUFIP bridges interactions between the core box C/D proteins in a partially reconstituted presnoRNP. Restructuring of the core complex probably reflects the conversion of the pre-snoRNP, where core protein-protein interactions are maintained by the bridging biogenesis factors, to the mature snoRNP.Three of the four eukaryotic rRNAs are cotranscribed as a large precursor RNA (pre-rRNA) in the nucleolus (2, 37, 46). The pre-rRNA undergoes a complex series of processing and modification steps (17) to generate the mature rRNAs. Small nucleolar RNAs (snoRNAs) are an evolutionarily conserved group of noncoding RNAs found in both eukaryotes and Archaea that are involved in the modification and processing of rRNAs (2, 9, 46). Based on conserved sequence elements, two classes of snoRNA have been defined; the H/ACA and box C/D snoRNAs. The majority of box C/D snoRNAs direct the 2Ј-O methylation of RNA by base pairing with target sequences (2,30,46). A subset of box C/D snoRNAs, including U3, U8, and U14, contain rRNA complementary regions that are proposed to act as chaperones in rRNA processing (reviewed in reference 46). Box C/D snoRNAs are present in the cell as small nucleolar ribonucleoprotein particles (snoRNPs) and are associated with a common set of four core proteins: 15.5K, fibrillarin (methyltransferase), NOP56, and NOP58 (reviewed in reference 46). Association with the core box C/D proteins is essential for the accumulation of the snoRNA, as well as snoRNA processing and nucleolar localization (29). The hierarchical assembly of the eukaryotic box C/D snoRNPs in nuclear extract first involves 15.5K binding to the highly conserved k-turn element present in the box C/D motif (48, 50). Binding of 15.5K and the conserved stem II of the box C/D motif are required for the recruitment of NOP56, NOP58, and fibrillarin (48). A similar assembly pathway has been observed for the Archaeal complexes, with the 15.5K homologue L7ae binding first followed by NOP5 and fibrillarin (40). The proteins are predicted to be distributed symmetrically with one copy of L7ae, NOP5, and fibrilla...

The box C/D small nucleolar RNPs (snoRNPs) are essential for the processing and modification of rRNA. TIP48 and TIP49 are two related AAA ؉ proteins that are essential for the formation of box C/D snoRNPs. These proteins are key components of the pre-snoRNP complexes, but their exact role in box C/D snoRNP biogenesis is largely uncharacterized. Here we report that TIP48 and TIP49 interact with one another in vitro, and only the TIP48/TIP49 complex, but not the individual proteins, possesses significant ATPase activity. Loss of TIP48 and TIP49 results in a change in pre-snoRNA levels and a loss of U3 snoRNA signal in the Cajal body. We show that TIP48 and TIP49 make multiple interactions with core snoRNP proteins and biogenesis factors and that these interactions are often regulated by the presence of ATP. Furthermore, we demonstrate that TIP48 and TIP49 efficiently bridge interactions between the core box C/D proteins NOP56 or NOP58 and 15.5K. Our data imply that the snoRNP assembly factor NUFIP can regulate the interactions between TIP48 and TIP49 and the core box C/D proteins. We suggest that snoRNP assembly involves an intricate series of interactions that are mediated/regulated by bridging factors and chaperones.In the eukaryotic nucleolus, small nucleolar RNAs (snoRNAs) are involved in the processing and modification of rRNA (1,20,38). The H/ACA snoRNAs and box C/D snoRNAs function as sequence-specific guides to direct the isomerization of uridine to pseudouridine and the 2Ј-O methylation of rRNA, (1,20,38). A subset of box C/D snoRNAs that includes U3, U8, and U14 box C/D snoRNAs is essential for rRNA processing. These snoRNAs base pair with specific regions of the pre-rRNA and have been proposed to function as RNA chaperones by regulating rRNA folding (36). Box C/D snoRNAs contain a conserved box C/D motif which is involved in the binding of the four core proteins, 15.5K, NOP56, NOP58, and the methyltransferase fibrillarin (10). The core proteins assemble onto the snoRNA in a stepwise manner with 15.5K first binding to the k-turn element in the box C/D motif followed by the recruitment of the remaining core proteins into the complex (41,45).Most box C/D snoRNAs are encoded within the introns of protein-coding genes and are processed from the spliced intron lariat (9,19,21,38). In contrast, a subset of box C/D snoRNAs, including U3, U8, and U13, are independently transcribed by RNA polymerase II (9, 28, 46). The initial transcripts of these genes contain an m 7 G cap and a short 3Ј extension. During snoRNP biogenesis the m 7 G cap is converted into an m 3 G cap and the 3Ј extension is removed by exonucleases (9, 28, 46). The maturation of the snoRNA and the assembly of the snoRNP is an intricate process involving the dynamic and temporal association of numerous factors in a large multiprotein pre-snoRNP complex (29,42,43). These factors include proteins linked to snoRNP assembly (TIP48, TIP49, NUFIP, TAF9, NOP17, and BCD1), molecular chaperones (HSP90 and HSC70), nucleocytoplasmic transport factors (PHAX, ...

The rate of ribosome biogenesis, which is downregulated in terminally differentiated cells and upregulated in most cancers, regulates the growth rate and is linked to the cell's proliferative potential. The U3 box C/D small nucleolar RNP (snoRNP) is an integral component of the small subunit (SSU) processome and is essential for 18S rRNA processing. We show that U3 snoRNP assembly, and therefore U3 snoRNA accumulation, is regulated through the U3-specific protein hU3-55K. Furthermore, we report that the levels of several SSU processome components, including the U3 snoRNA but not other box C/D snoRNAs, are specifically downregulated during human lung (CaCo-2) and colon (CaLu-3) epithelial cell differentiation. c-Myc is reported to play an integral role in regulating ribosome production by controlling the expression of many ribosome biogenesis factors. Our data, however, indicate that this regulation is not dependent on c-Myc since the level of this protein does not change during epithelial cell differentiation. In addition, depletion of c-Myc had only a mild affect on the levels of SSU processome proteins. CaCo-2 cells are colon adenocarcinoma epithelial cells that are believed to revert to their precancerous state during differentiation. This suggests a significant increase in the levels of specific SSU processome components during tumorogenesis.