Evidence that the AAA⁺Proteins TIP48 and TIP49 Bridge Interactions between 15.5K and the Related NOP56 and NOP58 Proteins during Box C/D snoRNP Biogenesis

Abstract

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 R2TP chaperone complex: its involvement in snoRNP assembly and tumorigenesis

“…It has been proposed that the assembly of box C/D snoRNP starts with the interaction between box C/D snoRNA and Snu13/15.5k, followed by the binding of Nop58 and then Nop1/fibrillarin and Nop56 are recruited (28,32). In this process, Nufip/Rsa1 (snoRNP assembly factor) and Rvbs facilitate the assembly of box C/D snoRNP by bridging the interactions of core components of box C/D snoRNP: between 15.5K and both Nop56 and Nop58 (29)(30)(31). Nufip is likely to regulate the interaction between Rvbs and the box C/D core proteins by tightly interacting with 15.5K (30).…”

“…This modification is essential for the subsequent endonucleotic cleavage and processing of the pre-rRNA to produce mature rRNAs (21,27). The assembly of snoRNP is hierarchical and its proper assembly is essential for both the activity and stability of snoRNP (6,16,17,(28)(29)(30)(31). It has been proposed that the assembly of box C/D snoRNP starts with the interaction between box C/D snoRNA and Snu13/15.5k, followed by the binding of Nop58 and then Nop1/fibrillarin and Nop56 are recruited (28,32).…”

“…In this process, Nufip/Rsa1 (snoRNP assembly factor) and Rvbs facilitate the assembly of box C/D snoRNP by bridging the interactions of core components of box C/D snoRNP: between 15.5K and both Nop56 and Nop58 (29)(30)(31). Nufip is likely to regulate the interaction between Rvbs and the box C/D core proteins by tightly interacting with 15.5K (30). An involvement of R2TP in this assembly process also have been identified in yeast and human (6,29), and very recently, it was shown that the yeast R2TP specifically associates with unassembled Nop58 which has yet to be assembled with other core factors such as Nop1, Snu13, and Nop56 (16).…”

Mechanism of the AAA+ ATPases pontin and reptin in the biogenesis of H/ACA RNPs

“…Several chaperones have been implicated in snoRNP biogenesis, in particular, the AAA+ ATPases pontin and reptin, the R2TP complex that additionally includes PIH1D1 and RPAP3 ( Fig. 1E), HSP90, and several others (Newman et al 2000;King et al 2001;Watkins et al 2002Watkins et al , 2004McKeegan et al 2007McKeegan et al , 2009Venteicher et al 2008;Zhao et al 2008;Boulon et al 2010). As AAA+ proteins are known for breaking protein-protein interactions, e.g., the prototypical AAA protein N-ethylmaleimide-sensitive factor (NSF) disassembles SNAP-SNARE complexes (Zhao et al 2012), we tested if pontin and reptin functioned similarly in the separation of NAP57 and SHQ1.…”

“…Genetic depletion and siRNA-mediated mRNA knockdown studies further supported their participation, however, without clue as to their mode of action (Gallant 2007;Jha and Dutta 2009;Huen et al 2010). Moreover, the role of the R2TP complex is much better documented for C/D than for H/ACA RNPs (Newman et al 2000;Watkins et al 2002Watkins et al , 2004Boulon et al 2008;McKeegan et al 2009). Pontin and reptin are evolutionary highly conserved proteins with homology with bacterial helicases (Iyer et al 2004;Ammelburg et al 2006).…”