Conserved stem II of the box C/D motif is essential for nucleolar localization and is required, along with the 15.5K protein, for the hierarchical assembly of the box C/D snoRNP.

The 5' stem-loop of the U4 snRNA and the box C/D motif of the box C/D snoRNAs can both be folded into a similar stem-internal loop-stem structure that binds the 15.5K protein. The homologous proteins NOP56 and NOP58 and 61K (hPrp31) associate with the box C/D snoRNPs and the U4/U6 snRNP, respectively. This raises the intriguing question of how the two homologous RNP complexes specifically assemble onto similar RNAs. Here we investigate the requirements for the specific binding of the individual snoRNP proteins to the U14 box C/D snoRNPs in vitro. This revealed that the binding of 15.5K to the box C/D motif is essential for the association of the remaining snoRNP-associated proteins, namely, NOP56, NOP58, fibrillarin, and the nucleoplasmic proteins TIP48 and TIP49. Stem II of the box C/D motif, in contrast to the U4 5' stem-loop, is highly conserved, and we show that this sequence is responsible for the binding of NOP56, NOP58, fibrillarin, TIP48, and TIP49, but not of 15.5K, to the snoRNA. Indeed, the sequence of stem II was essential for nucleolar localization of U14 snoRNA microinjected into HeLa cells. Thus, the conserved sequence of stem II determines the specific assembly of the box C/D snoRNP.