Characterization of the interaction between protein Snu13p/15.5K and the Rsa1p/NUFIP factor and demonstration of its functional importance for snoRNP assembly

Abstract

The yeast Snu13p protein and its 15.5K human homolog both bind U4 snRNA and box C/D snoRNAs. They also bind the Rsa1p/NUFIP assembly factor, proposed to scaffold immature snoRNPs and to recruit the Hsp90-R2TP chaperone complex. However, the nature of the Snu13p/15.5K–Rsa1p/NUFIP interaction and its exact role in snoRNP assembly remained to be elucidated. By using biophysical, molecular and imaging approaches, here, we identify residues needed for Snu13p/15.5K–Rsa1p/NUFIP interaction. By NMR structure determination and docking approaches, we built a 3D model of the Snup13p–Rsa1p interface, suggesting that residues R249, R246 and K250 in Rsa1p and E72 and D73 in Snu13p form a network of electrostatic interactions shielded from the solvent by hydrophobic residues from both proteins and that residue W253 of Rsa1p is inserted in a hydrophobic cavity of Snu13p. Individual mutations of residues in yeast demonstrate the functional importance of the predicted interactions for both cell growth and snoRNP formation. Using archaeal box C/D sRNP 3D structures as templates, the association of Snu13p with Rsa1p is predicted to be exclusive of interactions in active snoRNPs. Rsa1p and NUFIP may thus prevent premature activity of pre-snoRNPs, and their removal may be a key step for active snoRNP production.

Protein Hit1, a novel box C/D snoRNP assembly factor, controls cellular concentration of the scaffolding protein Rsa1 by direct interaction

“…GST pull down and Y2H experiments previously revealed a possible interaction between Rsa1p and Pih1p (21), and accordingly, we found that Rsa1p is required to tether Pih1p−Tah1p to a [C/D RNA−Snu13p] complex (29). Nop58p was initially shown to interact with Pih1p (31), and previous Y2H assays (21) and the present in vitro binding assays (Figure 1H) have revealed that Nop58p also associates with the purified Rsa1p 230−375 –Hit1p and Snu13p–Rsa1p 230−375 –Hit1p complexes.…”

“…First, as Snu13p is known to interact with C/D snoRNAs and with Rsa1p (29), we tested by electrophoresis mobility shift assays (EMSA) whether the Rsa1p−Hit1p interaction is compatible with binding of Rsa1p to a [C/D snoRNA–Snu13p] complex. As illustrated in Figure 1G, when incubated with a Snu13p–Rsa1p–Hit1p purified ternary complex, a radiolabeled C/D snoRNA is found in a complex displaying a slower mobility compared to the complex formed with the purified Snu13p–Rsa1p heterodimer.…”

Implication of the box C/D snoRNP assembly factor Rsa1p in U3 snoRNP assembly

“…These data are in agreement with previous mapping of the interaction sites at the surface of Snu13p/SNU13 (Supplementary Figure S7) and the recent 3D models of U3 snoRNP (48–51) showing that Rrp9p/55.5K and Rsa1p/NUFIP1 interact with independent regions on Snu13p. For example, arginine R 91 of the human SNU13 protein (R 89 for the S. cerevisiae Snu13p) was shown necessary for the recruitment of U3-55K onto the U3 snoRNP (69).…”

The Proteasome Subunit Rpn8 Interacts with the Small Nucleolar RNA Protein (snoRNP) Assembly Protein Pih1 and Mediates Its Ubiquitin-independent Degradation in Saccharomyces cerevisiae

“…Pih1 interacts directly with Nop58, one of the core protein subunits in box C/D snoRNP (11) and affects its stability (14). Pih1 also interacts with Rsa1, another snoRNP assembly factor through which the R2TP complex may interact with the snoRNP core protein Snu13 (15). Similar to the role of Tah1 in protecting Pih1, a small protein, Hit1, was also identified to interact with and protect Rsa1, thus revealing a complex regulatory network controlling snoRNP assembly in vivo (16).…”