In vitro, assembly of box C/D small nucleolar ribonucleoproteins (snoRNPs) involves the sequential recruitment of core proteins to snoRNAs. In vivo, however, assembly factors are required (NUFIP, BCD1, and the HSP90-R2TP complex), and it is unknown whether a similar sequential scheme applies. In this paper, we describe systematic quantitative stable isotope labeling by amino acids in cell culture proteomic experiments and the crystal structure of the core protein Snu13p/15.5K bound to a fragment of the assembly factor Rsa1p/NUFIP. This revealed several unexpected features: (a) the existence of a protein-only pre-snoRNP complex containing five assembly factors and two core proteins, 15.5K and Nop58; (b) the characterization of ZNHIT3, which is present in the protein-only complex but gets released upon binding to C/D snoRNAs; (c) the dynamics of the R2TP complex, which appears to load/unload RuvBL AAA(+) adenosine triphosphatase from pre-snoRNPs; and (d) a potential mechanism for preventing premature activation of snoRNP catalytic activity. These data provide a framework for understanding the assembly of box C/D snoRNPs.
Biogenesis of eukaryotic box C/D small nucleolar ribonucleoprotein particles (C/D snoRNPs) involves conserved trans-acting factors, which are proposed to facilitate the assembly of the core proteins Snu13p/15.5K, Nop58p/NOP58, Nop56p/NOP56 and Nop1p/Fibrillarin on box C/D small nucleolar RNAs (C/D snoRNAs). In yeast, protein Rsa1 acts as a platform, interacting with both the RNA-binding core protein Snu13 and protein Pih1 of the Hsp82-R2TP chaperone complex. In this work, a proteomic approach coupled with functional and structural studies identifies protein Hit1 as a novel Rsa1p-interacting partner involved in C/D snoRNP assembly. Hit1p contributes to in vivo C/D snoRNA stability and pre-RNA maturation kinetics. It associates with U3 snoRNA precursors and influences its 3'-end processing. Remarkably, Hit1p is required to maintain steady-state levels of Rsa1p. This stabilizing activity is likely to be general across eukaryotic species, as the human protein ZNHIT3(TRIP3) showing sequence homology with Hit1p regulates the abundance of NUFIP1, the Rsa1p functional homolog. The nuclear magnetic resonance solution structure of the Rsa1p317-352-Hit1p70-164 complex reveals a novel mode of protein-protein association explaining the strong stability of the Rsa1p-Hit1p complex. Our biochemical data show that C/D snoRNAs and the core protein Nop58 can interact with the purified Snu13p-Rsa1p-Hit1p heterotrimer.
We report the nearly complete (1)H, (15)N and (13)C resonance assignment of the complex formed by the C-terminal domains of Pih1 and Tah1 from S. cerevisiae and evidence the folding ability of Tah1 under complex formation.
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.
The ubiquitous Hsp90 chaperone participates in snoRNP and RNA polymerase assembly through interaction with the R2TP complex. This complex includes the proteins Tah1, Pih1, Rvb1, and Rvb2. Tah1 bridges Hsp90 to R2TP. Its minimal TPR domain includes two TPR motifs and a capping helix. We established the high-resolution solution structures of Tah1 free and in complex with the Hsp90 C-terminal peptide. The TPR fold is similar in the free and bound forms and we show experimentally that in addition to its solvating/stabilizing role, the capping helix is essential for the recognition of the Hsp90 (704)EMEEVD(709) motif. In addition to Lys79 and Arg83 from the carboxylate clamp, this helix bears Tyr82 forming a ?/S-CH3 interaction with Hsp90 M(705) from the peptide 310 helix. The Tah1 C-terminal region is unfolded, and we demonstrate that it is essential for the recruitment of the Pih1 C-terminal domain and folds upon binding.
Multiple RNA-guided pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10-L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21.
Controlling iron homeostasis is crucial for all aerobically grown living cells that are exposed to oxidative damage by reactive oxygen species (ROS), as free iron increases the production of ROS. Methionine sulfoxide reductases (Msr) are key enzymes in repairing ROS-mediated damage to proteins, as they reduce oxidized methionine (MetSO) residues to methionine. E. coli synthesizes two Msr, A and B, which exhibit substrate diastereospecificity. The bacterial iron-responsive small RNA (sRNA) RyhB controls iron metabolism by modulating intracellular iron usage. We show in this paper that RyhB is a direct regulator of the msrB gene that encodes the MsrB enzyme. RyhB down-regulates msrB transcripts along with Hfq and RNaseE proteins since mutations in the ryhB, fur, hfq, or RNaseE-encoded genes resulted in iron-insensitive expression of msrB. Our results show that RyhB binds to two sequences within the short 5UTR of msrB mRNA as identified by reverse transcriptase and RNase and lead (II) protection assays. Toeprinting analysis shows that RyhB pairing to msrB mRNA prevents efficient ribosome binding and thereby inhibits translation initiation. In vivo site directed-mutagenesis experiments in the msrB 5UTR region indicate that both RyhB-pairing sites are required to decrease msrB expression. Thus, this study suggests a novel mechanism of translational regulation where a same sRNA can basepair to two different locations within the same mRNA species. In contrast, expression of msrA is not influenced by changes in iron levels.
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