Research paperThe yeast C/D box snoRNA U14 adopts a “weak” K-turn like conformation recognized by the Snu13 core protein in solution
Introduction
In the cell, RNA molecules associate with proteins to form ribonucleoproteins (RNPs) such as ribosome and spliceosome to ensure cell viability. The assembly of these RNA-protein complexes relies on the ability of the RNA to adopt the correct bound conformation, an event that generally requires the intervention of co-factors. Understanding the sequence-structure-function relationships in RNPs requires three-dimensional structural information on RNA molecules, both in presence and absence of their binding partners (proteins) or cofactors (metal ions).
The kink-turn or K-turn motifs are universal structural elements found in ribosomal RNAs (rRNAs) [1], small nuclear RNAs (snRNAs) [2], small nucleolar RNAs (snoRNAs) [3], SelenoCystein Insertion Sequence (SECIS) of the 3′-untranslated regions of selenoprotein mRNA [4,5], and 5′ UTRs including several riboswitches [6,7]. They consist of a three-nucleotides internal loop flanked on its 3′ side by a G•A/A•G tandem pairs followed by one or two non-Watson-Crick pairs (NC), and on its 5′ side by a conventionally base-paired helix (C) (Fig. 1A). This peculiar arrangement induces a strong curvature of the phosphodiester backbone between the two helices leading to a compact structure thanks to near-universal A-minor tertiary interactions [8,9]. There are two classes of K-turn structure depending on whether the O2′-H of the ribose at the -2 position establishes hydrogen bonds with the N3 or N1 of the A5 cycle (Fig. 1A). The modification of the hydrogen bond network involves a different rotation of the A5 nucleobase that affects its conserved base-pairing with G6 and changes the whole shape of the K-turn structure. The first and second unpaired residues (N1 and N2, Fig. 1A) are stacked on the C and NC helices, respectively. The third unpaired residue (N3, Fig. 1A), which is fully exposed to the solvent, represents a key interaction determinant for K-turn binding proteins.
Small nucleolar RNAs found in Archaea and Eukaryotes guide chemical modifications of ribosomal RNAs (rRNA), transfer RNAs (tRNA) and small nuclear RNAs (snRNA). The C/D box snoRNAs are associated with 2′-O-methylation of rRNA. They are characterized by the presence of conserved sequences, termed boxes C (5′-RUGAUGA-3′, where R is a purine) and D (5′-CUGA-3′), located near the 5′ and 3′ ends of the snoRNA, respectively (Fig. 1B). The C/D box folds into a K-turn through pairing between regions upstream of the C box and downstream of the D box, that are brought in close proximity [10]. The C/D box K-turn is a key player in methylation and processing of rRNA, stability and localization of snoRNPs. In an early stage during box C/D snoRNP assembly, the recruitment of the L7Ae protein [11,12] or SNU13/Snu13 protein (Human/Yeast) initiates the assembly process by binding specifically the K-turn motif of snoRNAs [[13], [14], [15]]. In particular, SNU13/Snu13p binds specifically the U14 snoRNA, a prerequisite for the successive arrival of the other constituents of the ribonucleoparticle, namely in eukaryotes, the proteins NOP58/Nop58 and NOP56/Nop56 and the 2′-O-methyltransferase, Fibrillarin/Nop1p [14,15].
As U3 snoRNA, the U14 snoRNA is a conserved non-coding RNA found in yeast and vertebrates required for the pre-rRNA maturation. Genetic depletion studies in S. cerevisiae showed that C/D box snoRNA U3, and U14 (also named snR128) are essential for cell viability and required for the 35S pre-rRNA cleavages that release the 18S rRNA [16,17]. Analyses of U14 sequences revealed the presence of a conserved terminal helix and five universal sequence elements: C and D boxes corresponding to a K-turn sequence motif, and A, B, and Y domains (Fig. 1B). A second set of boxes, the C'/D', has been recently identified [18]. Base pairing of A, B and Y domains of U14 with the 18S rRNA is required for the synthesis of functional ribosomes in yeast [[19], [20], [21]] suggesting that U14 contributes directly to rRNA processing. Moreover, the B domain of both vertebrate and yeast U14 snoRNAs is complementary to a sequence in 18S rRNA region that contains validated 2′-O-methylation at nucleotide C414 (UMASS Amherst Yeast snoRNA Database). In Xenopus laevis, depletion of the U14 snoRNA prevents 2′-O-methylation of 18S rRNA and an intact B domain is essential for the methylation [22]. Altogether, the snoRNP U14 would therefore have the dual function in rRNA maturation and ribose methylation.
Here, we report the solution structure of the free U14 snoRNA K-turn motif determined by Nuclear Magnetic Resonance (NMR). We demonstrate that a major fraction of U14 adopts a pre-folded conformation similar to known bound K-turn, even in the absence of protein or divalent ions. Finally, we show that the structure of kt-U14 is stabilized upon Snu13p binding. This first structural characterization of a free canonical C/D motif can serve as a reference for the C/D box snoRNA family. Its comparison with other family members allows discussing the consequences of this pre-folded k-turn during the snoRNP assembly and the relevance of an external scaffold.
Section snippets
RNA samples preparation
Milligram quantities of RNAs were prepared both unlabeled and uniformly 13C/15N labeled by in vitro transcription with in-house T7 RNA polymerase from oligonucleotide templates [23]. The DNA templates were purchased from Eurogentec (France). Labeled NTPs (nucleotide triphosphate) were purchased from SIGMA-ALDRICH (USA). Transcription conditions were optimized with varying magnesium concentrations in 25 μL reaction mixtures in 40 mM TRIS-HCl pH 8, 1 mM spermidine, 0.01% Triton X-100, 5 mM DTT
Resonance assignment of kt-U14
To identify the conformation adopted by the free C/D box snoRNA U14 recognized by Snu13p, we set out to determine the solution structure of the 31 nucleotides RNA (Fig. 1B and C). A thermo-stable UUCG tetraloop, followed by a C:G base pair on its 5’ side, was added to ensure proper folding of the RNA (Fig. 1C) [47,48]. The terminal A:U and U:A base pairs present in the full-length RNA were substituted with two G:C base pairs to improve transcription yield (Fig. 1B and C).
The secondary structure
Conclusive remarks
In this work, we described the structure adopted by a major fraction of the kt-U14 RNA in the absence of protein and divalent ions. Our NMR studies reveal that free U14 RNA adopts a pre-folded conformation similar to known bound K-turn such as the one formed by the B/C motif in U3 snoRNA (kt-U3B/C) or in the archaeal C/D box. In contrast to these latter RNAs, the structure of the free kt-U14 is dominated by a weak hydrogen bond network in its central region. The sharp kink of the phosphodiester
Coordinates
Atomic coordinates have been deposited in the Protein Data Bank (PDB ID code: 6HYK) and the NMR data in the Biological Magnetic Resonance Bank (BMRB access code: 34321).
Acknowledgments
This work was supported by Agence Nationale de la Recherche [Grant ANR-16-CE11-0032-02], CNRS, University of Lorraine, University of Strasbourg and INSERM. The authors thank the UMS 2008 for ITC and NMR facilities.
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Present address: Benjamin Rothé, Ecole Polytechnique Fédérale de Lausanne (EPFL) SV ISREC, Station 19, CH-1015 Lausanne, Switzerland.