Elsevier

Biochimie

Volume 164, September 2019, Pages 70-82
Biochimie

Research paper
The yeast C/D box snoRNA U14 adopts a “weak” K-turn like conformation recognized by the Snu13 core protein in solution

https://doi.org/10.1016/j.biochi.2019.03.014Get rights and content

Highlights

  • Free U14 C/D box snoRNA folds into a K-turn structure.

  • kt-U14 adopts a prefolded conformation similar to L7Ae family protein bound K-turns.

  • U14 is the first example of free pre-formed K-turn of the canonical C/D boxes.

  • Both sequence and pre-bound state of U14 are optimal for promoting the RNP assembly.

Abstract

Non-coding RNAs associate with proteins to form ribonucleoproteins (RNPs), such as ribosome, box C/D snoRNPs, H/ACA snoRNPs, ribonuclease P, telomerase 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. K-turn motifs represent ubiquitous binding platform for proteins found in several cellular environment. This structural motif has an internal three-nucleotide bulge flanked on its 3′ side by a G•A/A•G tandem pairs followed by one or two non-Watson-Crick pairs, and on its 5′ side by a classical RNA helix. This peculiar arrangement induces a strong curvature of the phosphodiester backbone, which makes it conducive to multiple tertiary interactions. SNU13/Snu13p (Human/Yeast) binds specifically the U14 C/D box snoRNA K-turn sequence motif. This event is the prerequisite to promote the assembly of the RNP, which contains NOP58/Nop58 and NOP56/Nop56 core proteins and the 2′-O-methyl-transferase, Fibrillarin/Nop1p. The U14 small nucleolar RNA is a conserved non-coding RNA found in yeast and vertebrates required for the pre-rRNA maturation and ribose methylation. Here, we report the solution structure of the free U14 snoRNA K-turn motif (kt-U14) as determined by Nuclear Magnetic Resonance. We demonstrate that a major fraction of free kt-U14 adopts a pre-folded conformation similar to protein bound K-turn, even in the absence of divalent ions. In contrast to the kt-U4 or tyrS RNA, kt-U14 displays a sharp bent in the phosphodiester backbone. The U•U and G•A tandem base pairs are formed through weak hydrogen bonds. Finally, we show that the structure of kt-U14 is stabilized upon Snu13p binding. The structure of the free U14 RNA is the first reference example for the canonical motifs of the C/D box snoRNA family.

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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  • 1

    Present address: Benjamin Rothé, Ecole Polytechnique Fédérale de Lausanne (EPFL) SV ISREC, Station 19, CH-1015 Lausanne, Switzerland.

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