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. 2001 Dec 17;20(24):7250-8.
doi: 10.1093/emboj/20.24.7250.

Solution structure of conserved AGNN tetraloops: insights into Rnt1p RNA processing

I Lebars  1 , B Lamontagne, S Yoshizawa, S Aboul-Elela, D Fourmy
Affiliations

Solution structure of conserved AGNN tetraloops: insights into Rnt1p RNA processing

I Lebars et al. EMBO J. .

Abstract

Rnt1p, the yeast orthologue of RNase III, cleaves rRNAs, snRNAs and snoRNAs at a stem capped with conserved AGNN tetraloop. Here we show that 9 bp long stems ending with AGAA or AGUC tetraloops bind to Rnt1p and direct specific but sequence-independent RNA cleavage when provided with stems longer than 13 bp. The solution structures of these two tetraloops reveal a common fold for the terminal loop stabilized by non-canonical A-A or A-C pairs and extensive base stacking. The conserved nucleotides are stacked at the 5' side of the loop, exposing their Watson-Crick and Hoogsteen faces for recognition by Rnt1p. These results indicate that yeast RNase III recognizes the fold of a conserved single-stranded tetraloop to direct specific dsRNA cleavage.

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Figures

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Fig. 1. Illustration of Rnt1p model substrates. The conserved AGNN nucleotides are shown in bold. The mutations introduced in the wild-type sequence are shown in grey. The four heterologous base pairs used to stabilize the structure are shown as an outline. R31 represents the terminal stem–loop of the U5 snRNA. R32 represents the terminal stem–loop of snR47. R31U is R31 with a mutation that changes the conserved A residue in the tetraloop to U. R31A contains the R31 stem sequence but the loop sequence is changed to GAAA. R31L contains the R31 sequence and an insertion of 10 bp that extends the stem size to a total of 19 bp. The sites of cleavages by Rnt1p are indicated by the arrowheads.
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Fig. 2. Gel shift assay of different Rnt1p substrates. Increasing concentrations (µM) of Rnt1p were incubated with 2 fmol of R31 (A), R32 (B), R31L (C), R32U (D) and R31A (E) in 150 mM KCl. RNA incubated without enzyme under the same conditions is included as a control. The positions of the unbound RNA and the shifted RNA are indicated on the left. The protein concentration is indicated on the top.
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Fig. 3. Cleavage assay of Rnt1p substrates. The different RNA substrates were incubated with recombinant Rnt1p in the presence of Mg2+ and 150 mM KCl. After completion of the cleavage reactions, the RNA was fractionated using 20% denaturing PAGE and the bands visualized using Instant Imager. The positions of the different substrates (S31L–S32U) and the cleavage products (P1 and P2) are indicated on the right. The position of the 10 bp molecular weight marker is shown on the left. The (+) and (–) indicate the presence and absence of Rnt1p, respectively.
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Fig. 4. Imino proton region of the one-dimensional spectrum (A) and the HSQC spectrum (B) of the AGUC tetraloop RNA. The imino proton region of the one-dimensional spectrum (C) and the HSQC spectrum (D) of the AGAA tetraloop RNA. All one-dimensional imino and HSQC spectra were recorded at 5°C.
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Fig. 5. Best-fit superposition of the 20 final simulated annealing structures of AGUC (A) and AGAA (B) tetraloop RNAs. The heavy atoms of the RNA have been superimposed. Bases are shown in dark blue and the backbone in light blue.
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Fig. 6. Stereo views of single representative structures of AGUC (A) and AGAA (B) tetraloops. All heavy atoms are displayed. Bases are coloured in light blue, with nitrogen and oxygen atoms in dark blue and red, respectively. Ribose–phosphate backbones are coloured in yellow, and phosphate oxygen in red.
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