Solution structure of the pseudo-5' splice site of a retroviral splicing suppressor - PubMed (original) (raw)
Solution structure of the pseudo-5' splice site of a retroviral splicing suppressor
Javier Cabello-Villegas et al. RNA. 2004 Sep.
Abstract
Control of Rous sarcoma virus RNA splicing depends in part on the interaction of U1 and U11 snRNPs with an intronic RNA element called the negative regulator of splicing (NRS). A 23mer RNA hairpin (NRS23) of the NRS directly binds U1 and U11 snRNPs. Mutations that disrupt base-pairing between the loop of NRS23 and U1 snRNA abolish its negative control of splicing. We have determined the solution structure of NRS23 using NOEs, torsion angles, and residual dipolar couplings that were extracted from multidimensional heteronuclear NMR spectra. Our structure showed that the 6-bp stem of NRS23 adopts a nearly A-form duplex conformation. The loop, which consists of 11 residues according to secondary structure probing, was in a closed conformation. U913, the first residue in the loop, was bulged out or dynamic, and loop residues G914-C923, G915-U922, and U916-A921 were base-paired. The remaining UUGU tetraloop sequence did not adopt a stable structure and appears flexible in solution. This tetraloop differs from the well-known classes of tetraloops (GNRA, CUYG, UNCG) in terms of its stability, structure, and function. Deletion of the bulged U913, which is not complementary to U1 snRNA, increased the melting temperature of the RNA hairpin. This hyperstable hairpin exhibited a significant decrease in binding to U1 snRNP. Thus, the structure of the NRS RNA, as well as its sequence, is important for interaction with U1 snRNP and for splicing suppression.
Figures
FIGURE 1.
RSV NRS23 RNA is complementary to both U1 and U11 snRNAs. (A) The NRS23 secondary structure determined by nuclease probing and chemical modification. The pseudo 5′ splice site is indicated by a large arrow. The sequence highlighted in yellow is identical to the 5′ splice site consensus. Results of the chemical and enzymatic probing are indicated as follows: RNase V1 or T2 cleavages are indicated by small arrows linked to squares or circles, respectively, and lead cleavages by triangles. Colors indicate cleavage yields (green and orange for weak and medium). (B) NRS23 is shown interacting with complementary sequences from U1 snRNA (residues 1 to 10). (C) NRS23 can also base-pair with U11 snRNA (residues 4 to 18).
FIGURE 2.
Solution structures of NRS23. The solid model drawing of the NRS23 RNA structure (A) and the stereo plot of the 15 lowest-energy structures calculated with RDCs (B). The structures are aligned with the stem residues. The structures refined with RDCs are more extended than those refined without RDCs. The tilt of the bases flanking U913 is ~13°. Color code in A: purple, stem region before the U913 bulge; blue, 3 bp in loop; light red, the UUGU tetraloop. For clarity, the bulged U913 is drawn as a space-filling model.
FIGURE 3.
Schematic representation of NOE data collected on NRS23 (only residues 910–926 are shown). Thin lines represent interresidue NOEs. The ribose pucker is represented by dark filling for C2′ endo, gray for mixed 2C2′/C3′ endo, and open for C3′ endo. An extremely weak NOE is represented by a broken line.
FIGURE 4.
HNN-COSY spectrum of NRS23 showing the A H2 to U N3 region (A). 15N-1H HMQC optimized for long-range correlations showing the H5/N1 region of U (B). The horizontal line connects the interresidue correlation between A921 H2 and U916 N3 (circled) and the intrabase correlation between U916 H5 and N3. The H8 to N9 and G N3 correlations are seen in the HNN-COSY; some N9-H1′ and N1-H5/H1′ correlations are seen in the HMQC.
FIGURE 5.
The loop residues 914–923 from the minimized average structure of NRS23. This structure was calculated from the 15 lowest-energy NRS23 structures. Note that the top of the loop is closed by 3 bp. The UUGU tetraloop adopts no particular structure and presumably is ready for intermolecular interaction.
FIGURE 6.
Melting profiles show that NRS23 is less stable than NRS23ΔU913. Melting curves (A) and their first derivatives (B) of NRS23 and the NRS23ΔU913 mutant, respectively. Dark blue and red, experimental; pink and light blue, fit curves. The thermodynamic data are listed in the embedded table. The melting temperature has an error of ±0.5°C.
FIGURE 7.
Binding of U1 snRNP to the NRS requires destabilizing the double-stranded helix. (A) Northern analysis of U1 snRNA affinity selected from HeLa nuclear extract, with wild-type (WT) NRS RNA, the UU917/918AA mutant that is capable of splicing (AA), the U913 deletion mutant, and the nonfunctional UU917/918CC mutant (CC). (B) Relative U1 snRNP affinities of the wild-type NRS and the mutants. The background binding to the beads was subtracted, and each trial was normalized to the WT levels. The average of three experiments is presented.
FIGURE 8.
Histogram of the RDC distribution (A) and calculated versus experimental RDCs for NRS23 (Pearson’s plot, B). The one-bond C-C and C-H RDCs are normalized to the NH couplings. Pearson’s plot of experimental RDC vs. the values calculated after best fitting the alignment tensor to the lowest-energy structure refined without RDC constraints. The Pearson’s coefficient is ~0.83. The RDCs of the tetraloop residues were excluded because of their dynamic behavior.
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