The snRNP 15.5K protein folds its cognate K-turn RNA: a combined theoretical and biochemical study

Abstract

The human 15.5K protein binds to the 5' stem-loop of U4 snRNA, promotes the assembly of the spliceosomal U4/U6 snRNP, and is required for the recruitment of the 61K protein and the 20/60/90K protein complex to the U4 snRNA. In the crystallographic structure of the 15.5K-U4 snRNA complex, the conformation of the RNA corresponds to the family of kink-turn (K-turn) structural motifs. We simulated the complex and the free RNA, showing how the protein binding and the intrinsic flexibility contribute to the RNA folding process. We found that the RNA is significantly more flexible in the absence of the 15.5K protein. Conformational transitions such as the interconversion between alternative purine stacking schemes, the loss of G-A base pairs, and the opening of the K-turn occur only in the free RNA. Furthermore, the stability of one canonical G-C base pair is influenced both by the binding of the 15.5K protein and the nature of the adjacent structural element in the RNA. We performed chemical RNA modification experiments and observed that the free RNA lacks secondary structure elements, a result in excellent agreement with the simulations. Based on these observations, we propose a protein-assisted RNA folding mechanism in which the RNA intrinsic flexibility functions as a catalyst.

FIGURE 1.

Simulated RNA constructs. (A) The K-turn motif found in the crystal structure of the 5′ stem–loop of U4 snRNA bound to 15.5K protein (K1 RNA); the C-stem has three Watson-Crick base pairs (G26–C47, G46–C27, and G45–C28), the internal loop contains three unpaired nucleotides (A29, A30, U31) and two tandem-sheared G-A base pairs (G32–A44, G43–A33), and the NC-stem consists of two Watson-Crick base pairs (G34–C42 and G35–C41). Guanines are shown in blue, cytosines in orange, adenines in red, and the uracils in yellow. φ is the angle between the P atoms of C47, U31, and G35. (B) K2 RNA (naturally occurring): the pentaloop UUUAU (shown in red) is attached to the NC-stem of K1 RNA. (C) K3 RNA: the tetraloop UGAA was attached to the NC-stem of K1 RNA. (D) K4 RNA: the hexaloop UUAAUU was attached to the NC-stem of K1 RNA. (E) K5 RNA: the C-stem and the NC-stem of K1 RNA were extended with seven and six Watson-Crick base pairs. The core of the K-turn RNA observed in the crystal structure is encircled in B–E.

FIGURE 2.

General analysis of MD simulations (K2 RNA). (A) B-factors per residue for the RNA structure during the MD-LES trajectories of the bound (red curve) and unbound RNA (black curve). (B) Root mean square deviations (RMSD) of the RNA backbone from the initial structure for the bound and unbound RNA during the MD-LES (red and black curves) and MD+LES trajectories (green and blue curves). (C) φ angle of the bound and unbound RNA during the MD-LES (red and black curves) and MD+LES trajectories (green and blue curves). (D) Closed conformation of the K-turn (φ = 20°–40°). (E) Open conformation of the K-turn (φ = 50°–80°). In C and D the loop attached to the NC-stem is not shown and the φ angle is shown in black in C and D (see Fig. 1A).

FIGURE 3.

Conformational states of A30 (K2 RNA). (A) “3 + 1” stacking scheme formed by adenines A29, A30, A44, and A33 in the bound RNA, A30 being in a syn/C2′ endo conformation. (B) “2 + 2” stacking scheme formed by adenines A29, A30, A44, and A33 during the trajectories of the unbound RNA, A30 being in a high-anti, anti/C3′ endo conformation.

FIGURE 4.

Interstrand contacts established in the RNA (K2 RNA). (A) The interstrand contact formed between the O2′ atom of A29 and the N1 atom of A44. (B) Hydrogen-bonding distance between the O2′ atom of A29 and the N1 atom of A44 in the bound (red curve) and unbound RNA (black curve) during the MD-LES trajectories. (C) The interstrand contact formed between the N2 atom of G45 and the O3′ atom of A33. (D) Hydrogen-bonding distance between the N2 atom of G45 and the O3′ atom of A33 in the bound and unbound RNA during the MD-LES (red and black curves) and MD+LES (green and blue curves) trajectories.

FIGURE 5.

The G32–A44 base pair (K2 RNA); hydrogen-bonding distance between the N2 atom of G32 and the N7 atom of A44 in the bound and unbound RNA during the MD-LES (red and black curves) and MD+LES (green and blue curves) trajectories. The hydrogen bond is pointed with a double arrowhead.

FIGURE 6.

Interactions between the 15.5K protein and the external loop. (A) Asn40 and Lys44 of the 15.5K protein establish a bridge between G43 and the sugar-phosphate backbone of the external loop of the 5′ stem–loop of U4snRNA. (B) Bridging distance between the O6 atom of G43 and the O2P atom of C41 for the unbound K2 RNA (black curve) and for the bound K2 (red curve), K3 (green curve), and K4 (blue curve) and K5 (brown curve) RNAs during the MD-LES trajectories; the bridge is shown in A. (C) B-factors per residue for the nucleotides of the external loop for the unbound K2 RNA (black curve) and for the bound K2 (red curve), K3 (green curve), and K4 (blue curve) RNAs during the MD-LES trajectories. (D) Asn64 and Lys68 of the ribosomal L7AE protein establish a similar bridge between G43 and the sugar-phosphate backbone of the Kt15 RNA.

FIGURE 7.

Chemical RNA structure probing. (A) Primer extension analysis of U4 snRNA after DMS treatment of the RNA either in the absence or presence of recombinant 15.5K protein (lanes 2,3); (lanes 1,4) control lanes, no DMS modification. (B) Primer extension analysis of U4 snRNA after Kethoxal treatment either in the absence (lanes 2,3,6,7) or presence (lanes 4,5,8,9) of 15.5K protein; (lanes 6–9) RNA modification after Proteinase K digestion; (lanes 1,10) control lanes, no Kethoxal treatment; the modification of an RNA base results in a stop of the reverse transcriptase 1 nt before the site of attack (Ehresmann et al. 1987); modified nucleotides are indicated by an arrowhead; nucleotides that are clearly protected from chemical modification in the presence of bound 15.5K protein are marked by asterisks; the presence or absence of the 15.5K protein is indicated by “+” or “-,” respectively. C, U, A, and G refer to dideoxysequencing reactions and correspond to the sequence of human U4 snRNA; 0 indicates a control primer extension with unmodified U4 snRNA where no ddNTPs were added to the reaction; and the position of every tenth nucleotide of the U4 snRNA is indicated on the left in panels A and B, respectively.