Analysis of four-way junctions in RNA structures

Abstract

RNA secondary structures can be divided into helical regions composed of canonical Watson-Crick and related base pairs, as well as single-stranded regions such as hairpin loops, internal loops, and junctions. These elements function as building blocks in the design of diverse RNA molecules with various fundamental functions in the cell. To better understand the intricate architecture of three-dimensional (3D) RNAs, we analyze existing RNA four-way junctions in terms of base-pair interactions and 3D configurations. Specifically, we identify nine broad junction families according to coaxial stacking patterns and helical configurations. We find that helices within junctions tend to arrange in roughly parallel and perpendicular patterns and stabilize their conformations using common tertiary motifs such as coaxial stacking, loop-helix interaction, and helix packing interaction. Our analysis also reveals a number of highly conserved base-pair interaction patterns and novel tertiary motifs such as A-minor-coaxial stacking combinations and sarcin/ricin motif variants. Such analyses of RNA building blocks can ultimately help in the difficult task of RNA 3D structure prediction.

Figure 1

(a) 2D diagram of a 4-way junction element composed of four helices labeled and color-coded by H₁ (red), H₂ (blue), H₃ (green) and H₄ (magenta), and the corresponding single stranded loop regions labeled J1/2 to J4/1 with nucleotides color-coded in yellow. Helices and loop regions are labeled in a unique way according to the 5′ to 3′ orientation of the entire RNA structure, by labeling H₁ as the first helix encountered, while entering the junction region, as one moves along the nucleotide chain in the 5′ to 3′ direction and so forth. Lines inside the helices represent the canonical WC basepairs G-C, A-U, and the wobble basepair G-U. (b) 3D diagrams containing two pairs of helices: H₁ with H₄, and H₂ with H₃, which are coaxial stacked. The 4-way junction illustrated corresponds to the 23S rRNA 1S72_2678 from Table 1.

Figure 2

Network interaction diagrams for the nine families of 4-way junctions. Family H, cH and cL contains two coaxial helices; family cK and π contains one coaxial stacking; while families cW, ψ, X and cX contains no coaxial stacking. The “c” before the capital letter in the family name denotes the crossing observed at the point of strand exchange. The network symbology follows the Leontis-Westhof notation (see inset boxes).

Figure 2

Network interaction diagrams for the nine families of 4-way junctions. Family H, cH and cL contains two coaxial helices; family cK and π contains one coaxial stacking; while families cW, ψ, X and cX contains no coaxial stacking. The “c” before the capital letter in the family name denotes the crossing observed at the point of strand exchange. The network symbology follows the Leontis-Westhof notation (see inset boxes).

Figure 2

Network interaction diagrams for the nine families of 4-way junctions. Family H, cH and cL contains two coaxial helices; family cK and π contains one coaxial stacking; while families cW, ψ, X and cX contains no coaxial stacking. The “c” before the capital letter in the family name denotes the crossing observed at the point of strand exchange. The network symbology follows the Leontis-Westhof notation (see inset boxes).

Figure 3

A-minor interactions within junction domains. a) Secondary structure diagram for the most common interaction. b) Motif consensus for the most common interaction. c) Consensus motif for the less common interaction. N and M represent 0–3 and 2–3 nucleotides respectively. Often, a WC (GC) interaction appears next to the consecutive adenines at the n+2 position.

Figure 4

Interactions similar to sarcin/ricin motif (left box). The interactions are part of the junctions 23S rRNA 2AW4_267 and 23S rRNA 2J01_1832 of family ψ, and 16S rRNA 2AVY_942 of family cX. The interaction in 2AW4_267 was previously observed by Leontis et al. RNA-Protein interactions (red font) are denoted by protein name followed by amino acid type and residue number.

Figure 5

Anatomy of a 4-way junction.