Identification of a GUAAY Pentaloop Sequence Involved in a Novel RNA Loop-Helix Interaction

Abstract

Large RNAs often utilize GNRA tetraloops as structural elements to stabilize the overall tertiary fold. These tetraloop-receptor (TR) interactions have a conserved geometry in which the tetraloop docks into the receptor at an angle of ~15° from the helix containing the receptor. Here, we show that the conserved GUAAY pentaloop found in domain III of group IIB1 introns participates in a novel class of RNA tertiary interaction with a geometry and mode of binding that are significantly different from that found in GNRA TR interactions. This pentaloop is highly conserved within the IIB1 class and interacts with the minor groove of the catalytic domain V. The base planes of the loop and receptor nucleotides are not coplanar and greatly deviate from standard A-minor motifs. The helical axis of the GUAAY stem loop diverges ~70° from the angle of insertion found in a typical GNRA TR interaction. Therefore, the loop architecture and insertion orientation are distinctive, with in vitro splicing data indicating that a GNRA tetraloop is incompatible at this position. The GUAAY pentaloop-receptor motif is also found in the structure of the eukaryotic thiamine pyrophosphate riboswitch in the context of a hexanucleotide loop sequence. We therefore propose, based on phylogenetic, structural, and biochemical data, that the GUAAY pentaloop-receptor interaction represents a novel structural motif that is present in multiple structured RNAs.

Keywords: RNA structure; ribozyme.

Figure 1. DV acts as a receptor to two tertiary interactions

A) Secondary structure shows the three tertiary interactions involving DV. μ and κ insert five adenosines into the minor groove of DV. B) The tertiary structure of the minor groove of DV. The five adenosines form an extended base stack, which is perpendicular to the direction of the minor groove. The μ-μ′ interaction positions three nucleotides into the minor groove of DV. The base planes of the μ loop are approximately perpendicular to that of the receptor helix.

Figure 2. Comparison of the GNRA tetraloop versus the GUAAY pentaloop

A) The GNRA tetraloop (left) and the GUAAY pentaloop (right) are connected to a three base-pair helix and aligned to the bottom of the stem. Central panel shows the alignment of the two loops. This alignment shows that the the GUAAY pentaloop extends approximately one base pair higher and at a different orientation compared to the GNRA tetraloop. Nucleotides of the GNRA tetraloop and GUAAY pentaloop that interact with their cognate receptor are colored in orange and green, respectively. B) The GNRA tetraloop (left) nucleotides insert parallel to the base planes of the receptor, while the GUAAY pentaloop (right) nucleotides are nearly perpendicular to the receptor base planes. This allows a greater number of nucleotides to insert into the minor groove. The alignment of the two structures (central panel) shows a ~70° deviation in terms of the angle of attack of the loop for the receptor. C) The structure of the equivalent GUAAY-containing hexanucleotide loop-receptor (left) from the thiamine pyrophosphate riboswitch (PDB 2CKY). The alignment with the GUAAY pentaloop-receptor from the group IIB intron (right) shows an almost identical mode of recognition between the two components of the interaction.

Figure 3. Secondary structure of P.li.LSUI2 D3a mutants

Mutants were grouped into four categories: a) loop point mutants, b) loop mutants, c) helical length mutants, and d) helical length/tetraloop mutants. Red letters indicate changes to the wild-type sequence. Red lines indicate where helical deletions took place. Values below the mutants correspond to Figure 4 for in vitro splicing assays at 2.5mM (blue) and 10mM (orange) MgCl₂.

Figure 4. In vitro splicing assay of P.li.LSUI2 D3a mutants

a) Point mutants made either in the closing base pair of the stem or in the pentaloop. The single point mutants retained activity comprable to wild-type. However, mutating the loop entirely to adenosines resulted in a drastic reduction in activity. b) Mutagenesis of the loop to any of the tetraloop sequences results in severe defects in splicing activity. c) Changing the helical length from the native seven base-pairs perturbs activity. d) The defects caused by the GAAA-tetraloop cannot be rescued with different helical length mutations. Thirty minute time points taken for all splicing assays. *<0.05 by two-tailed Student’s t-test. **< 0.001 by two-tailed Student’s t-test.