INTRODUCTION
Rnt1p is a member of the RNAse III family of double-stranded RNA endonucleases. It is a key component of the S. cerevisiae RNA-processing machinery. Most RNAses III cleave dsRNA in a non-specific way. The Rnt1p double-stranded RNA binding domain (dsRBD) is believed to target its RNA substrate by recognizing an AGNN tetraloop RNA fold, since the AGNN tetraloop is a strong determinant for binding and cleavage.

dsRBDs are involved in many biological processes and are able to recognize specific double-stranded RNAs. This site illustrates the AGNN tetraloop, the Rnt1p dsRBD as well as the protein-RNA complex.

THE AGNN TETRALOOP
The double stranded RNA substrates of RNase III (Rnt1p) are capped by tetraloops that have a consensus sequence of AGNN. This tetraloop region acts as the primary docking site for RNase. This consensus sequence is located 13-16 base pairs away from the cleavage site.

Click to show the AGNN tetraloop. This button will zoom in on the AGNN (here it is AGAA) tetraloop region. You can see the AGNN tetraloop region in the bend of the RNA helix.

The AGNN tetraloop acts as an independently folding unit, and it has been shown that when a short hairpin is capped with this sequence, it coaxially stacks against a long helix where the cleavage occurs.

In the AGNN tetraloop, the G is universally conserved, the A is highly conserved, and no C is ever found in the tetraloop. The universally conserved G is very important, and substituting the Rp non-bridging phosphate O at 3 side with a phosphorothioate dramatically reduces the binding affinity of the dsRBD.

The universally conserved G is found in a syn glycosidic torsion angle conformation immediately before the backbone turns.

This causes the bases of the tetraloop to stack. In the AGAA tetraloop, A15 and G16 are stacked over C14, and A17 and A18 are stacked over G19 in the stem.

The tetraloop is stabilized by hydrogen bonds between A18 amino and A15 N3 (forming a sheared A-A base pair), and G16 amino proton with one of the 5 O of its phosphate group.

The best substrate for Rnt1p is AGAA (with 90% cleavage after one minute) followed by UGAA and AGCA (75%), mutants with an ACAA hairpin are cleaved less than 10% after one minute. When C is in place of G, it is not in the syn position, causing the backbone to turn after the 1st nucleotide instead of the 2nd. This indicates that the backbone turn structure is recognized by the RNase III.

THE dsRBD

There are two types of dsRBDs, both of which are approximately 70 amino acids long. Type A have a strong homology along the entire length, while Type B are highly conserved only at their C-termini and bind double-stranded RNA poorly compared to Type A. Both types show a common ±1¾1¾2¾3±2 structure where the ± helices lie on the face of the antiparallel ¾ sheet. This protein dsRBD has 3 alpha helical regions, (one more than usual) and an antiparallel beta sheet made up of 3 beta strands. The ± helices are colored pink, and the ¾ sheet is yellow. The alpha helices are amphipathic. Click to stop the rotation.

You can see helix ±1, helix ±2, and helix ±3 as well as ¾ strand 1 , ¾2 , and ¾3.

Most dsRBDs lack helix ±3. In this molecule, the hydrophobic face of helix ±3 interact with the C terminus of helix ±1 and the ±1-¾1 loop.This orientation is thought to stabilize helix ±1. Click to show where they interact.

THE COMPLEX

The Rnt1p dsRBD here is complexed with the 5' terminal hairpin of one of its small nucleolar RNA substrates. The RNA/protein complex is the first example of a nonspecific double stranded RNA that can bind specifically to RNAs. The presence of the AGNN sequence is a strong determinant for binding and cleavage. Although the A is highly conserved and the G is universally conserved, it appears that the dsRBD does not recognize these bases specifically, but instead recognizes the shape. It is likely that the Rnt1p dsRBD recognizes and binds to the dsRNA substrate at the tetraloop nonspecifically, positioning the nuclease domain at the cleavage site. Click to display amino acids contacting the successive minor, major, and minor grooves. The RNA is shown in white, helix ±1 is cyan, N-terminal end of Helix ±2 and the ¾3-±2 loop are yellow, and the ¾1-¾2 is green.

The Rnt1p dsRBD binds on one face of the RNA over almost its entire length, and interacts primarily with the sugar-phosphate backbone in three regions. The residue side chains insert into successive minor and major grooves. This button will highlight Helix 1 in cyan. You can see that helix ±1 lies in the minor groove of the RNA.

The N-terminal end of Helix ±2 and the ¾3-±2 loop contacts the sugar phosphate backbone of the major groove. Click to color the N-terminal end of the ±2 helix and the ¾3-±2 loop yellow.
N419 backbone CO contacts residues U5, A6 through water-mediated H-bonds, while the sidechain NH forms a direct H-bond with U5 phosphate's non-bridging O. K421 H-bonds directly with G23 phosphate group and I425 contacts A21 via van der Waals interactions.

The ¾1-¾2 loop contact the minor groove 10-13bp away from the tetraloop. Click to illustrate this interaction.

You can see that the spacing between the ¾1-¾2 loop and the N-terminal end of helix ±1 is ideal for interacting with the successive minor grooves for an RNA A form helix.

Three peptide backbone groups interact with 3 ribose groups and one base. P393 CO, T394 NH, A395 CO interact with U5 2'OH, C30 2'OH and U31 O4' as well as C30 O2 via direct and water mediated H-bonds.

In addition, A395, V396 and P398 contact U29, C30 and U5 respectively via van der Waals interactions.
The Rnt1p dsRBD for AGNN hairpins binds with 5-fold greater affinity compared to a GUGA hairpin. This is likely due to the increased number of interactions between amino acid residues (6 instead of 2) with nucleotides (7 instead of three).

DISCUSSION
Rnt1p is a endonuclease which binds and cleaves double-stranded RNA with a dsRBD that specifically recognizes the AGNN tetraloop. It specifically recognizes this RNA fold due to structure specific interactions. Helix ±1 recognizes the RNA by fitting into the minor groove side and interacting with the non-conserved bases. The other helices and sheets interact with the RNA as well and also function to stabilize the interaction with helix ±1.

REFERENCES
Haihong Wu, Anthony Henras, Guillaume Chanfreau, and Juli Feigon. Structural basis for recognition of the AGNN tetraloop RNA fold by the double-stranded RNA-binding domain of Rnt1p RNase III. 2004 8307-8312

Haihong Wu, Pok K. Yang, Samuel E. Butcher, Sundeep Kang, Guillaume Chanfreau and Juli Feigon. A novel family of RNA tetraloop structure forms the recognition site for Saccharomyces cerevisiae RNase III. 2001 7240-7249

Doyle M. and Jantsch M.F. (2002) J. Structural Biol. 140, 147-153.