Home
This Title All WIREs
WIREs RSS Feed
How to cite this WIREs title:
WIREs RNA
Impact Factor: 4.928

Structure and function of Rnt1p: An alternative to RNAi for targeted RNA degradation

Full article on Wiley Online Library:   HTML PDF

Can't access this content? Tell your librarian.

The double‐stranded RNA‐binding protein (dsRBP) family controls RNA editing, stability, and function in all eukaryotes. The central feature of this family is the recognition of a generic RNA duplex using highly conserved double‐stranded RNA‐binding domain (dsRBD) that recognizes the characteristic distance between the minor grooves created by the RNA helix. Variations on this theme that confer species and functional specificities have been reported but most dsRBPs retain their capacity to bind generic dsRNA. The ribonuclease III (RNase III) family members fall into four classes, represented by bacterial RNase III, yeast Rnt1p, human Drosha, and human Dicer, respectively. Like all dsRBPs and most members of the RNase III family, Rnt1p has a dsRBD, but unlike most of its kin, it poorly binds to generic RNA helices. Instead, Rnt1p, the only known RNase III expressed in Saccharomyces cerevisiae that lacks the RNAi (RNA interference) machinery, recognizes a specific class of stem‐loop structures. To recognize the specific substrates, the dsRBD of Rnt1p is specialized, featuring a αβββααα topology and a sequence‐specific RNA‐binding motif at the C‐terminus. Since the discovery of Rnt1p in 1996, significant progress has been made in studies of its genetics, function, structure, and mechanism of action, explaining the reasons and mechanisms for the increased specificity of this enzyme and its impact on the mechanism of RNA degradation. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition RNA Processing > Processing of Small RNAs RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes
Double‐stranded RNA‐binding domains (dsRBDs) of ribonuclease III (RNase III) enzymes in the dsRNA‐binding protein family. (a) Structure‐based sequence alignment of bacterial RNase III (PDB entry 2EZ6, residues 150–220), yeast Rnt1p (PDB entry 4OOG, residues 368–457), human Dicer (PDB entry 5ZAK, residues 1851–1913), and human Drosha (PDB entry 5B16, residues 1259–1333). Secondary structural elements are indicated, but the β1 strand in human Dicer is not well‐defined. Also indicated are RNA‐binding motifs (RBMs). Conserved amino acid residues are boxed, while nucleotide base‐recognizing residues (Q157 and Q161 in RNase III; R445, I448, and S453 in Rnt1p) are indicated with red triangles. (b) Superimposition of the four dsRBDs, RNase III in cyan, Rnt1p in green, Dicer in yellow, and Drosha in pink. (c, d) In bacterial RNase III, RBM1 residues T154, Q157, E158, and Q161 form hydrogen bonds to 2′‐hydroxyl groups in the minor groove. In addition, Q157 and Q161 form base‐specific hydrogen bonds (highlighted in red)
[ Normal View | Magnified View ]
Model for the substrate‐loaded complex of Rnt1p showing a stem‐loop RNA capped with an NGNN tetraloop (G2‐loop) properly aligned in the catalytic valley of the Rnt1p dimer. The model was built based on the crystal structure of the substrate‐loaded complex of Rnt1p, which contains a nonhydrolysable substrate RNA (Figure b; PDB entry 5T16), in four steps including: (1) the tetraloop that contacts double‐stranded RNA‐binding domain 2 (dsRBD2) was removed; (2) the stem of the remaining stem‐loop RNA was extended; (3) the stem‐loop RNA (left) and the RNA duplex (right) were connected, and (4) each cleavage site was indicated with one of the two Mg2+ ions seen in the post‐cleavage complex (Figure c; PDB entry 4OOG). This model shows distinct RNA‐binding modes of the two dsRBDs. dsRBD1 recognizes the Gua nucleotide in second position of the tetraloop of substrate RNA with RNA‐binding motif 0 (RBM0), and also binds to the RNA stem with RBM1 and RBM2. In contrast, dsRBD2 only binds to the RNA stem with RBM1 and RBM2, where RBM0 does not interfere with binding. The specialized RNase III catalytic domain (RIIID) dimer (in cyan and orange) and the N‐terminal domain (NTD) dimer (in pink and light blue) are illustrated as molecular surfaces. The dsRBDs (in cyan and orange) are shown as ribbon diagrams (helices as spirals, strands as arrows, and loops as tubes). The RNA substrate is represented with a tube‐and‐stick model and outlined with a transparent molecular surface. The RBMs in dsRBD1 are colored in blue and those in dsRBD2 are in red
[ Normal View | Magnified View ]
Cleavage site assemblies of prokaryotic and eukaryotic ribonuclease III (RNase III) enzymes. (a) Structure‐based sequence alignment of human Dicer (PDB entry 5ZAL), human Drosha (PDB entry 5B16), yeast Rnt1p (PDB entry 4OOG), and bacterial RNase III, (PDB entry 2NUG). The conserved amino acid residues in the cleavage site are highlighted in red. Residues N5 and K6 are unique for eukaryotic enzymes. (b) Based on cleavage site arrangements as revealed by the crystal structures of post‐cleavage complex of RNase III (PDB entry 2NUG, upper‐right panel) and that of Rnt1p (PDB entry 4OOG, lower‐right panel), models for the reaction intermediates (the two panels in the middle) and pre‐cleavage complexes (the two panels on the left) are readily derived. Both the protein and RNA are illustrated as stick models in atomic color scheme (nitrogen in blue, carbon in gray, oxygen in red, phosphorus in orange, and magnesium in black). Water molecules are shown as spheres. Hydrogen bonds are indicated with dashed lines, while coordination bonds with solid lines. (Some components of this figure were adapted from (Gan et al., 2008; Song et al., 2017))
[ Normal View | Magnified View ]
The functional cycle of Rnt1p. (a) The apo‐Rnt1p structure by small‐angle X‐ray scattering (SAXS) shows that the ligand‐free protein adopts an open conformation in solution. (b) Model of the substrate‐selected complex, in which one of the two double‐stranded RNA‐binding domains (dsRBDs) of Rnt1p recognizes a stem‐loop RNA capped with an NGNN tetraloop (G2‐loop). (c) The crystal structure of the substrate‐loaded complex (PDB entry 5T16) shows that the RNA substrate is properly loaded into the catalytic valley where the stem is held in place and the N‐terminal domain dimer moves in to contact the tetraloop. (d) Model of the pre‐cleavage complex, illustrating that the active cleavage assembly is formed in the presence of two Mg2+ ions at each cleavage site. (e) The crystal structure of the post‐cleavage complex (PDB entry 4OOG), showing the architectural arrangement of the cleavage assembly immediately after the hydrolysis of the two phosphodiester bonds. (This figure was originally published in (Song et al., 2017))
[ Normal View | Magnified View ]
Distinct modes of double‐stranded RNA recognition by the dsRNA‐binding domains (dsRBDs) of Rnt1p (a) and RNase III (b). The specialized RNase III catalytic domains (RIIIDs) are illustrated as molecular surfaces, dsRBDs as cartoons, and RNAs as tube‐and‐stick models. Protein domains are color‐coded. The span of the two dsRBDs along dsRNA is highlighted with a double‐headed arrow. (c) Superposition of the RIIID2:MgRNA structures of Rnt1p (in cyan and blue, PDB entry 4OOG) and RNase III (in orange and red, PDB entry 2NUG) demonstrates the same RIIID2:MgRNA architecture. (A slightly different version of panels a and b was previously published in (Liang et al., 2014))
[ Normal View | Magnified View ]
The double‐ruler mechanism of Rnt1p. (a) Gua16, the nucleotide residue in the second position of the terminal tetraloop of G2‐loop RNA, is specifically recognized by Rnt1p. The Gua16 base is sandwiched between the side chains of R445 and R450 of double‐stranded RNA‐binding domain 1 (dsRBD1). Residue Y46 of N‐terminal domain 1 (NTD1) and residues I448, S453, and R445 of dsRBD1 form a total of four specific hydrogen bonds with the Gua16 base. (b) The cleavage sites are defined by two rulers. Ruler 1, the specialized RNase III catalytic domain 1 (RIIID1) in conjunction with dsRBD1, measures 16 nucleotides from the tetraloop. Ruler 2, the RIIID2 in conjunction with the N‐terminal domain dimer (NTD1/NTD2), measures 14 nucleotides from the tetraloop. As such, the double‐ruler architecture ensures the cleavage accuracy. The protein is illustrated as a molecular surface. The RNA is shown as a cartoon. Gua16 is highlighted as either a stick (in panel a) or a ball‐and‐stick (in panel b) model. (Panel b was originally published in (Liang et al., 2014))
[ Normal View | Magnified View ]
Three‐dimensional structures of yeast Rnt1p. (a) Solution structure of apo‐Rnt1p by small‐angle X‐ray scattering (SAXS) exhibits the open conformation of the protein (Song et al., ). (b) Crystal structure of Rnt1p in complex with a nonhydrolysable substrate analog (PDB entry 5T16), exhibiting the closed conformation, represents the substrate‐loaded complex of the protein. (c) Crystal structure of Rnt1p in complex with the cleavage product of a G2‐loop (PDB entry 4OOG), representing the post‐cleavage complex, is overlapped with the SAXS envelop of the same complex in solution (Song et al., ). The structures are illustrated as ribbon diagrams (helices as spirals, strands as arrows, loops as tubes, RNAs as tube‐and‐stick models, and Mg2+ ions as spheres). The protein domains are color coded. The largest dimension of the structures is indicated
[ Normal View | Magnified View ]
Three‐dimensional structures of double‐stranded RNA‐binding domain (dsRBD) in complex with RNA reveal distinct fashions of substrate recognition by dsRBD in the absence or presence of other domains of Rnt1p. (a) NMR structure of isolated dsRBD in complex with the AGAA tetraloop (PDB entry 1T4L) exhibits the normal mode of dsRNA recognition by dsRBDs via RNA‐binding motifs 1 and 2 (RBM1 and RBM2). (b) NMR structure of isolated dsRBD in complex with the AAGU tetraloop (PDB entry 2LBS) also exhibits the normal mode of dsRNA recognition. (c) Crystal structure of Rnt1p in complex with the AGUC tetraloop reveals the unique, productive mode of dsRNA recognition, featuring sequence‐specific recognition of NGNN tetraloops by Rnt1p via RBM0, a new RBM near the C‐terminus of dsRBD (PDB entry 4OOG). Other domains of the 4OOG structure, not shown here for clarity, will be illustrated later in this review. The protein is illustrated as a cartoon model, the RNA as a transparent molecular surface, and the nucleotide residue in the second position of the tetraloop as a ball‐and‐stick model
[ Normal View | Magnified View ]
Representative members of the RNase III family. (a) Domain structures of bacterial RNase III, yeast Rnt1p, human Drosha, and human Dicer. Whereas RNase III and Rnt1p contain a specialized RNase III catalytic domain (RIIID) that is followed by a double‐stranded RNA‐binding domain (dsRBD), Drosha and Dicer contain two RIIIDs followed by a dsRBD. Every eukaryotic RNase III has an N‐terminal extension beyond RIIID(s). The N‐terminal extension of Rnt1p consists of a single N‐terminal domain (NTD), whereas those of Drosha and Dicer consist of multiple domains including: Connector, PAZ, Platform, RS‐rich, and P‐rich domains in Drosha; and Connector, PAZ, Platform, DUF283, and DExD/H helicase domains in dicer. (b) Crystal structure of RNase III in complex with cleavage products of stem‐loop RNA (PDB entry 2EZ6). (c) Crystal structure of RNase III in complex with cleavage products of long dsRNA (PDB entry 2NUG), showing the typical length of small RNA product. (d) Crystal structure of RNase III in complex with cleavage products of dsRNA (PDB entry 2NUG), explaining the typical 22‐nucleotide length of small interfering RNA (siRNA) product of the inside‐out cleavage. (e) Crystal structure of Rnt1p in complex with cleavage products governed by the double‐ruler mechanism (PDB entry 4OOG). (f) Crystal structure of RNA‐free human Drosha from Platform to dsRBD (PDB entry 5B16). (g) Cryo‐EM structure of human Dicer in complex with a pre‐let‐7 substrate (PDB entry 5ZAL), in which the RNA is not bound by dsRBD and the distance between the RNA and the cleavage sites is ~40 Å
[ Normal View | Magnified View ]

Browse by Topic

RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms
RNA Processing > Processing of Small RNAs
RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes
RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition

Access to this WIREs title is by subscription only.

Recommend to Your
Librarian Now!

The latest WIREs articles in your inbox

Sign Up for Article Alerts