Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay

Abstract

Tudor staphylococcal nuclease (TSN) is an evolutionarily conserved ribonuclease in eukaryotes that is composed of five staphylococcal nuclease-like domains (SN1-SN5) and a Tudor domain. TSN degrades hyper-edited double-stranded RNA, including primary miRNA precursors containing multiple I•U and U•I pairs, and mature miRNA during miRNA decay. However, how TSN binds and degrades its RNA substrates remains unclear. Here, we show that the C. elegans TSN (cTSN) is a monomeric Ca²⁺-dependent ribonuclease, cleaving RNA chains at the 5'-side of the phosphodiester linkage to produce degraded fragments with 5'-hydroxyl and 3'-phosphate ends. cTSN degrades single-stranded RNA and double-stranded RNA containing mismatched base pairs, but is not restricted to those containing multiple I•U and U•I pairs. cTSN has at least two catalytic active sites located in the SN1 and SN3 domains, since mutations of the putative Ca²⁺-binding residues in these two domains strongly impaired its ribonuclease activity. We further show by small-angle X-ray scattering that rice osTSN has a flexible two-lobed structure with open to closed conformations, indicating that TSN may change its conformation upon RNA binding. We conclude that TSN is a structure-specific ribonuclease targeting not only single-stranded RNA, but also unstructured regions of double-stranded RNA. This study provides the molecular basis for how TSN cooperates with RNA editing to eliminate duplex RNA in cell defense, and how TSN selects and degrades RNA during microRNA decay.

Keywords: RNA editing; RNA silencing; TSN; microRNA decay; ribonuclease.

FIGURE 1.

Full-length TSN is a monomeric protein. (A) The domain structure of Caenorhabditis elegans cTSN. The two putative Ca²⁺-binding residues in the SN1 and SN3 domains are shown at the top. (B) The SDS–PAGE of the purified recombinant cTSN from Caenorhabditis elegans, hTSN from Homo sapiens, and osTSN from Oryza sativa. (C) The molecular weights of cTSN, osTSN, and hTSN were estimated by ultracentrifugation. (D) TSN proteins were subjected to SEC–MALS (size-exclusion chromatography coupled with multiangle light scattering) and are represented as elution profiles.

FIGURE 2.

cTSN is a Ca²⁺-dependent endonuclease cleaving at the 5′-side of phosphodiester bonds. (A) cTSN (100 nM) degraded the 5′-fluorescein-labeled pre-miR142 RNA (500 nM) in the presence of Ca²⁺ at concentrations of 0.1–1 mM. The sizes of pre-miR142 (68 nt) and an RNA marker (28 nt) were labeled in the left of the gel. (B) cTSN cleaved at the 5′-side of phosphodiester bonds to produce degraded fragments with 3′-phosphate and 5′-OH ends that could be labeled by T4 polynucleotide kinase (T4 PNK) but not by T4 RNA ligase (T4 ligase).

FIGURE 3.

cTSN preferentially binds double-stranded RNA containing I•U and U•I pairs. cTSN (0–2.0 µM) was incubated with 5′-P³²-labeled RNA for filter-binding assays. Binding percentages from three measurements were calculated to derive the apparent K_d values between cTSN and RNA by a one-site binding curve, with R² (all greater than 0.95) shown in parentheses.

FIGURE 4.

cTSN degrades single-stranded RNA and mismatched double-stranded RNA with or without inosines. (A) cTSN (100 nM) degraded stem–loop 5′-fluorescein-labeled RNAs (500 nM) containing mispaired or loop regions (the percentages of the remained substrates are listed at the bottom of the gel). (B) cTSN (100 nM) degraded single-stranded RNA (500 nM) with or without inosines, as well as double-stranded RNA containing mispaired base pairs, including AAUC/CGCC and IIUI/UIUU. In contrast, cTSN could not degrade paired double-stranded RNA with or without inosines. The RNA sequences are listed at the bottom of the gel.

FIGURE 5.

The catalytic residues of cTSN are located in the SN1 and SN3 domains. (A) Sequence alignment of the SN1, SN2, SN3, and SN4 domains of cTSN from Caenorhabditis elegans, osTSN from Oryza sativa, and hTSN from Homo sapiens. The putative metal ion-binding residues (M) are marked in red. (B) With long degradation time (3 h), cTSN cleaved the RNA substrates, pre-miR142 and pre-miR142AtoI, into small RNA fragments of about 10–20 nt. (C) cTSN and its mutants D35A, D354A, and D35A/D354A (100 nM) were incubated with 5′-fluorescein-labeled pri-mR142-AtoI (500 nM). Wild-type cTSN, D35A, and D354A degraded the RNA substrate. The cTSN double mutant D35A/D354A exhibited greatly reduced ribonuclease activity.

FIGURE 6.

Solution SAXS analysis of rice osTSN reveals a flexible two-lobed structure. (A) Small-angle X-ray scattering (SAXS) curves are represented as logarithmic scattering intensities. Theoretical scattering intensities generated from ensemble models are fitted to experimental data with a χ value of 1.94. Right panel is the distance distribution function with a maximum diameter (D_max) of 191 Å and a radius of gyration (R_g) of 52 Å. (B) The R_g-based dimensionless Kratky plot with curve peak at a q × R_g of greater than √3, indicating flexible conformations. (C) Three major ensemble models, with the number of times each conformation existed within the ensemble represented as fractions (%) below each conformation.