Structure of Schlafen13 reveals a new class of tRNA/rRNA- targeting RNase engaged in translational control

Abstract

Cleavage of transfer (t)RNA and ribosomal (r)RNA are critical and conserved steps of translational control for cells to overcome varied environmental stresses. However, enzymes that are responsible for this event have not been fully identified in high eukaryotes. Here, we report a mammalian tRNA/rRNA-targeting endoribonuclease: SLFN13, a member of the Schlafen family. Structural study reveals a unique pseudo-dimeric U-pillow-shaped architecture of the SLFN13 N'-domain that may clamp base-paired RNAs. SLFN13 is able to digest tRNAs and rRNAs in vitro, and the endonucleolytic cleavage dissevers 11 nucleotides from the 3'-terminus of tRNA at the acceptor stem. The cytoplasmically localised SLFN13 inhibits protein synthesis in 293T cells. Moreover, SLFN13 restricts HIV replication in a nucleolytic activity-dependent manner. According to these observations, we term SLFN13 RNase S13. Our study provides insights into the modulation of translational machinery in high eukaryotes, and sheds light on the functional mechanisms of the Schlafen family.

Fig. 1

Structure of rSLFN13 N′-domain. a Schematic representation showing the organisation of crystallised rSLFN13_14–353 based on full-length rSLFN13. NE denotes the N-terminal extension coded by the DNA sequence from the vector; BD denotes bridging domain. Elements for rSLFN13_14–353 are assigned according to the structure. Borders of each element are indicated by residue numbers. b Structure of rSLFN13_14–353. Domains of rSLFN13_14–353 are indicated and coloured as in a. Disordered loops are shown as dashed lines. The Zn²⁺ ion in the zinc finger is shown as a grey sphere. α-helices and β-strands of each domain are specified. The size of the valley is indicated. c The topology diagram of rSLFN13_14–353. Secondary structural elements were not drawn to scale. Elements of rSLFN13_14–353 are named and coloured as in a. A dashed line is drawn to indicate the pseudo-symmetry of rSLFN13_14–353. d Interactions between BD and N-lobe. Side chains of involved residues are shown in the same colour as the domains they belong to. e Interactions between BD and C-lobe. Note the similarity with d. f Extra interactions between BD and C-lobe. Note the CCCH-type zinc finger

Fig. 2

Endoribonuclease activity of SLFN13-N. a Dose-dependent and time-dependent tRNA digestion by rSLFN13-N in the presence of Mg²⁺. rSLFN13_1–353 of indicated concentrations were incubated with [α-³²P]-labelled human tRNA^Gly, human tRNA^Ser or rat tRNA^Ser (rtRNA^Ser) for 5, 15 and 30 min. b rSLFN13-N preferably cleaves tRNA. ssDNA/ssRNA single-stranded DNA/RNA, dsDNA/dsRNA double-stranded DNA/RNA. c Analysis of divalent cations as cofactor of rSLFN13-N for tRNA digestion. 500 nM rSLFN13_1–353 was incubated with [α-³²P]-labelled tRNA^Ser for 30 min. Four different divalent cations were individually tested. d Cleavage assay for different SLFN-N proteins. 250 nM rSLFN13_1–353, mSLFN8_1–359, hSLFN13_1–355 or hSLFN5_12–334 was individually incubated with [α-³²P]-labelled tRNA^Ser. Note that hSLFN13-N shows highest nucleolytic efficiency over human tRNA^Ser. e Cleavage assay for rSLFN13-N on acceptor stem variants. Modifications of the variants are specified. 500 nM rSLFN13_1–353 was used in each reaction. f tRNA cleavage pattern of SLFN13-N. The primary cleavage products from tRNA^Ser by rSLFN13_1–353 and hSLFN13_1–355 were compared with in vitro transcribed tRNA^Ser that lacks 3′-terminal 10 nt (tRNA^Ser−3′-Δ10 nt) or 11 nt (tRNA^Ser−3′-Δ11 nt), and portrayed to the left. The migration of two truncated tRNA^Ser markers is indicated by dashes lines. g Proposed primary cleavage site of SLFN13-N. The sequence of human tRNA^Ser is used here as representative

Fig. 3

Putative catalytic site and cleavage model of SLFN13-N. a Structural comparison between the C-lobe of rSLFN13_14–353 and the DNase I subdomain of RNase E (PDB ID 2BX2). For RNase E, the portion that is structurally aligned to rSLFN13_14–353 C-lobe is shown in blue, and the rest part in light blue. b The electrostatic surface potentials of rSLFN13_14–353, coloured from red (negative) to blue (positive). Two negatively charged patches and the conserved positively charged area are indicated. Positions of the residues tested in c are specified. c tRNA cleavage assay for rSLFN13_1–353 mutants of conserved charged residues in the presence of Mg²⁺. For each sample, 500 nM protein was used. d Comparison between the putative nucleolytic active site of rSLFN13-N and the actives sites of RNase E (2BX2) and RNase III (2EZ6). Note the similar tripod architecture of the three residues for each protein, which are shown as ball-and-stick models. e Cleavage assay of hSLFN13_1–355 mutants at the putative active site. E208A, E213A and D251A of hSLFN13-N correspond to E205, E210 and D248 of rSLFN13-N, respectively. f Schematic drawing of SLFN13 cleaving a tRNA. The U-pillow-shaped SLFN13 N′-domain embraces the acceptor stem of the tRNA. The 3′ tail that is to be cleaved off the tRNA by SLFN13-N is coloured purple. g Structural model of SLFN13-N manipulating tRNA. rSLFN13_14–353 is shown as surface representations and the subdomains are colour-specified. The putative active site is coloured red. The coordinate of tRNA^Gly (5E6M, excerpted) is used and coloured orange. SLFN13-N clamps the acceptor stem of tRNA and may have no contact with other parts of the tRNA. h Close view of SLFN13-N accommodating the acceptor stem. The surface positively charged residues of rSLFN13_14–353 tested in c are coloured blue. Part of the tRNA is removed for clarity. The 5′ and 3′ ends of the tRNA are indicated. The phosphate atom of the 66th nucleotide of tRNA is shown as a sphere

Fig. 4

SLFN13-N cleaves native tRNA and rRNA. a, b Cleavage assay of hSLFN13-N (a) and rSLFN13-N (b) on native tRNAs in the presence of Mg²⁺. Small RNA-293T denotes total small RNA extracted from 293T cells that is mainly constituted of tRNAs. tRNA^Ser and tRNA^Gly were used as controls to indicate molecular weight. c Cleavage assay of different SLFN-Ns on native tRNAs. d–f Cleavage assay on specific native tRNAs. The substrates and products were identified by Northern blot using [γ-³²P]-labelled probes targeting tRNA^Ser (d), tRNA^Gly (e) or tRNA^Lys (f). g Cleavage assay for hSLFN13-N on 5S rRNA. 5S rRNA and products were identified by Northern blot. h Cleavage assay on native rRNAs. 4 μg total RNA extracted from 293T cells was used for each reaction, which mainly contains rRNAs

Fig. 5

hSLFN13 disrupts translational machinery. a Subcellular localisation of hSLFN13. b hSLFN13 restricts tRNAs and rRNAs in cells. The small RNA pool is mainly constituted of tRNAs and 5S/5.8S rRNA (See Supplementary Fig. 8f). Expression of hSLFN13 WT and hSLFN13(E213A) was confirmed by Western blot. Note the prominent restriction of cellular RNAs caused by relatively low level of overexpressed hSLFN13. Error bar indicates s.d. (n = 3). c The experimental process of the FUNCAT-click chemistry assay. d FUNCAT assay and subsequent flow cytometry examination for Cy5 fluorescence showing that hSLFN13 inhibits protein synthesis in living cells. e hSLFN13 inhibits protein synthesis. Upper: fluorescent image of the SDS-PAGE gel showing the newly synthesised proteins labelled by FUNCAT assay. Lower: same gel stained with Coomassie Blue showing the loading consistency

Fig. 6

hSLFN13 restricts HIV production. a The experimental process of titre assay for VSV-G pseudotyped HIV (HIV^VSV-G). b Viral production was assayed by titrated infection and the GFP level. Expression of hSLFN13 WT and mutants was confirmed by Western blot. Error bar indicates s.d. (n = 3). c Knock-down of endogenous SLFN13 increased viral titre of 293 cells. d Viral particle content in supernatants was analysed by p24 ELISA. e, f Extracellular vRNA concentration was determined by qPCR for total vRNA (e) and unspliced vRNA (f). g hSLFN13 suppressed GFP production from the viral plasmids. h The experimental process of the viral infection assay. i Examination for the infection efficiency of HIV^VSV-G in cells pre-transfected with hSLFN13 WT or mutants. j Proposed protein synthesis inhibition and anti-HIV mechanism of hSLFN13