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

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.

a Domain architecture of human and mouse SLFN proteins, as well as rat SLFN13, which are classified as three subgroups. SLFNL1: Schlafen-like protein 1. The predicted nuclear localization signal (NLS) for mouse SLFNs 1 is indicated by a white vertical bar.
b Schematic representation showing the composition of rSLFN13 14-353 , rSLFN13 1-353 and hSLFN13  that were used in this study. E1, E2 and E3 denote the sequences of three N'-terminal extensions to corresponding SLFN constructs that are encoded by vector residues. Residues that are observable in the rSLFN13 14-353 structure are indicated by an underscore. c Superposition of the N-lobe and C-lobe of rSLFN13  showing the consistent folding of the two subdomains. d X-ray fluorescence scan over the rSLFN13 14-353 crystal. Emission of characteristic fluorescent X-ray at Zn-K edge confirmed the zinc incorporation in rSLFN13  .  Subgroup Ⅲ 1 Supplementary Figure 2: Dimerization of rSLFN13 14-353 .
a Dimerization properties of different rSLFN13-N constructs were assayed in analytical gel filtration coupled to RALS. Calculated molecular mass of each sample at the absorption peak of 280 nm are plotted in red. mAU, milli-absorption units. b The putative dimeric interface of rSLFN13 14-353 in the crystal structure. The two monomers are coloured teal and wheat, and their vector-coded N'-terminal extensions are specified in green and yellow, respectively. The cleavage sites of the PreScission protease are indicated by spheres. Residues involved in the dimerization interface are shown as ball-and-stick models. His 6 -tagged rSLFN13  Untagged rSLFN13  Retention volume ( a-c Cleavage assays for rSLFN13-N and hSLFN13-N on different types of in vitro transcribed tRNAs in the presence of Mg 2+ . tRNA substrates are: human tRNA His , tRNA Sec , mitochondrial tRNA mt-Met (tRNA His , tRNA Sec , tRNA mt-Met ), tRNA Sec denotes the transfer tRNA for selenium cysteine; Pyrococcus abyssi tRNA Phe (Pa-tRNA Phe ); Saccharomyces cerevisiae tRNA Gly , tRNA His , tRNA Ser , tRNA Ala (Sc-tRNA Gly , Sc-tRNA His , Sc-tRNA Ser , Sc-tRNA Ala ); Bacillus paralicheniformis tRNA Leu (Bp-tRNA Leu ); Mycobacterium tuberculosis tRNA Tyr , tRNA Lys , tRNA Gln (Mt-tRNA Tyr , Mt-tRNA Lys , Mt-tRNA Gln ); Thermus thermophiles tRNA Leu (Tt-tRNA Leu ); Escherichia coli tRNA Glu (Ec-tRNA Glu ); rabbit tRNA Ser (rtRNA Ser ). and Dicer (2FFL), are shown as ribbon-typed models. For rSLFN13-N, EndA, Drosha and Dicer, the dimeric or pseudodimeric regions that possess the nucleolytic activity are coloured salmon and olive, and the rest portions of Drosha and Dicer are coloured grey. Colicin E5, PaT and angiogenin are coloured salmon.
b Top 10 hits of the existing structures homologous to rSLFN13-N, output from the Dali server.
c Structural superposition of rSLFN13-N C-lobe and the DNase I-like subdomain of RNase E (2BX2). Cɑ atoms of 78 residues are aligned with a rmsd of 3.14 Å. Colour as in Fig. 3a. d Comparison of the structural topology between rSLFN13-N C-lobe and the DNase I-like subdomain of RNase E.
e The structure of RNase E catalytic domain tetramer in complex with RNA (2VRT). A dimerized DNase I-like subdomain pair is coloured either salmon or olive for each protomer. One chain is coloured light orange, and other portions of the tetramer are coloured grey. Residues with a conservation of 100% are in red shades, greater than 80% in green shades and 60% in grey shades, respectively. α-helices are shown as cylinders and β-strands as arrows for rSLFN13 above the sequences. The secondary structure signs are coloured and labelled as in Fig. 1c. Regions not resolved in the crystal structure are indicated by dashed lines. Important residues that are discussed in the paper are specified by different symbols according to their structural and functional roles. mSLFN8 was predicted to carry an NLS 1 . The sequences of the selected SLFNs are aligned at the NLS of mSLFN8 which is indicated by a black box. Sequences at this region are not very conserved, and no apparent NLS is found for rSLFN13 or hSLFN13.