Abstract
Velcrin compounds kill cancer cells expressing high levels of phosphodiesterase 3A (PDE3A) and Schlafen family member 12 (SLFN12) by inducing complex formation between these two proteins, but the mechanism of cancer cell killing by the PDE3A–SLFN12 complex is not fully understood. Here, we report that the physiological substrate of SLFN12 RNase is tRNALeu(TAA). SLFN12 selectively digests tRNALeu(TAA), and velcrin treatment promotes the cleavage of tRNALeu(TAA) by inducing PDE3A–SLFN12 complex formation in vitro. We found that distinct sequences in the variable loop and acceptor stem of tRNALeu(TAA) are required for substrate digestion. Velcrin treatment of sensitive cells results in downregulation of tRNALeu(TAA), ribosome pausing at Leu-TTA codons and global inhibition of protein synthesis. Velcrin-induced cleavage of tRNALeu(TAA) by SLFN12 and the concomitant global inhibition of protein synthesis thus define a new mechanism of apoptosis initiation.
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Data availability
Ribosome profiling, RNA-seq and tRNA-seq datasets were deposited to the GEO database (GSE185828). Analyzed ribosome profiling data can be found in Supplementary Data 1–3. Raw data, original images and PDB files are provided in Source data and Supplementary Data 4. The reads from tRNA sequencing were mapped using GtRNAdb (http://gtrnadb.ucsc.edu/). Source data are provided with this paper.
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Acknowledgements
This work is supported by Bayer A.G. (S.L., S.H., X.W., C.G., J.M., A.C., M.M., H.G.), National Cancer Institute grant R35 CA197568 (to M.M.) and a Broad Institute NextGen award (to H.G.). We thank S. Goldoni and H. Faus for helpful discussions.
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Contributions
The project was conceptualized by S.L., X.W., M.M. and H.G. All experiments were designed and performed by S.L. and J.M. S.H. and A.D.C. analyzed the Ribo-seq data. C.G. prepared the purified SLFN12 proteins. M.T. and X.W. made the SLFN12-knockout cell lines. M.S. performed the computational analysis of the SLFN12–tRNA structure. M.G.R. analyzed the correlation between SLFN11 expression and DNMDP response. M.M. provided key input on several experiments. H.G. assisted with experimental design, supervised the experiments and led the project. The manuscript was written by S.L. and H.G. with input from all authors.
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All authors received research funding from Bayer A.G. M.M. also received research funding from Janssen and Ono; consulting fees from Bayer, Interline and Isabl; royalties from LabCorp; patents relevant to abstract presentation; and additional patents not relevant to research funding. In addition, X.W., M.M. and H.G. receive an inventor’s share of license revenue as part of their employment for certain patent filings, including US-2016-0016913 and US-2018-0235961, which relate to aspects of the work described in this manuscript. The co-owners of those patent filings are The Broad Institute, Inc., Dana-Farber Cancer Institute, Inc. and Bayer Pharma A.G.
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Extended data
Extended Data Fig. 1 Down-regulation of tRNA-Leu-TAA upon DNMDP treatment.
(a) The effect of DNMDP on ribosomal RNA integrity. Three velcrin-sensitive cell lines (HeLa, A2058, and SKMEL3) and insensitive SLFN12-depleted HeLa were treated with 3 µM DNMDP for the indicated time. SLFN12-depleted HeLa cells were generated by prolonged incubation with DNMDP2, and serve as a negative control in this experiment. Total RNA purified from these cell lines was analyzed on a 6% urea-polyacrylamide gel with RNA ladders (n = 4 replicates for HeLa and n = 2 for others). (b) Leucine tRNA abundance in a panel of cancer cell lines treated with DMSO or 1 mM DNMDP3. After demethylation of tRNA, cDNA was synthesized and leucine tRNA levels were measured using qPCR. tRNA-Leu-TAA-1 level is plotted relative to tRNA-Leu-TAG-2 as a control (n = 3 replicates per condition). Data are represented as mean ± SEM. P values were calculated by two-tailed unpaired Student’s t test. not significant (ns). Sensitive cell lines are arranged left to right in order of greatest to least DNMDP response3, from HeLa at the top left to CAL51 at the bottom right.
Extended Data Fig. 2 SLFN12 induces down-regulation of tRNA-Leu-TAA.
(a-c) Average intact tRNA-Leu-TAA-1 abundance measured by qPCR (n = 3 replicates) was plotted against DNMDP response3, (a) PDE3A protein expression3, and (c) SLFN12 mRNA levels (b) from the DepMap dataset (https://depmap.org/portal/). P-values were calculated by linear regression model (a-c). (c) The A549 cell line lacking expression of SLFN12 and the UACC257 cell line lacking expression of AIP3 were excluded in Extended Data Fig. 1b. (d) Leucine tRNA abundance in SLFN12 knockout cell lines treated with DMSO or 1 µM DNMDP was measured by qPCR. The level of tRNA-Leu-TAA-1 was plotted relative to control tRNA, tRNA-Leu-TAG-2 (n = 3 replicates per condition). Data are represented as mean ± SEM. P values were calculated by two-tailed unpaired Student’s t test. (e) Western blots of SLFN12 and GAPDH in wild-type and SLFN12 knockout cell lines treated with 1 µM DNMDP for 18 hours. The SLFN12 band is marked with an arrow to distinguish it from the upper nonspecific band (n = 2 replicates).
Extended Data Fig. 3 The effect of SLFN11 expression on DNMDP response.
(a) Correlation between DNMDP response1 and expression of 19217 genes from DepMap dataset (https://depmap.org/portal/) in 674 cell lines. SLFN11, SLFN12 and PDE3A correlation coefficients are indicated with green, red and blue, respectively (center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers). (b) In 14 cancer cell lines, SLFN11 mRNA level was plotted against DNMDP response3. (c) Immunoblots of the SLFN11 and GAPDH in the indicated cancer cell lines (n = 2 replicates). (d) The effects of SLFN11 expression on DNMDP response. Untransduced HeLa cells or HeLa cells stably expressing SLFN11 were treated with the indicated DNMDP concentrations. Viable cells were measured by CellTiter-Glo (n = 2 replicates). The protein expression of SLFN11 and GAPDH was measured by immunoblotting in untransduced HeLa cells or HeLa cells stably expressing SLFN11 (n = 2 replicates). (e) HeLa, A2058, and IGR37 cells were transfected with SLFN11 siRNA or control siRNA. Equal numbers of cells were treated with 0.5 µM DNMDP for 2 days after transfection. Cell viability was assessed by CellTiter-Glo (n = 3 replicates). Data are represented as mean ± SEM. P-values were calculated by a two-tailed unpaired Student’s t test (ns, not significant). The protein expression of SLFN11 and GAPDH was measured in IGR37 (data are representative of n = 2 replicates). HeLa and A2058 cells express no SLFN11 as shown in (c) and were therefore not included in the knockdown immunoblot. (f) Leucine tRNA abundance in HeLa cells treated with DMSO, 25 μM cisplatin or 0.1 μM camptothecin (CPT) for 48 hours was measured by qPCR. tRNA-Leu-TAA-1 levels are plotted relative to control tRNA, tRNA-Leu-TAG-2 (n = 3 replicates). Data are represented as mean ± SEM. Statistical significance were calculated by two-tailed unpaired Student’s t test (ns, not significant).
Extended Data Fig. 4 SLFN12 selectively digests tRNA-Leu-TAA in vitro.
(a) Indicated tRNAs were synthesized using a T7 RNA polymerase-mediated transcription reaction. Synthetic tRNAs were treated with the indicated concentrations of SLFN12 at 37 °C for 40 minutes (n = 2 replicates for Leu-TAA-1, Leu-TAA-3, Ser-CGA and Cys-GCA; n = 3 replicates for others). (b) 0.25 µM PDE3A and SLFN12 proteins pre-treated with 1.25 µM DNMDP were incubated with synthetic tRNAs at 37 °C for 40 minutes (n = 3 replicates for Leu-TAA-1 and Leu-TAA-3; n = 2 replicates for others). (c) 0.5 µM ΔSLFN12 (amino acids 1–347) or PDE3A proteins pre-treated with DMSO, 2.5 µM DNMDP, 2.5 µM trequinsin or 2.5 µM estradiol were incubated with synthetic tRNAs at 37 °C for 40 minutes. After incubation, RNA samples were analyzed on a 15% denaturing polyacrylamide gel (n = 2 replicates). Representative figures are shown here. Intact tRNA is indicated with an arrow. The relative amounts of intact tRNA were quantified using ImageJ software and are shown at the bottom. (d) Leucine tRNA abundance was measured by qPCR in purified total RNA treated with 0.25 μM SLFN12 recombinant protein. tRNA-Leu-TAA-1 levels are plotted relative to control tRNA, tRNA-Leu-TAG-2 (n = 3 replicates). Data are represented as mean ± SEM. Statistical significance was calculated by a two-tailed unpaired Student’s t test.
Extended Data Fig. 5 Leucine and serine tRNA sequences are conserved except in the acceptor and variable loop regions.
(a) Sequences of the indicated tRNAs were aligned in R using the msa package. The consensus logo is shown at the top of the figure, with more conserved positions appearing larger. Positions where the sequences do not agree are blank in the consensus. Structural regions of the tRNA are labeled on the bottom of the alignment. (b) The computational model of the structure of SLFN12 bound to tRNA was generated using the HADDOCK software. Both the catalytic SLFN12 monomer (green) and the adaptor SLFN12 monomer (magenta) interact with a single molecule of tRNA-Leu-TAA-3 (orange). The catalytic SLFN12 monomer interacts with the tRNA variable loop region (yellow) and the adaptor SLFN12 monomer interacts with the tRNA acceptor stem (cyan). The active site residues of the catalytic SLFN12 monomer are close to the primary tRNA cleavage site in this model.
Extended Data Fig. 6 Ribo-seq analysis.
(a) Distribution of read lengths of ribosome-protected fragments (RPF) upon treatment with DMSO (gray) or DNMDP (green). (b) RPF read densities near the translation initiation site (left) and translation termination site (right). (c) Scatter plot showing A-site ribosome occupancy at individual codons in DMSO or DNMDP treated cells. The TTA codon is indicated with a red dot; all other codons are grey. (d-e) RNA motifs (d) and RNA secondary structures (e) of the 50 bp upstream and downstream sequences from TTA-pause or TTA-non pause sites identified from Ribo-seq and RNA-seq (n = 2) were examined. RNA motifs at TTA-pause sites were identified compared to TTA non-pause sites using the MEME suite35 in Differential Enrichment mode. The most significant three RNA motifs are shown for upstream and downstream sequences. The minimum free energy (MFE) of the RNA structure was calculated using RNAFold36 and was plotted in a box plot (center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers). The p-values were calculated with the two-sided Wilcoxon Rank Sum test.
Extended Data Fig. 7 Genes containing TTA codons.
(a) Pie chart showing leucine codon usage frequency. (b) Histogram showing the distribution of the number of TTA codons per gene.
Extended Data Fig. 8 Upregulation of stress response upon DNMDP treatment.
(a) Immunoblots of the indicated proteins from HeLa cells treated with DMSO or DNMDP for the indicated time. Data are representative of n = 2 replicates. (b) Gene set enrichment analyses of up-regulated mRNAs in DNMDP-treated HeLa cells for 18 hours compared to DMSO-treated HeLa cells. The p-value was calculated by Fisher’s exact test.
Extended Data Fig. 9 Effects of leucine tRNA ectopic expression on DNMDP response.
HeLa cells stably expressing wild-type leucine tRNA-Leu-CAG or hybrid tRNA-Leu-[CAG:TAA] were treated with the indicated DNMDP concentrations for 2 days. Cell viability was assessed by CellTiter-Glo (n = 3 replicates). Data are represented as mean ± SEM.
Supplementary information
Supplementary Information
Supplementary Figs. 1–3 and Supplementary Tables 1–6.
Supplementary Data 1
Pause scores from Ribo-seq analysis.
Supplementary Data 2
Genes with TTA pause sites from Ribo-seq analysis.
Supplementary Data 3
Fold change of pause scores for Fig. 4.
Supplementary Data 4
The computational model of the structure of SLFN12 bound to tRNA.
Supplementary Data 5
Statistical source data for Supplementary Fig. 2.
Supplementary Data 6
Statistical source data for Supplementary Fig. 3.
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Lee, S., Hoyt, S., Wu, X. et al. Velcrin-induced selective cleavage of tRNALeu(TAA) by SLFN12 causes cancer cell death. Nat Chem Biol 19, 301–310 (2023). https://doi.org/10.1038/s41589-022-01170-9
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DOI: https://doi.org/10.1038/s41589-022-01170-9
