Journal: Nature chemical biology

Article Title: Velcrin-induced selective cleavage of tRNA Leu (TAA) by SLFN12 causes cancer cell death

doi: 10.1038/s41589-022-01170-9

Figure Lengend Snippet: a , Sequence analysis of tRNAs extracted from HeLa cells treated with DMSO or 1 μM DNMDP for 18 h ( n = 2 replicates), based on the mapping to the genomic tRNA database, GtRNAdb 35 . Scatter plot (left), tRNA abundance in DMSO-treated ( X axis) and DNMDP-treated cells ( Y axis). Red dots, tRNA Leu (TAA) isodecoders. b , Leucine tRNA abundance in a panel of cancer cell lines treated with DMSO or 1 μM DNMDP 3 . 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 ± s.e.m. P values were calculated by unpaired two-tailed Student’s t -test.

Article Snippet: RNAs were synthesized from PCR products using the Transcript Aid T7 High Yield Transcription Kit (ThermoFisher) and purified by extracting full-length tRNAs from 15% TBE-urea gels (ThermoFisher).

Techniques: Sequencing, Synthesized, Control, Two Tailed Test

Journal: Nature chemical biology

Article Title: Velcrin-induced selective cleavage of tRNA Leu (TAA) by SLFN12 causes cancer cell death

doi: 10.1038/s41589-022-01170-9

Figure Lengend Snippet: a , b , Preferential digestion of tRNA Leu (TAA) by SLFN12. 0.5 μg of the indicated tRNAs were digested with ( a ) 2 μM SLFN12 ( n = 2 replicates for tRNA Leu (TAA)-1, tRNA Leu (TAA)-3, tRNA Ser (CGA) and tRNA Cys (GCA), and n = 3 replicates for others) or ( b ) 0.25 μM PDE3A and SLFN12 proteins preincubated with 1.25 μM DNMDP ( n = 3 replicates for tRNA Leu (TAA)-1 and tRNA Leu (TAA)-3, and n = 2 replicates for others). The relative amounts of intact tRNAs were measured and plotted. c , The variable loops or acceptor stems of tRNA Leu (TAG)-2 and tRNA Leu (TAA)-3 were swapped to generate six different hybrid leucine tRNAs. Sequences of tRNA Leu (TAG)-2 and tRNA Leu (TAA)-3 are indicated by red and black colors, respectively. d , Each leucine tRNA synthesized by in vitro transcription was digested with 0.25 μM PDE3A and 0.25 μM SLFN12 proteins preincubated with 1.25 μM DNMDP. Cleaved tRNA was analyzed on 15% TBE-Urea gels. The results were reproducible and representative images are shown ( n = 2 for hybrids 2, 3, 5 and 6; n = 3 replicates for others). e , The relative amount of intact tRNA, quantified using ImageJ software, is shown at the bottom of the gels and plotted ( n = 2 for hybrids 2, 3, 5 and 6; n = 3 replicates for others). Data are represented as mean ± s.e.m. P values were calculated by the unpaired two-tailed Student’s t -test.

Article Snippet: RNAs were synthesized from PCR products using the Transcript Aid T7 High Yield Transcription Kit (ThermoFisher) and purified by extracting full-length tRNAs from 15% TBE-urea gels (ThermoFisher).

Techniques: In Vitro, Synthesized, Software, Two Tailed Test

Journal: Nature chemical biology

Article Title: Velcrin-induced selective cleavage of tRNA Leu (TAA) by SLFN12 causes cancer cell death

doi: 10.1038/s41589-022-01170-9

Figure Lengend Snippet: a , Construction of resistant tRNA Leu (CAG:TAA) by mutating the anticodon of tRNA Leu (CAG)-1 to TAA. b , Indicated tRNAs were digested with 0.25 μM PDE3A and 0.25 μM SLFN12 proteins preincubated with 1.25 μM DNMDP. Intact tRNAs are marked with an arrow and were quantified by ImageJ (1.8.0), as reported at the bottom of each lane ( n = 2 replicates). c , HeLa cells were transfected with plasmids expressing tRNA Leu (CAG)-1, tRNA Leu (TAA)-3 or variant tRNA Leu (CAG:TAA) followed by DMSO or 0.5 μM DNMDP treatment. Forty-eight hours after drug treatment, cell viability was assessed by CellTiter-Glo ( n = 3 replicates). Data are represented as mean ± s.e.m. P values were calculated by the unpaired two-tailed Student’s t -test (NS, not significant).

Article Snippet: RNAs were synthesized from PCR products using the Transcript Aid T7 High Yield Transcription Kit (ThermoFisher) and purified by extracting full-length tRNAs from 15% TBE-urea gels (ThermoFisher).

Techniques: Expressing, Transfection, Variant Assay, Two Tailed Test

Journal: Nature chemical biology

Article Title: Velcrin-induced selective cleavage of tRNA Leu (TAA) by SLFN12 causes cancer cell death

doi: 10.1038/s41589-022-01170-9

Figure Lengend Snippet: ( 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.

Article Snippet: RNAs were synthesized from PCR products using the Transcript Aid T7 High Yield Transcription Kit (ThermoFisher) and purified by extracting full-length tRNAs from 15% TBE-urea gels (ThermoFisher).

Techniques: In Vitro, Synthesized, Incubation, Software, Purification, Recombinant, Control, Two Tailed Test

Journal: Nature chemical biology

Article Title: Velcrin-induced selective cleavage of tRNA Leu (TAA) by SLFN12 causes cancer cell death

doi: 10.1038/s41589-022-01170-9

Figure Lengend Snippet: ( 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.

Article Snippet: RNAs were synthesized from PCR products using the Transcript Aid T7 High Yield Transcription Kit (ThermoFisher) and purified by extracting full-length tRNAs from 15% TBE-urea gels (ThermoFisher).

Techniques: Labeling, Generated, Software