Supplemental Figure S1. Sequence alignment of all human ADAT substrate tRNAs.

1. Supplemental Figure S1. Sequence alignment of all human ADAT substrate tRNAs.

2. Supplemental Figure S2. Sequence alignment a selection of 50 representative tRNAs, including ADAT substrates and non-ADAT substrates (all of them encoding for amino acids T, A, P, S, L, I, V, R). The selected tRNAs containing either A34, C34, or U34 (no G34-tRNAs encoding for T, A, P, S, L, I, V, R are present in human). T-coffee was used to select the 50 tRNAs with the highest sequence variability from a total of 180.

3. Supplemental Figure S3. Sequence alignment of non-ADAT substrate tRNAs (all of them encoding for amino acids T, A, P, S, L, I, V, R). Aligned sequences of non-ADAT substrate tRNAs with C34 (upper panel) and U34 (lower panel) (G34 tRNAs encoding for T, A, P, S, L, I, V, R are not found).

4. Supplemental Figure S4. SDS-PAGE (A) and Western blot (B) analysis of the E. coli expressed and affinity-purified human heterodimeric ADAT2-ADAT3 . 75 – 48 – B A 75 35 – 48 25 – ADAT3 35 25 α-Strep α-His ADAT2 17 – 17 75 – 48 – 35 – 25 – 17 –

5. Supplemental Figure S5. Analysis of ADAT-mediated deamination of tRNAAlaAGC and tRNA mini-substrates derived from tRNAAlaAGC after 1 hour incubation at 37ºC. LC-MS analysis of the tRNA fragments after incubation with 800 nM ADAT for 1 hour at 37ºC. Extracted ion chromatograms (XICs) from m/z 322.0446 [C-H]-, m/z 323.0286 [U-H]-, m/z 346.0558 [A-H]-, m/z 347.0398 [I-H]- and m/z 362.0507 [G-H]-. IMP was only detected in the full-length tRNAAlaAGC and tRNAAlaAGC lacking the CCA. +ADAT

6. +ADAT +ADAT +ADAT +ADAT

7. +ADAT

8. tRNAArgACG tRNAAlaAGC Supplemental Figure S6. Prediction of the secondary structure of the chimeric tRNAs. Both chimeric tRNAs display the typical cloverleaf secondary structure. tRNAAlaAGC tRNAArgACG

9. Supplemental Figure S7. Sequences and secondary structures of the acceptor arms and anticodon arms of the tRNAs used in the study. tRNAArgACG acceptor stem tRNAArgACG anticodon stem tRNAAlaAGC acceptor stem tRNAAlaAGC anticodon stem tRNAProAGG acceptor stem A C C C G-C G-C C-G U-A C-G G-C U-A A C C G G-C G-U G-C C-G C-G A-U G-C U-A C-G U-A G-C A-U A C C A G-C G-C G-U G-C G-C U-A G-C C-G G-C U-A G-C C-G

10. [tRF] (µM) Supplemental Figure S8. Dose-response curves for 5’-half and 5’-tRF derived from tRNAAlaAGC inhibitory effect on ADAT-mediated deamination of tRNAArgACG and tRNAAlaAGC. A Representative RFLP experiments to assess the inhibitory effect of 5’-half derived from tRNAAlaAGC and 5’-tRF derived from tRNAAlaAGC on ADAT-mediated deamination of tRNAArgACG (upper panel) and tRNAAlaAGC (lower panel). B Percentage of I34 modification upon varying concentrations of the tRFs from three independent experiments and averaged. Error bars reflect the standard deviation. 0.5 + + + + 50 + + 5 + + - + - - A34 I34 15 nM ADAT BlpI A34 I34 A B tRNAArgACG deamination 200 [tRF] (µM) 0.5 + + 50 + + 5 + + - + 200 - - + + 150 15 nM ADAT 150 100 BlpI 100 75 75 50 50 tRNAAlaAGC deamination

11. Supplemental Figure S9. Synthetic HsADAT genes optimized for E. coli expression. Scheme: HsADAT2 (blue) with stop codon (red) - RBS (from pETDUET-1, black lowercase) - HsADAT3 (green) (without stop codon). Gene sequences were obtained using the amino acid sequences annotated in Uniprot for human ADAT2 (Q7Z6V5-1) and ADAT3 (Q96EY9). The cDNA was optimized for expression in E. coli. ATGGAAGCAAAAGCAGCACCGAAACCGGCAGCAAGCGGTGCATGTAGCGTTAGTGCCGAAGAAACCGAAAAATGGATGGAAGAGGCAATGCATATGGCAAAAGAAGCACTGGAAAATACCGAAGTTCCGGTTGGTTGTCTGATGGTGTATAATAACGAAGTTGTTGGCAAAGGTCGCAATGAAGTTAATCAGACCAAAAATGCAACCCGTCATGCAGAAATGGTTGCAATTGATCAGGTTCTGGATTGGTGTCGTCAGAGCGGTAAAAGCCCGAGCGAAGTTTTTGAACATACCGTTCTGTATGTTACCGTTGAACCGTGTATTATGTGTGCAGCAGCACTGCGTCTGATGAAAATTCCGCTGGTTGTTTATGGTTGTCAGAATGAACGTTTTGGTGGTTGTGGTAGCGTTCTGAATATTGCAAGTGCCGATCTGCCGAATACCGGTCGTCCGTTTCAGTGTATTCCGGGTTATCGTGCGGAAGAGGCCGTTGAAATGCTGAAAACCTTCTATAAACAAGAGAATCCGAATGCACCGAAAAGCAAAGTGCGTAAAAAAGAATGTCAGAAAAGCTAAtgcttaagtcgaacagaaagtaatcgtattgtacacggccgcataatcgaaattaatacgactcactataggggaattgtgagcggataacaattccccatcttagtatattagttaagtataagaaggagatatacatATGGAACCGGCACCGGGTCTGGTTGAACAGCCGAAATGTCTGGAAGCAGGTAGTCCGGAACCGGAACCTGCACCGTGGCAGGCACTGCCGGTTCTGAGCGAAAAACAGAGCGGTGATGTTGAACTGGTTCTGGCATATGCAGCACCGGTTCTGGATAAACGTCAGACCAGCCGTCTGCTGAAAGAAGTTAGCGCACTGCATCCGCTGCCTGCACAGCCGCATCTGAAACGTGTTCGTCCGAGCCGTGATGCAGGTTCACCGCATGCACTGGAAATGCTGCTGTGTCTGGCAGGTCCGGCAAGCGGTCCGCGTAGCCTGGCAGAACTGCTGCCTCGTCCGGCAGTTGATCCGCGTGGTCTGGGTCAGCCGTTTCTGGTTCCGGTGCCTGCCCGTCCGCCTCTGACCCGTGGTCAGTTTGAAGAAGCACGCGCACATTGGCCGACCAGCTTTCATGAAGATAAACAGGTTACCAGCGCACTGGCAGGCCGTCTGTTTAGCACCCAAGAACGTGCAGCAATGCAGAGCCATATGGAACGTGCCGTTTGGGCAGCACGTCGTGCAGCAGCACGTGGTCTGCGTGCAGTTGGTGCCGTTGTTGTTGATCCGGCAAGTGATCGTGTTCTGGCAACCGGTCATGATTGTAGCTGTGCAGATAACCCGCTGCTGCATGCCGTTATGGTTTGTGTTGATCTGGTTGCACGCGGTCAGGGTCGTGGCACCTATGATTTTCGTCCGTTTCCGGCATGTAGCTTTGCACCGGCAGCCGCACCGCAGGCAGTTCGTGCTGGTGCAGTTCGTAAACTGGATGCAGATGAAGATGGTCTGCCGTATCTGTGTACCGGTTATGATCTGTATGTTACCCGTGAACCGTGTGCAATGTGTGCCATGGCCCTGGTTCATGCACGTATTCTGCGTGTTTTTTATGGTGCACCGAGTCCGGATGGTGCGCTGGGCACCCGTTTTCGTATTCATGCGCGTCCGGATCTGAATCATCGTTTTCAGGTTTTTCGTGGTGTTCTGGAAGAACAGTGTCGTTGGCTGGATCCGGATACC