
TRNT1 Does Not Repair CCA-Ends in Cellulo After ANG Cleavage
In vitro, TRNA Nucleotidyl Transferase 1 (TRNT1) can repair CCA ends after ANG-mediated cleavage (Czech et al., 2013). In order to assess whether a similar mechanism occurs in cellulo, we examined whether the CCA-adding enzyme TRNT1 is involved in CCA repair following ANG treatment. To do so, we used TRNT1 depletion approach using siRNA targeting TRNT1. We reproducibly achieved an approximately 85% knockdown of TRNT1 protein (Figure 2A), which mimics previously reported hypomorphic phenotype of TRNT1 deficiency (Sasarman et al., 2015). RNAi mediated knockdown of TRNT1 caused no detectable difference in ANG-mediated tiRNA production ( Figure 2B). Under these conditions, we generated RNA-seq libraries using gel-purified tiRNA fractions. To further confirm that TRNT1 knockdown affected TRNT1 activity, we calculated %CCA of mitochondrial tRNA Ser-GCT, because mitochondrial tRNA Ser-GCT is most susceptible to TRNT1 hypofunction (Sasarman et al., 2015). In patients with TRNT1 hypomorphic mutations, CCA addition to mitochondrial tRNA Ser-GCT is significantly affected (Sasarman et al., 2015). As expected, the total number of 3′-fragment reads derived from mitochondrial tRNA Ser-GCT was significantly decreased by ANG treatment (59.0 PMMR and 21.4 PMMR for siControl-NC and siControl-ANG, respectively. p = 0.0068), because of the marked increase of tiRNAs derived from cytoplasmic tRNAs. However, ANG treatment did not induce significant cleavage of mitochondrial tRNA Ser-GCT ( Figure 2C; Supplementary Table S4A). In the non-treated samples (NC column in Supplementary Table S4A), the number of CCAadded 3′-fragments was significantly decreased by TRNT1 knockdown (36.6 PMMR and 17.5 PMMR for NC-siControl and NC-siTRNT1, respectively, p = 0.0409). The proportion of CCA-added 3′-fragments was also significantly decreased by TRNT1 knockdown (61.4 and 45.5% for NC-siControl and NC-siTRNT1, respectively. p = 0.0066). These data demonstrate that TRNT1 knockdown was effective enough to have a functional impact on CCA addition. We then calculated the proportion of CCA-added and CCterminating 3′-tiRNAs derived from cytoplasmic tRNAs in control and TRNT1 knockdown cells treated or not treated with ANG ( Figure 2D; Supplementary Table S4B). As expected, ANG treatment increased levels of 3′-tiRNAs significantly both in control and TRNT1 knockdown cells. However, the percentage of CCA-terminating cytoplasmic 3′-tiRNAs did not change significantly following TRNT1 knockdown (98.98 and 97.77% for siControl and siTRNT1, respectively; p = 0.9731). In addition, the percentage of CC-  terminating 3'-tiRNAs was not affected by TRNT1 knockdown in ANG-treated cells (0.77 and 0.79% for siControl and siTRNT1, respectively. p = 0.9998) ( Figure 2D; Supplementary Table S4B).
These results show that TRNT1 does not play a role in regulating ANG-dependent cleavage of tRNAs in cellulo.

Probing Integrity of tRNAs' CCA-Ends in Cellulo Using CCA-specific Ligation Method
Since tRNA sequencing approaches have own limitations, we complemented our findings by alternative approaches. We examined the effect of ANG on tRNAs' CCA-ends by biochemical approaches based on ligation of oligos specific to tRNAs possessing intact 3′-CCA termini. In this method named "CCA-specific ligation", oligos which specifically anneal tRNAs possessing intact 3′-CCA termini are ligated resulting in longer ligated tRNA products ( Figure 3A). First, we optimized the condition of ligation reaction using DNA ligase (Dnl) or RNA Ligase 2 (Rnl 2 ) since the ligation efficiency can significantly differ depending on the combination of oligos and ligases (Bullard and Bowater 2006). In this method, CCA-intact tRNAs and CCAdeactivated tRNAs would be detected as longer ligation products (ligated tRNAs) and unligated tRNAs, respectively. Therefore, if ligation efficiency was low, it would be impossible to distinguish between CCA-deactivated tRNAs and unreacted tRNAs just due to low ligation efficiency. We also chose two substrates that were previously used to study the integrity of CCA ends, namely hairpin oligo (Dittmar et al., 2006;Czech et al., 2013) and double-stranded oligo [ds-oligo (Shigematsu et al., 2017)] ( Figure 3A). As shown in Figures 3B,C, the combination of the Rnl 2 ligase with ds-oligo resulted in the most efficient capture and ligation of mature CCA-intact tRNAs as judged by total tRNA shift by SYBR Gold staining ( Figure 3B) and by the northern blotting against individual tRNA species ( Figure 3C). Under this condition, most of mature tRNAs were ligated to dsoligo and detected as "ligated tRNAs", suggesting that almost all tRNAs were CCA-intact under this condition ( Figure 3B). On the other hand, "Ligation 1″ method (combination of Dnl and hairpin oligo) showed the lowest ligation efficiency. Under this condition, most of mature tRNAs still remained as unligated tRNAs although most of them must have intact 3′-CCA termini. We conclude that "Ligation 3″ method is the best way to detect CCA-deactivated tRNAs. We used this approach to determine whether SA and/or ANG promote cleavage of the 3′-CCA termini in cellulo (Figure 4). We treated U2OS cells with SA or recombinant ANG, purified total RNA from the cells and proceeded with CCA-specific ligation using Rnl 2 and ds-oligo containing biotin ("ligation 3″ in Figure 3A). RNA samples from all conditions were efficiently ligated as judged by SYBR Gold staining ( Figure 4A, CCAligation) and immuno-blotting against biotin-containing oligo ( Figure 4B). Figures 4A,B also showed that mature tRNAs were exclusively ligated, demonstrating excellent specificity of this method toward mature tRNAs. Using probes against specific tRNA species (tRNA Gly-GCC and tRNA iMet-CAT, Figure 4C), we saw no difference in the amount of ligated tRNAs between control and ANG-treated samples. However, slight yet reproducible increase of unligated tRNAs was detected in the SA-induced samples ( Figure 4C; Supplementary Figure S4, unligated tRNA). This unligated tRNAs in SA-induced samples seemed slightly shorter than those in NC and ANG groups, suggesting that SA treatment affected CCA cleavage. While the amount of unligated tRNA Gly-GCC was slightly but significantly increased by SA treatment, that of unligated tRNA iMet-CAT was not significantly increased (Supplementary Figure S4). These data suggest that even though SA treatment can induce CCA-deactivation, the proportion of CCA-deactivated tRNAs remains very low. It should also be noted that treatment of the purified total RNA with SA at various concentration in vitro did not trigger degradation, cleavage or shortening of tRNAs ( Supplementary  Figures S5A, S5B) thus rejecting the possibility that SA chemically triggers tRNA cleavage. Taken together, these data demonstrate that ANG does not efficiently cleave 3′-CCA termini of tRNAs in cellulo.

CC-specific Ligation Shows That SA Promotes Cleavage of tRNAs' CCA-Termini in Cellulo
To further complement these findings, we have developed an alternative approach that aims on the ligation of only CCterminating tRNAs, named "CC-specific ligation" ( Figure 5A). In contrast to "CCA-specific ligation", only CC-terminating tRNAs will be detected as ligation products by this method. Because ds-oligo has 5′-monophosphate and can only be ligated by Rnl 2 to the CC-terminating tRNAs with 3′-hydroxyl group, if SA induces an RNase that cleaves tRNAs' CCA termini to produce tRNAs terminating with CC-end with 2′, 3′-cyclic phosphate, such substrate will not be ligated ( Figure 5A). To examine this possibility, we investigated the effect of pretreatment of T4 PNK, which removes both a 3′-phosphate and 2′, 3′-cyclic phosphate to form a 3′-hydroxyl end, on CC-specific ligation products. As shown in Figures 5B,C, Rnl 2 more efficiently ligates tRNAs from SA-treated cells that were pretreated with T4 PNK. However, unless pre-treated with PNK, even tRNAs from SA-treated cells were not ligated to the oligo, suggesting that CC-terminating tRNAs contained 2′, 3′-cyclic phosphates at their 3′-ends. These findings indicate that an enzyme that triggers CCA cleavage under SA stress may belong to the RNase A superfamily members.
Interestingly, when total RNAs were subjected to in vitro ANG digestion, tRNAs were cleaved at both anticodon and 3′-CCA termini, and CCA-deactivated tRNAs were detected by CCAspecific ligation (Supplementary Figures S6A, S6B) and CCspecific ligation (Supplementary Figures S6C, S6D), which was in marked contrast to that from in cellulo ANG experiments (Figures 1, 4). These results clearly indicate that the specificity of ANG is significantly different between in