Intron-Dependent or Independent Pseudouridylation of Precursor tRNA Containing Atypical Introns in Cyanidioschyzon merolae

Eukaryotic precursor tRNAs (pre-tRNAs) often have an intron between positions 37 and 38 of the anticodon loop. However, atypical introns are found in some eukaryotes and archaea. In an early-diverged red alga Cyanidioschyzon merolae, the tRNAIle(UAU) gene contains three intron coding regions, located in the D-, anticodon, and T-arms. In this study, we focused on the relationship between the intron removal and formation of pseudouridine (Ψ), one of the most universally modified nucleosides. It had been reported that yeast Pus1 is a multiple-site-specific enzyme that synthesizes Ψ34 and Ψ36 in tRNAIle(UAU) in an intron-dependent manner. Unexpectedly, our biochemical experiments showed that the C. merolae ortholog of Pus1 pseudouridylated an intronless tRNAIle(UAU) and that the modification position was determined to be 55 which is the target of Pus4 but not Pus1 in yeast. Furthermore, unlike yeast Pus1, cmPus1 mediates Ψ modification at positions 34, 36, and/or 55 only in some specific intron-containing pre-tRNAIle(UAU) variants. cmPus4 was confirmed to be a single-site-specific enzyme that only converts U55 to Ψ, in a similar manner to yeast Pus4. cmPus4 did not catalyze the pseudouridine formation in pre-tRNAs containing an intron in the T-arm.


Introduction
Eukaryotic transfer RNA (tRNA) genes are transcribed by RNA polymerase III, and the transcript then goes through a complex maturation pathway that involves trimming of the 5 and 3 ends, the addition of a CCA terminus, removal of intronic sequences, and modification of multiple nucleosides [1]. The order of these processes, in particular, that of intron removal and nucleoside modification, is variable among different tRNA genes and specific nucleoside modifications [2]. Modified nucleosides that are insensitive to introns include dihydrouridine at position 17 (D17), N 2 ,N 2 -dimethylguanosine at position 26 (m 2 2 G26), pseudouridine at position 39 (Ψ39), 7-methylguanosine at position 46 (m 7 G46), 5-methylcytidine at position 49 (m 5 C49), 5-methyluridine at position 54 (m 5 U54), and Ψ55 [3,4].
In the most notable cases, tRNA modifications are introduced prior to the removal of the intron and added to a specific tRNA processing intermediate [6,10]. For example, Figure 1. An inferred secondary structure of pre-tRNA Ile (UAU) of C. merolae. Three introns, shown in lowercase letters, interrupt D-arm, anticodon arm, and T-arm of the mature tRNA Ile (UAU) nucleotide, shown in uppercase letters. In yeast tRNA Ile (UAU), U34/U36 and U55 are pseudouridylated by yeast Pus1 and Pus4, respectively. U34, U36, and U55, which may be pseudouridylated by cmPus1 and cmPus4, are indicated in red.
In this article, we first demonstrate that cmPus1 unexpectedly mediates Ψ modification in intronless tRNA Ile (UAU), and that the Ψ modification was at 55 instead of being at 34 and 36. To some pre-tRNA Ile (UAU) variants that contain one or more intron(s), cmPus1 introduced Ψ at positions 34, 36, and 55, but not to the others. cmPus4 converted U55 to Ψ55 in intron-less and -containing tRNA Ile (UAU), but not in pre-tRNA Ile (UAU) containing an intron in the T-arm. All these demonstrate a novel relationship between tRNA introns and the Ψ syntheses. Figure 1 shows an inferred secondary structure of pre-tRNA Ile (UAU) from C. merolae [23]. This pre-tRNA contains three introns in the D-arm, anticodon-loop, and T-arm. U34, U36, and U55, which would be pseudouridylated by yeast Pus1 and Pus4, are indicated in red. Pseudouridylations of U34 and U36 by Pus1 are intron-dependent, and Ψ55 synthesized by Pus4 is insensitive to the presence of an intron in yeast [3,19]. By what mechanisms do cmPus1 and cmPus4 act on the pre-tRNA species, and how does the presence or absence of the intron affect the activity of cmPus1 and cmPus4?

Analysis of Pseudouridine Formation in Intronless tRNA Ile (UAU) by cmPus1 Candidate
Initially, we investigated whether purified cmPus1 possesses pseudouridine synthase activity. Internally 32 P-UTP-labeled tRNA Ile (UAU) transcript, in which introns were not included (Figure 2A), was treated with purified cmPus1 ( Figure S1A). Because it was reported that yeast Pus1 did not synthesize Ψ into mature size tRNA without an intron [15], we performed this experiment as a negative control. Unexpectedly, however, 2D-TLC analysis of modified nucleotides in this sample showed the formation of 32 P-labeled pΨ ( Figure 2B, panel 1). Thus, this result reveals that the substrate RNA specificity of Figure 1. An inferred secondary structure of pre-tRNA Ile (UAU) of C. merolae. Three introns, shown in lowercase letters, interrupt D-arm, anticodon arm, and T-arm of the mature tRNA Ile (UAU) nucleotide, shown in uppercase letters. In yeast tRNA Ile (UAU), U34/U36 and U55 are pseudouridylated by yeast Pus1 and Pus4, respectively. U34, U36, and U55, which may be pseudouridylated by cmPus1 and cmPus4, are indicated in red.

Inferred Secondary
Structure of Pre-tRNA Ile (UAU) from C. merolae Figure 1 shows an inferred secondary structure of pre-tRNA Ile (UAU) from C. merolae [23]. This pre-tRNA contains three introns in the D-arm, anticodon-loop, and T-arm. U34, U36, and U55, which would be pseudouridylated by yeast Pus1 and Pus4, are indicated in red. Pseudouridylations of U34 and U36 by Pus1 are intron-dependent, and Ψ55 synthesized by Pus4 is insensitive to the presence of an intron in yeast [3,19]. By what mechanisms do cmPus1 and cmPus4 act on the pre-tRNA species, and how does the presence or absence of the intron affect the activity of cmPus1 and cmPus4?

Analysis of Pseudouridine Formation in Intronless tRNA Ile (UAU) by cmPus1 Candidate
Initially, we investigated whether purified cmPus1 possesses pseudouridine synthase activity. Internally 32 P-UTP-labeled tRNA Ile (UAU) transcript, in which introns were not included (Figure 2A), was treated with purified cmPus1 ( Figure S1A). Because it was reported that yeast Pus1 did not synthesize Ψ into mature size tRNA without an intron [15], we performed this experiment as a negative control. Unexpectedly, however, 2D-TLC analysis of modified nucleotides in this sample showed the formation of 32 P-labeled pΨ ( Figure 2B, panel 1). Thus, this result reveals that the substrate RNA specificity of cmPus1 is completely different from that of yeast Pus1.    (C,D) A complementary primer as shown with the primer I blue (C) or primer II green (D) arrow in A was used for reverse transcription. Primer extensions were performed on the wild type tRNA Ile (UAU) transcripts treated with cmPus1. The transcripts were treated with CMCT for 2, 10, or 20 min plus or minus alkaline (OH − ) treatment (+ or −). Dideoxy sequencing was performed with the same primer.

Intronless tRNA
We also performed a nearest neighbor analysis. Intronless tRNA Ile (UAU) transcript was 32 P-labeled using α-32 P-CTP or α-32 P-ATP, and then the labeled tRNA was incubated with purified cmPus1. The modified tRNA was digested with RNase T2 ( Figure 2B, panels 2 and 3). Ψp was only detected when the tRNA was CTP-labeled ( Figure 2B, panels 3). These results indicate that the Ψ formation occurs 5 -adjacent to cytidine and are limited to the positions at U3, U12, U31, U41 U47, U55, and U62 in the intronless tRNA Ile (UAU).
Furthermore, we investigated Ψ formation using 32 P-UTP-labeled-mutant tRNAs in which U at positions 34 and/or 36 was substituted with C because C is not subject to the Ψ formation ( Figure 2B, panels 4-6). The result of the 2D-TLC showed that Ψ formations occurred in both the tRNA Ile U34C and U36C mutants ( Figure 2B, panels 4 and 5). At the start of this experiment, we predicted that cmPus1 would produce Ψ modifications at positions 34 and 36 in intronless tRNA Ile (UAU). However, Ψ formation could also be detected in the tRNA Ile U34C-U36C double mutant ( Figure 2B, panel 6). On the contrary, 2D-TLC analysis using tRNA Ile U55C mutants showed that cmPus1 did not mediate the formation of Ψ ( Figure 2B, panel 7). Moreover, we monitored the pseudouridylation activity of cmPus1 against 32 P-UTP-labeled-tRNA Ile U34C-U55C, U36C-U55C, and U34C-U36C-U55C mutants. The pΨ spot was not observed in these samples ( Figure S2).
In addition, the modification position(s) was analyzed by a combination of 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT)-chemical modification and primer extension [24]. We performed the reverse transcription with tRNA samples treated with cmPus1 using a DNA primer complementary to the sequence from position 60 to 40 (Primer I) or 76 to 59 (Primer II) of tRNA Ile (UAU) (Figure 2A and Table S1). Alkaline incubation allows the hydrolysis of U and Ψ adducts, whereas Ψ-CMC adducts are insensitive, and Ψ-CMC stops reverse transcription. Thus, reverse transcription stops indicate the position of Ψ-CMC. Guanosine is also modified with CMCT, but less efficiently than U and Ψ, and the G adduct is easily removed by the alkaline treatment. No stops in the reverse transcription with primer I at either position 34 or 36 were observed ( Figure 2C). On the other hand, the reverse transcription with primer II stopped at position 56 ( Figure 2D), and the Ψ position was predicted to be at 55 but not at 34 and 36. These results are consistent with the 2D-TLC results. At least in our in vitro experiment, pseudouridylation by cmPus1 occurs at position 55 and not at positions 34 and 36 in intronless tRNA Ile (UAU).
To investigate whether the Ψ formation at position 55 occurs only in tRNA Ile (UAU), we also analyzed cmPus1 activity for tRNA Ile (GAU), which possesses an intron in the anticodon-loop ( Figure S3A). Although Ψ formations were detected in both the intronless and intron-containing tRNA Ile (GAU), the activities diminished in the U55C mutant ( Figure S3B). Thus, the pseudouridylation at position U55 by cmPus1 is not limited to tRNA Ile (UAU).

CmPus4 Possesses a tRNA Ψ55 Synthase Activity
Our experimental results show that cmPus1 possesses a tRNA Ψ55 synthase activity. In yeast, Ψ55 is synthesized by Pus4. Therefore, we investigated the activity of cmPus4. As with cmPus1, wild type and the U55C mutant tRNA Ile (UAU) transcripts were treated with purified cmPus4 ( Figure S1B). The modified nucleotides were analyzed by 2D-TLC, and pΨ was detected in the wild type tRNA sample ( Figure 3A
Subsequently, for the pre-tRNAs that accepted pseudouridylation, pre-tRNA mutant transcripts were prepared to allow the determination of the Ψ positions. The results are described in order as follows.
Pre-tRNA Ile (UAU) Containing the D-AC-introns: Ψ formation was detected in the pre-tRNA U34C-U36C mutant ( Figure 5A). This predicted that the position of Ψ would be at position 55. With the pre-tRNA U55C sample treated with cmPus1, a pΨ spot appeared in the 2D-TLC analysis. Since the position(s) of Ψ was predicted to be at 34 and/or 36, pre-tRNA U34C-U55C and U36C-U55C mutants were prepared and analyzed. A very weak but detectable Ψ formation was observed for the pre-tRNA mutants ( Figure 5B), whereas it was not detectable in the nearest neighbor analysis of the ATP-labeled pre-tRNA ( Figure S4). Furthermore, with the pre-tRNA U34C-U35C-U55C mutant, no Ψ spot was detected ( Figure S5). These results suggest that the cmPus1 accepts pre-tRNA containing the D-AC introns, and Ψ formation occurs mainly at position 55 and slightly at positions 34 and 36.   Pre-tRNA Ile (UAU) containing the AC-intron or the full-introns: Ψ formation was detectable in the pre-tRNA U34C-U36C mutants ( Figure 5A). However, no pΨ spots appeared with the pre-tRNA U55C mutants ( Figure 5A). Thus, cmPus1 forms Ψ only at position 55 in the pre-tRNAs containing the AC-intron or the full-introns. The result of the AC-intron was in agreement with that of pre-tRNA Ile (GAU) carrying the AC-intron ( Figure S3).
Pre-tRNA Ile (UAU) containing the D-intron or the D-T-introns: Ψ formation was detectable in pre-tRNA U34C-U36C and U55C mutants ( Figure 5A). In addition, the cmPus1 did not accept the pre-tRNA U34C-U35C-U55C mutant ( Figure S5). Thus, the pre-tRNAs have the potential to have Ψ at positions 34 and/or 36 and 55. However, since the pre-tRNAs Ile (UAU) containing the D-intron and the D-T-introns were not detected in C. merolae RNA fraction [23], the intermediate pre-tRNAs might only barely be present in cells.
Based on the experimental results, we conclude that the site specificity of cmPus1 is regulated by the presence of introns, at least in vitro. When all introns are absent, cmPus1 acts only on U55. When AC-intron is present, cmPus1 does not act on U34 and U36. Thus, AC-intron is a negative element for Ψ34 and Ψ36 modifications by cmPus1. However, this negative effect of AC-intron on Ψ34 and Ψ36 modifications is abolished by the existence of D-intron (results of D-AC-introns). Furthermore, the presence of T-intron negatively regulates the Ψ55 formation activity of cmPus1. However, this effect by T-intron is abolished by the presence of D-intron (results of full introns and D-T-introns). Moreover, U34 and U36 can be modified under the T-and D-intron regulation (results of D-T-introns). In addition, the presence of D-intron is essential for Ψ34 and Ψ36 modifications.

Analysis of cmPus4 Mediated Pseudouridine Formation in Pre-tRNA Ile (UAU) In Vitro
As for cmPus1, the pseudouridine formation assay with cmPus4 was performed with pre-tRNA Ile (UAU) transcripts as shown in Figure 6A. cmPus4 accepted the pre-tRNAs Ile (UAU) containing the D-, AC-, and D-AC-introns ( Figure 6A). Since aminoacylstem is close to position 55 in tRNA, the same experiment was performed for the intronless and intron-containing tRNAs with CCA attached to the 3 end, considering the effect of the presence of the CCA terminal on the cmPus4 activity. These results were consistent with those against the tRNA variants without a CCA terminal ( Figure S6). In the three pre-tRNA U55C mutants, no pΨ spots were detected in the three pre-tRNA U55C mutants ( Figure 6B). Thus, the cmPus4 forms Ψ only at position 55 in the pre-tRNAs Ile (UAU) without T-intron, in addition to the intronless tRNA Ile (UAU).

No RT-PCR Products Derived from Pre-tRNA Ile (UAU) Containing the D-Intron and D-T-Intron Were Detected
In a previous report by Soma et al. [23], it was demonstrated that no product was detected for an intermediate in which anticodon intron was removed before the D-arm intron and the T-arm intron on the RT-PCR analysis of C. merolae total RNA. Thus, it is supposed that the processing of D-and T-introns precedes that of anticodon intron [23]. We performed RT-PCR again using another primer set to detect the intermediates (Figure 7). The RT-PCR products derived from pre-tRNA Ile (UAU) variants containing D-AC introns and full-introns were detected. Annotation of each PCR product was based on sequencing analysis of gel purification of the bands followed by TA-cloning. In our experiment, RT-PCR products derived from either pre-tRNA Ile (UAU) containing the D-intron or D-T-intron could not be detected. Thus, these experimental results show that the Ψ modification patterns in the three pre-tRNAs (full-introns, D-AC-introns, and AC-intron) and the fully spliced intronless form reflect the physiological phenomena in the living cells. Two bands derived from D-AC-intron are indicated by the arrows in Figure 7 because these PCR products had the same sequence.

Position of Ψ in Native tRNA Ile (UAU)
To clarify whether Ψ modifications are introduced at positions 34, 36, and 55 of the tRNA Ile (UAU) in the cell, a reverse transcription-based analysis was performed for an isolated class I tRNA from C. merolae. The primer was designed to be complementary from position 76 to position 59 in the tRNA ( Figure 8A). The reverse transcription was paused at position 55, whereas no stop was detected at either position 34 or 36 ( Figure 8B). Since reverse transcription also paused at positions 18, 26, and 37 regardless of CMCT treatment, it is considered that some modifications were introduced in the positions. In yeast tRNA, Gm18, N 2 -methylguanosine (m 2 G26) or N 2 , N 2 -dimethylguanosine (m 2 2 G26), and N 6 -threonylcarbamoyladenosine (t 6 A37) are introduced in the positions [26]. As these modified nucleosides stop the reverse transcriptase reaction, Gm18, m 2 G26 (or m 2 2 G26), and t 6 A37 may also be introduced in the tRNA Ile (UAU) of C. merolae.

Position of Ψ in Native tRNA Ile (UAU)
To clarify whether Ψ modifications are introduced at positions 34, 36, and 55 of the tRNA Ile (UAU) in the cell, a reverse transcription-based analysis was performed for an isolated class I tRNA from C. merolae. The primer was designed to be complementary from position 76 to position 59 in the tRNA ( Figure 8A). The reverse transcription was paused at position 55, whereas no stop was detected at either position 34 or 36 ( Figure 8B). Since reverse transcription also paused at positions 18, 26, and 37 regardless of CMCT treatment, it is considered that some modifications were introduced in the positions. In yeast tRNA, Gm18, N 2 -methylguanosine (m 2 G26) or N 2 , N 2 -dimethylguanosine (m 2 2G26), and N 6 -threonylcarbamoyladenosine (t 6 A37) are introduced in the positions [26]. As these modified nucleosides stop the reverse transcriptase reaction, Gm18, m 2 G26 (or m 2 2G26), and t 6 A37 may also be introduced in the tRNA Ile (UAU) of C. merolae.

Discussion
In this study, we determined substrate and site specificities of cmPus1 and cmPus4 for pre-tRNA containing multiple introns using various pre-tRNA Ile (UAU) transcripts in vitro. A summary of the results is shown in Figure 9. cmPus1 can modify U34, U36, and U55 in some specific intron-containing pre-tRNA Ile (UAU) variants and can only form Ψ55 in intronless-tRNA Ile (UAU). The formation of Ψ34 and Ψ36 by cmPus1 occurs in very limited tRNA forms, and its activity is weak. The main pseudouridylation position of cmPus1 is U55, and the Ψ55 is formed except when tRNA Ile (UAU) has only T-intron with or without AC-intron. cmPus4 only modifies U55 but not in the presence of the T intron. Yeast Pus1 catalyzes Ψ formation at positions 1,26,27,28,34,35,36,65, and 67 in tRNAs [12,14,15,17,18] and at position 44 of U2 snRNA [27]. Therefore, Pus1 is a multiplesites-specific enzyme and acts on multiple substrate RNAs. Yeast Pus1 is responsible for the Ψ formation at 34 and 36 of tRNA Ile (ΨAΨ), while the modification is dependent on the presence of an intron in the anticodon arm. We compared the amino acid sequences of cmPus1 and yeast Pus1 as well as human and mouse Pus1 ( Figure S7). cmPus1 contains two Ψ synthase domains, each of which has 32% (96-276 aa) and 29% (510-607 aa) identity with yeast Pus1 (https://www.ncbi.nlm.nih.gov accessed on 1 August 2021). The identity is not extremely low. However, over 40 amino acid residues are present between domains III and IV in cmPus1, and the amino acid identity of domain IV is low ( Figure S7). Our in vitro experimental results demonstrate that the site specificity of cmPus1 is regulated by the existence of the introns. Because we used purified cmPus1 and a pre-tRNA transcript throughout the experiments, the presence of other protein factors and guide RNA should not be considered [28]. When all introns are absent, cmPus1 acts only on U55. In the pre-tRNA Ile (UAU) containing AC-intron, cmPus1 does not act on U34 and U36. In addition, cmPus1 does not catalyze Ψ formation at least at position 27 from the experimental results using tRNA Ile (GAU) ( Figure S3). This characteristic is completely different from yeast Pus1. The effect of D-and T-introns on the site specificity of cmPus1 is unprecedented. To understand this effect, structural studies of pre-tRNA and cmPus1 are required. Furthermore, the pre-tRNAs corresponding to D-and D-T-introns ( Figure 7) were not detected in our current and previous [23] analyses of pre-tRNAs from C. merolae cells. Therefore, the Ψ formation in D-and D-T-introns are only in vitro experimental results.
In contrast to Pus1, Pus4 catalyzes Ψ formation only at position 55 and is a single-sitespecific Ψ synthase. Ψ55 is commonly found in eubacteria, archaea, and eukaryotes [29,30]. The archaeal Ψ55 synthases are Cbf5 and Pus10 [31][32][33][34]. Since the archaeal Pus10 can convert U54 and U55 to Ψ modifications, Pus10 is a multiple-site-specific pseudouridine synthase. cmPus4 mediates Ψ modification at position 55 in intronless tRNA Ile (UAU) and pre-tRNA Ile (UAU) variants except when pre-tRNAs contain a T-intron. Although Ψ55 has been proposed to be introduced at a fairly early stage after the tRNA gene is transcribed [6], cmPus4 cannot form Ψ modification in pre-tRNA containing full introns. However, another pseudouridine synthase cmPus1 catalyzes Ψ55 formation in the pre-tRNA.
The means of RNA recognition by Ψ synthetases allows the enzymes to be split into three classes. The first class consists of single-site-specific enzymes [16,35,36], the second consists of region-specific enzymes capable of modifying several neighboring positions [37,38], and the third consists of multiple-site and multiple-substrate-specific enzymes that modify distinct positions in different classes of RNAs [39]. In the current study, cmPus1 and cmPus4 are identified as belonging to the multiple-site-specific group and the single site-specific group, respectively. With regard to cmPus1, it is clear that U55 is converted to Ψ. Thus, the relationship between cmPus1 and cmPus4 is similar to that of yeast Pus1 and Pus7 [40,41]. Yeast Pus1 catalyzes Ψ formations at positions 34 and 36 in pre-tRNA Ile (UAU) and also at position 35 in pre-tRNA Tyr in vitro. In addition, Ψ at position 35 in pre-tRNA Tyr is also formed by the action of yeast Pus7 which also introduces Ψ at positions 35 of U2 snRNA [40,41].
Recently, in a study of S. cerevisiae mutant strains in which tRNA introns were removed from intron-containing tRNA genes, tRNA Ile (UAU) was analyzed. The tRNA Ile (UAU) intronless strain lacked the Ψ34 and Ψ36 modifications, and instead had a 5-carbamoylmethyluridine (ncm 5 U) modification at position 34 [42]. The six-Elongator protein subunit (Elp1-Elp6) complex is required for ncm 5 U synthesis [43], and the Elp complex is considered to catalyze the formation of ncm 5 U34 after intron splicing. In other words, Ψ is not required for the removal of the intron, and it is proposed that the presence of the intron prevents the introduction of the ncm 5 U modification [42]. Since the ncm 5 U34 allows for base-paring with G [44], tRNA Ile (UAU) carrying ncm 5 U34 may be used to decode the methionine AUG codon. In order to prevent misreading, Pus1 may form Ψ34 into tRNA Ile (UAU) containing an intron in yeast. Our experiments using UTP-labeled tRNA showed that Ψ formation at positions 34 and 36 by cmPus1 occurs very weakly in pre-tRNA Ile (UAU) transcript containing the D-AC-introns. Ψ34 and Ψ36 also were undetectable in the nearest neighbor experiment using ATP-labeled tRNA. In a reverse transcription-based analysis for the native tRNA Ile (UAU), no pauses of the reverse transcriptase were observed at positions 34 and 36. These results indicate that in the native tRNA Ile (UAU), Ψ modification might be absent or only partially introduced at positions 34 and/or 36. The experimental result using RNA from C. merolae cells is consistent with that pseudouridylation activity for U34 and U36 by cmPus1 was very weak in the in vitro experiment. We also suggest that the pseudouridylation in the pre-tRNA Ile (UAU) is unlikely to be a trigger for the AC-intron cleavage. This study clarifies the relationship between introns and pseudouridylation in tRNA Ile (UAU) which harbors multiple introns and provides important insights into the processing system of tRNA in C. merolae.

Construction of the Expression Vector
The CYME_CMH251C and CYME_CMF022C genes from C. merolae have been annotated as "similar to tRNA-pseudouridine synthase I" and "tRNA pseudouridine 55 synthase", respectively [20]. The CYME_CMH251C gene and CYME_CMF022C gene products were named cmPus1 and cmPus4 to represent candidate homologs of yeast Pus1 and Pus4, respectively. The C. merolae genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) extraction method [46]. The two genes were amplified by PCR from the genomic DNA of C. merolae using KOD Fx neo (Toyobo, Japan) and primers (Table S1). The obtained PCR product was assembled into a pET22b PCR fragment using NEBuilder HiFi assembly (New England BioLabs, United States). The resulting expression plasmids were designated as pET22b-cmPus1-His and pET22b-His-cmPus4. E. coli Rosetta 2 (DE3) transformed with the pET22b-His-pus4 was pre-cultured at 37 • C for 16 h, and then the pre-culture medium (50 mL) was seeded into MM9 medium (1000 mL) at 37 • C for 3.5 h (~0.8 OD 590 ). The expression of recombinant cmPus4 was performed by the addition of 1 mM (final concentration) isopropyl-β-D-1-thiogalactopyranoside and then further cultured at 37 • C for 2 h. The cmPus4 protein was purified by the same protocol for the cmPus1 purification described above. Briefly, the cmPus4 fraction was prepared using a Ni Sepharose High-Performance column. The cmPus4 fraction eluted with buffer A containing 20-150 mM imidazole was further purified using a Q Sepharose Fast Flow column. cmPus4 was eluted in buffer B containing 500 mM NaCl, and the fraction was dialyzed against buffer C [50 mM Tris-HCl (pH 8.0), 5 mM MgCl 2, 6 mM β-mercaptoethanol, 50 mM NaCl].
The fraction containing cmPus1 or cmPus4 was concentrated using an Amicon Ultra-15 (Millipore, United States) and then added to an equivalent volume of 100% glycerol and stored at −30 • C. Protein concentration was measured with a Bio-Rad protein assay kit (Quick Start TM Bradford Dye Reagent, Bio-Rad Laboratories, United States) and analyzed by 12% SDS-PAGE.

tRNA Preparation
Template DNAs for T7 RNA polymerase-mediated transcription reactions for the preparation of C. merolae tRNA Ile (UAU) variants and tRNA Ile (GAU) were prepared using the primers (Table S1). To prepare a transcriptional template for pre-tRNA Ile (UAU) containing full introns, C. merolae genome was used as a PCR template. Internally labeled tRNA Ile (GAU) and tRNA Ile (UAU) variants were prepared by transcription with α-32 P-UTP, α-32 P-CTP, or α-32 P-ATP (PerkinElmer, Waltham, MA, USA). The tRNA transcripts were purified using a Q Sepharose Fast Flow column and then analyzed by 10% PAGE (7 M urea). The 32 P internal labels in the transcripts were monitored using a Typhoon FLA 7000 imager (GE Healthcare, United States).

Detection of Pseudouridine Formation by Two-Dimensional Thin Layer Chromatography (2D-TLC)
The internally labeled tRNA transcript (containing 32 P at around 3000 cpm) and 150 ng of the purified cmPus1 or cmPus4 in 50-µL buffer D (100 mM Tris-HCl (pH 8.0), 100 mM ammonium acetate, 5 mM MgCl 2 , 2 mM DTT, 0.1 mM EDTA) were incubated at 37 • C for 1 or 2 h. The tRNA was extracted with phenol-chloroform and then recovered by ethanol precipitation. 0.8 A 260 units total RNA from Lactobacillus plantarum was added before ethanol precipitation. The tRNA pellet was dissolved and digested overnight at 37 • C with 3 µL (1 unit) nuclease P1 (Wako, Tokyo, Japan) in 50 mM ammonium acetate (pH 5.0) or RNase T2 in 20 mM ammonium acetate (pH 5.0). Nuclease P1 or RNase T2 was used for UTP-or ATP-and CTP-labeled tRNA transcripts, respectively. An aliquot of the sample was spotted onto a thin layer plate (Merck, Darmstadt, Germany, TLC Cellulose F; 10 cm × 10 cm) and separated as described previously [47]. The 32 P-labeled nucleotides were monitored using a Typhoon FLA 7000 imager (GE Healthcare, United States). Standard nucleotides were marked by UV 260nm irradiation.

RNA Isolation
Small RNA fraction was extracted using ISOGEN II (Nippon Gene, Tokyo, Japan) according to the manufacturer's manual. 140 µg of small RNA was isolated from 1 g of C. merolae 10D cells. Class I tRNA fraction was further purified by 10% PAGE (7 M urea).

CMCT Modification and Reverse Transcription
To detect the Ψ positions in tRNA, a combination of chemical modification and reverse transcription was performed. The tRNA Ile (UAU) transcript was incubated with the recombinant cmPus1 or cmPus4 as described above. The tRNAs were extracted with phenol-chloroform and then collected by ethanol precipitation. The Ψ modifications of the tRNA transcripts and isolated class I tRNA were chemically modified using a CMCT according to the literature [24]. The positions of CMCT-chemical modifications were determined by reverse transcription and DNA sequencing. Reverse transcriptions were performed using 5 -32 P-labeled primers (Table S1), 1 mM dNTPs, and ReverTraAce (Toyobo, Osaka, Japan) as described [48].

Reverse Transcription Polymerase Chain Reaction (RT-PCR) and DNA Sequencing
The analysis of RT-PCR and DNA sequencing were performed following previously described methods [21,23].