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1 Supplementary Information Precursors of trnas are stabilized by methylguanosine cap structures Takayuki Ohira and Tsutomu Suzuki Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Bldg Hongo, Bunkyo-ku, Tokyo , Japan. 1

2 Supplementary Results Supplementary Table 1. Sequences and molecular mass of 5' terminal fragments from pre-trnas (digested by RNase A). Sequences and teminal modifications Molecular mass Calculated Monoisotopic m/z trna Ala AGC, trna Ile AAU, trna Leu CAA, trna Leu UAG, and trna Trp CCA (Type a) Charge state pppaacp 1, , ppaacp 1, , paacp 1, , GpppAACp 1, , m 7 GpppAACp 1, , DMGpppAACp 1, , TMGpppAACp 1, , trna Ile UAU pppgaaaaup 2, , ppgaaaaup 2, , pgaaaaup 2, , GpppGAAAAUp 2, , m 7 GpppGAAAAUp 2, , DMGpppGAAAAUp 2, , TMGpppGAAAAUp 2, , trna Lys UUU pppgaagaacp 2, , ppgaagaacp 2, , pgaagaacp 2, , GpppGAAGAACp 2, , m 7 GpppGAAGAACp 2, , DMGpppGAAGAACp 2, , TMGpppGAAGAACp 2, , trna Trp CCA (Type b) pppaagaup 1, ppaagaup 1, paagaup 1, GpppAAGAUp 2, , m 7 GpppAAGAUp 2, , DMGpppAAGAUp 2, , TMGpppAAGAUp 2, ,

3 Supplementary Table 2a. 3' terminal fragments of pre-trnas isolated from the POP4-repressed cells (digested by RNase T 1 ). trnas Forms Sequences of RNA fragments Calculated Observed trna Ala AGC Pre UCCACCA OH (Mature) 2, , , trna Ile AAU Pre ACCACCA OH (Mature) 2, , , trna Ile UAU Pre 1 CAcuuucuc OH * 2, , , CAcuuucu OH 2, , , trna Ile UAU Pre 3 CACCA OH (Mature) 1, trna Leu CAA Pre 1 CAACCAu OH 2, , , CAACCA OH 1, CAACCACCA OH (Mature) 2, , , trna Leu CAA Pre 3 CAACCACCA OH (Mature) 2, , , trna Lys UUU Pre 1 uucuuuac OH 2, , , uucuuu OH 1, Pre 3U uucuuua OH 2, , , Pre 3L uucuu OH 1, CCA OH (Mature) trna Trp CCA_a Pre 1a UUUCAauuua OH 3, , , Pre 3a UUUCACCA OH (Mature) 2, , , trna Trp CCA_b Pre 1b UUUCAu OH 1, UUUCA OH 1, Pre 3b UUUCACCA OH (Mature) 2, , , * 3' trailer sequences are shown in small letters. Molecular mass (Da) Monoisotopic m/z Charge state Supplementary Table 2b. 3' terminal fragments of pre trnas isolated from the wild type cells (digested by RNase T 1 ). trnas Forms Sequences of RNA fragments Calculated Observed trna Leu CAA Pre 1 CAACCAu OH 2, , , CAACCACCA OH (Mature) 2, , , trna Leu UAG Pre 1 CUCUCAuuauuuu OH 3, , , CUCUCACCA OH (Mature) 2, , , trna Trp CCA_a Pre 1a UUUCACCA OH (Mature) 2, , , trna Trp CCA_b Pre 1b UUUCAuuu OH * 2, , , UUUCAuu OH 2, , , UUUCAu OH 1, trna Trp CCA_b Pre 3b UUUCA OH 1, * 3' trailer sequences are shown in small letters. Molecular mass (Da) Monoisotopic m/z Charge state UUUCACCA OH (Mature) 2, , ,

4 Supplementary Table 3. List of primers and probes used in this study. Primers for gene disruption Ceg1_KS_F Ceg1_SK_R Tgs1_KS_F Tgs1_SK_R Swm2_KS_F Swm2_SK_R Met22_KS_F Met22_SK_R Xrn1_KS_F Xrn1_SK_R Msn5_KS_F Msn5_SK_R Primers for check the gene disruption Ceg1_check_F Ceg1_check_R Tgs1_check_F Tgs1_check_R Swm2_check_F Swm2_check_R Met22_check_F Met22_check_R Xrn1_check_F Xrn1_check_R Msn5_check_F Msn5_check_R Primers for CEG1 gene cloning Ceg1_ORF_HindIII_F Ceg1_ORF_NotI_R Primers for quick change Ceg1_335CtoT_F Ceg1_335CtoT_R Ceg1_454GtoA_F Ceg1_454GtoA_R DNA probes for trna isolation trna Ala AGC trna Ile AAU trna Ile UAU trna Ile UAU 3' biotin trna Leu CAA trna Leu UAG trna Lys UUU trna Trp CCA DNA probes for nothern blotting trna Ala AGC trna Ile AAU trna Ile UAU trna Leu CAA trna Leu UAG trna Lys UUU trna Trp CCA U4 snrna human trna Ile UAU human trna Leu CAA_probe #1 human trna Leu CAA_probe #2 human U1 snrna Sequences (5' - 3') AGTTCTCCATGATGTTCGGGTCAGTCGCTCCGAAGCGTAACCTAGTCGAGGTCGACGGTATC ACAAAATTCTTACACCGATATATGTCGATACGAACACTTCTTAGCCGCTCTAGAACTAGTGGATC CAGACAGAAGTAGAAGAAAAAATTTTTGTTGATTTAACTGAAGTGTCGAGGTCGACGGTATC ATAACATAGTCATTGCATTTTCTATCATACGATTCAACTTGAACACGCTCTAGAACTAGTGGATC AGGAGACTAAATGCATAAGCAAAGAGAAGAGGATCTCCTCGGATGTCGAGGTCGACGGTATC GGTAGGCTCCGTGCATATTAGATGAGTGAACACACTTTAGGTTTACGCTCTAGAACTAGTGGATC TAAGAAGTTTAAAGACAACTCAGAAGACATCAGCACTTTACTATGTCGAGGTCGACGGTATC CTCATATATTTATGTCTATCAATAAAGTAAAATATATGTTATTTACGCTCTAGAACTAGTGGATC ACACTTGTAACAACAGCAGCAACAAATATATATCAGTACGGTATGTCGAGGTCGACGGTATC AACCCATTGTAAACCTTCAACGTAGTCTTTAGCAAGATCTCTCACCGCTCTAGAACTAGTGGATC AGAATCTGAAATTTTTTTTCTAACGTTGATTGGAAGAAAAGTAATTCGAGGTCGACGGTATC AACATAGAGACAACTGATCATATCCACGAACTTTTTAATTATAGGCGCTCTAGAACTAGTGGATC TGACCAGCTGAAAGGCAGATGATC TTTCCTAAGTGGTAACGGTAACC AGTTTTTCGTTGTATTGATTGCC ACGTAAGCGATCCTCTTTATGTGC AATCTGGTTTTACTCGTAGCCTTTT GGGAAAATACCTAGGTTGGTTAATG CAGTGAAGAGTTAAAACGTAAGGGA TTACAGATACCTTCCAATTCGTTTT ACAAACAAGAAGAGGTTAGAAAGCA AATGAGATCAATGAGAAGAAAGTGC TCGTGTAGCAATTGTTACTGTGA TAATCCGCTAACGATTAAGCTTGG GGGAAGCTTATGGTTTTAGCAATGGAAAGTAGAG GGGGCGGCCGCCTAATCCGACCAATCATCCTCGTCTA CCCAGATTACTCCAAAAGAAGAAAGAAGAGCTGC CTTCTTTTGGAGTAATCTGGGAAACCTAAATCCAT TTTAATGTTCAATTGTCTTGCTATCAATGGTAGATG GCAAGACAATTGAACATTAAATAACGCAACTCTTG 5' EC amino linker-gacctctcccatgctaagggagcgcgctac 5' EC amino linker-gatccccgcgttattagcacggtgccttaa 5' EC amino linker-ataagcacgaagctctaaccactgagctac ATAAGCACGAAGCTCTAACCACTGAGCTAC-3' bio 5' EC amino linker-tggttgctaagagattcgaactcttgcatc 5' EC amino linker-gagagctaagggattcgaacccttgcatcc 5' EC amino linker-gccgaacgctctaccaactgagctaacaag 5' EC amino linker-tgaaacggacaggaattgaacctgcaaccc TGGACGAGTCCGGAATCGAACCGGAG GGTCTCTAGCGGGATCGAACCGCTG GTGGGGATTGAACCCACGACGGTCGCG GAACTCTTGCATCTTACGATACCTGAG GAGAGCTAAGGGATTCGAACCCTTG TGGCTCCTCATAGGGGGCTCGAAC TGAAACGGACAGGAATTGAACCTGC CTCGGACGAATCCTCACTGATATGCG GCTCCAGGTGAGGCTCGAACTCACA CCTTAGACCACTCGGCCATCCTGAC TGTCAGAAGTGGGATTCGAACCCA GGTATCTCCCCTGCCAGGTAAGTAT 4

5 Supplementary Table 4. List of yeast strains used in this study. Strains Genotype Comments BY4742 MATα his3-1 leu2-0 lys2-0 ura3-0 EUROSCARF Δlos1 BY4742 los1::kanmx EUROSCARF Δmsn5 BY4742 msn5::kanmx EUROSCARF Δlos1/Δmsn5 BY4742 los1::kanmx msn5::leu2 this study teto 7 -POP4 R1158 ppop4::kanr-teto 7 -TATA GE Helthcare CEG1 + / teto7-pop4 teto 7 -POP4 ceg1::his3 pad4ceg1 WT this study ceg1 ts / teto7-pop4 teto 7 -POP4 ceg1::his3 pad4ceg1 ts this study Δtgs1 / teto7-pop4 teto 7 -POP4 tgs1::his3 this study Δswm2 / teto7-pop4 teto 7 -POP4 swm2::his3 this study Δmet22 / teto7-pop4 teto 7 -POP4 met22::his3 this study Δxrn1 / teto7-pop4 teto 7 -POP4 xrn1 ::LEU2 this study W303 MATa leu2-3, 112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 GE Helthcare MW1029 MATa ade2-101 lys2-801 leu2-1 his3-200 ura3-52 trp1-63 rpc160-1::his3 pc [TRP1 CEN4 rpc ] Thuillier et al. (1996) EMBO J 15: Supplementary Data Set 1 RLE values of CAGE analysis in trna genes. This data set is provided by a separated spread sheet. The RLE (Relative Log Expression) values of CAGE analyses for 10 human trna genes and 6 mouse trna genes in various cell lines obtained from FAMTOM5 database. 5

6 Supplementary Figures Supplementary Figure 1. Secondary structures of trna precursors accumulated in 6

7 the POP4-repressed cells. Supplementary Figure 1. Secondary structures of trna precursors accumulated in the POP4-repressed cells. Secondary structures of primary transcripts (top panels), Pre (middle panels), Pre 1 (middle panels), and Pre 3 (bottom panels) for each trna are represented. Oligo Us of primary transcripts were predicted based on their trna genes. Splice sites for intron removal are indicated by arrows. Sequences of 5 leader, 3 trailer and intron are shown in small letters. 5 and 3 termini of each pre-trna were determined by mass spectrometric analysis (Supplementary Figs. 5 and 6, Supplementary Tables 1 and 2a). Internal modifications are not shown. A region indicated by blue line in each trna 7

8 represents a DNA probe for Northern blotting. Supplementary Figure 2. Time course accumulation of pre-trnas and their 5 -capping. (a) Northern blotting of trna Ala AGC, trna Trp CCA, trna Lys UUU, and trna Leu UAG in POP4-repressed cells, harvested at the indicated period of time (0-10h) after doxycycline (Dox) addition. DNA probes for Northern blotting are listed in Supplementary Table 4. (b). Northern blotting of trna Trp CCA and trna Leu UAG immunoprecipitated with the anti-methyl-g cap antibody from the total RNA prepared from the cells as described above. 8

9 Supplementary Figure 3. Isolation of pre-trnas from the POP4-repressed cells by RCC. The isolated trnas were resolved by electrophoresis on 10% polyacrylamide gel containing 7 M urea, and stained by SYBR Gold. Precursors (Pre 1, Pre 2 and Pre 3) and mature (M) trnas are indicated. 9

10 Supplementary Figure 4. LC/MS co-injection analysis to confirm 5 capping of pre-trna Ile UAU. (a) Extracted ion chromatogram (XIC) of synthetic m 7 GpppG (200 fmol) analyzed by HILIC/ESI-MS and detected as a proton-adduct (m/z 803.1). (b) Total nucleoside of Pre 3 of trna Ile UAU isolated from POP4-repressed cells analyzed by HILIC/ESI-MS with (right panels) or without (left panels) synthetic m 7 GpppG. From top to bottom, XICs for monovalent positive ions of GpppG (m/z ), m 7 GpppG (m/z ), DMGpppG (m/z ), and TMGpppG (m/z ), respectively. The increased peak in the XIC by adding synthetic m 7 GpppG is indicated by red arrow. 10

11 Supplementary Figure 5. CID spectra of 5 terminal RNA fragments derived from pre-trna Trp. Divalent negative ions of the seven different 5 terminal fragments from pre-trna Trp digested by RNase A (Supplementary Table 1) were used as precursor ions for CID. The product ions in the CID spectrum are assigned on the sequences. 11

12 Supplementary Figure 6. CID spectra of 3 terminal RNA fragments derived from pre-trna Trp. Divalent negative ions of the five different 3 terminal fragments from pre-trna Trp digested by RNase T 1 (Supplementary Table 2b) were used as precursor ions for CID. The product ions in the CID spectrum are assigned on the sequences. 12

Supplementary Figure 7. Quantification of 5 terminal fragments in pre-trnas. Five species of pre-trnas isolated from naturally-growing wild cells were detected by LC/MS analyses (Figure 4b).

13 Supplementary Figure 7. Quantification of 5 terminal fragments in pre-trnas. Five species of pre-trnas isolated from naturally-growing wild cells were detected by LC/MS analyses (Figure 4b). XIC peak areas of 5 terminal fragments bearing triphosphate (ppp), diphosphate (pp), monophosphate (p), guanosine cap, m 7 G-cap, DMG-cap, and TMG-cap in each pre-trna were quantified. For each pre-trna, relative amount of each fragment was calculated and shown in the bar graph. Basically, in negative mode of ESI, ionization efficiencies of the RNA fragments bearing the same sequence but different modification do not differ largely, because ESI ionization relies mainly on numbers of phosphate groups, not on type of base modifications. Thus, we can relatively quantify abundance of 5 terminal fragments with different chemical structures from their intensities of XICs. However, relative ionization efficiencies of RNA fragments with 5 p, 5 pp and 5 ppp are found to be 100:43:34, according to our measurement of authentic fragments with different 5 termini. XIC peak area of each fragment was multiplied by these factors for compensation. 13

14 Supplementary Figure 8. The capped pre-trnas are transcribed by RNA pol III. (a) Northern blotting of trna Trp CCA, trna Leu UAG, U6 snrna (pol III transcript), and U4 snrna (pol II transcript) in wild type (W303) and pol III ts (MW1029) strains after heat induction. Total RNAs (1.4 μg each) from W303 and MW1029 strains cultured at non-permissive temperature (37 C) for 0h, 5h, and 10h were subjected to Northern blotting with DNA probes as shown in Supplementary Table 4. Precursors (Pre 1a, Pre 1b, Pre 1, and Pre 2) and mature (M) trnas are indicated. Unassigned band is indicated by the asterisk. (b) Northern blotting of trna Trp CCA and U4 snrna immunoprecipitated with the anti-methyl-g cap antibody from the total RNA (28 μg) prepared from W303 and MW1029 strains cultured at non-permissive temperature (37 C) for 0h and 10h. 14

15 Supplementary Figure 9. Pre-tRNA capping protects trna precursors from 5 exonucleolytic degradation. (a) Steady-state levels of precursors of three trna species detected by Northern blotting of total RNAs from a series of POP4-repressed strains, wild-type (-) (left lanes), CEG1 + (middle lanes), and ceg1 ts (right lanes), respectively. Upon POP4 repression, precursors (Pre, Pre 1, and Pre 3) accumulated markedly. M, mature trna. (b) Steady-state levels of precursors of three trna species detected by Northern 15

16 blotting of total RNAs from the POP4-repressed strains, wild-type (-) (left lanes) and Δmet22 (right lanes). (c) Quantification of the Northern blotting for pre-trnas accumulated in the POP4-repressed strain (black bars) and Δmet22 strain with POP4-repressed background (gray bars). Relative abundance of pre-trna (%) was normalized with that of mature trna. Data represent average values with s.d. of triplicate. *P < 0.05 versus control by Student s t-tests. 16

Supplementary Figure 10 Detection of pre-trna capping in human cells. (a) Immunoprecipitation and Northern blotting to detect pre-trna capping in human cells.

17 Supplementary Figure 10 Detection of pre-trna capping in human cells. (a) Immunoprecipitation and Northern blotting to detect pre-trna capping in human cells. Total RNA from HeLa cells was immunoprecipitated with anti-methyl-g cap antibody or control IgG, followed by Northern blotting to detect pre-trna Ile UAU (left panel), and U1 snrna (right panel, positive control). The middle panel represents longer exposure of the boxed area in the left panel. Unassigned band is indicated by the asterisk. (b) Immunoprecipitation of human pre-trnas by anti-methyl-g cap antibody immobilized on agarose beads, followed by Northern blotting to detect pre-trna Ile UAU (left panel), pre-trna Leu CAA (middle panels), and U1 snrna (right panel, positive control). Two different probes (#1 and #2, listed in Supplementary Table 4) were used for pre-trna Leu CAA. Precursors (Pre 1 and Pre 3) and mature (M) trnas are indicated. Input, FT, IP, and IP 19 represent total RNA of HeLa cells, flow-through fraction, one-volume of immunoprecipitant, and 19 volumes of immunoprecipitant, respectively. 17

18 Supplementary Figure 11. CAGE data in trna genes. 18

19 Supplementary Figure 11. CAGE data in trna genes. Mapping the result of CAGE by FANTOM5 project on human (a) and mouse (b) trna genes with flanking regions 1. Chromosome number and genomic locus of each trna gene is represented. Direction of each trna gene is shown by arrow. The RLE (Relative Log Expression) values of CAGE analysis at each individual site in various cell lines (Supplementary Data Set 1) are integrated and shown by black bar. 19

20 Supplementary Figure 12. Northern blotting of trna Ile UAU in trna exportin deletion strains. Total RNAs from Δlos1, Δmsn5, and Δlos1/Δmsn5 strains were subjected to Northern blotting with a probe specific to trna Ile UAU (Supplementary Table 4). Precursors (Pre 1, Pre 2 and Pre 3) and mature (M) trnas are indicated. Unassigned band is indicated by the asterisk. 20

21 Supplementary Figure 13. Full-size images for Figures as indicated The cut region of each image for use in the figure is red-boxed. 21

22 Supplementary References 1. Forrest, A.R. et al. A promoter-level mammalian expression atlas. Nature 507, (2014). 22

Supplementary Figure 1

Supplementary Figure 1 Isolation of mt-trnas and RNA-MS analysis of mt-trna Asn from M. nudus (a)m. nudus mt-trnas were isolated by RCC and resolved by 10% denaturing PAGE. The gel was stained with SYBR