#METABOLOMICS WORKBENCH atsai_20250207_210246 DATATRACK_ID:5605 STUDY_ID:ST003728 ANALYSIS_ID:AN006116 PROJECT_ID:PR002314 VERSION 1 CREATED_ON February 13, 2025, 5:23 pm #PROJECT PR:PROJECT_TITLE Comprehensive Nucleoside Analysis of Archaeal RNA Modification Profiles Reveals PR:PROJECT_TITLE a m7G in the Conserved P-loop of 23S rRNA PR:PROJECT_SUMMARY Extremophilic archaea employ diverse chemical RNA modifications, providing a PR:PROJECT_SUMMARY rich source of new enzymes for biotechnologically valuable RNA manipulations. PR:PROJECT_SUMMARY Our understanding of the modified nucleoside profiles in Archaea, as well as the PR:PROJECT_SUMMARY functions and dynamic regulation of specific RNA modifications is far from PR:PROJECT_SUMMARY complete. Here, we established an extensive profile of nucleoside modifications PR:PROJECT_SUMMARY in thermophilic and mesophilic Archaea through highly sensitive LC-MS/MS PR:PROJECT_SUMMARY analysis and rigorous non-coding RNA depletion, identifying - with high PR:PROJECT_SUMMARY confidence - at least four previously unannotated modifications in archaeal PR:PROJECT_SUMMARY mRNAs. Nucleoside quantification analysis conducted on total, large, small, and PR:PROJECT_SUMMARY mRNA-enriched subfractions of the model hyperthermophilic archaeon Thermococcus PR:PROJECT_SUMMARY kodakarensis revealed a series of modifications whose abundance is dynamically PR:PROJECT_SUMMARY responsive to growth temperatures, implying that specific RNA modifications are PR:PROJECT_SUMMARY fitness relevant under specific growth conditions. To predict the RNA-modifying PR:PROJECT_SUMMARY enzymes most likely to generate the new and dynamic RNA modifications, we PR:PROJECT_SUMMARY leveraged a bioinformatics analysis of open-access databases to annotate likely PR:PROJECT_SUMMARY functional domains of archaeal proteins. Putative enzyme activities were PR:PROJECT_SUMMARY confirmed in vitro and in vivo by assessing the presence of the target RNA PR:PROJECT_SUMMARY modification in genetic deletion strains of T. kodakarensis. Our approach led to PR:PROJECT_SUMMARY the discovery of a methyltransferase-encoded gene responsible for m7G PR:PROJECT_SUMMARY modification in the P-loop of 23S rRNA peptidyl transferase center and validates PR:PROJECT_SUMMARY a novel and effective platform for discovering RNA-modifying enzymes through PR:PROJECT_SUMMARY LC-MS/MS analysis that will accelerate efforts of the community towards PR:PROJECT_SUMMARY uncovering the complex and dynamic roles of RNA modifications. PR:INSTITUTE New England Biolabs PR:LAST_NAME Tsai PR:FIRST_NAME Yueh-Lin PR:ADDRESS 44 Dunham Ridge, Beverly, Massachusetts, 01915, USA PR:EMAIL atsai@neb.com PR:PHONE 978-380-6587 #STUDY ST:STUDY_TITLE Identification and quantification of a m7G modification in T. kodakarensis ST:STUDY_TITLE ribosomal RNA ST:STUDY_SUMMARY To investigate the activity of the protein encoded by TK0008, we conducted a ST:STUDY_SUMMARY comprehensive nucleoside analysis comparing the total RNA from the TS559 ST:STUDY_SUMMARY wild-type strain with that of the TK0008-deletion strain. ST:INSTITUTE New England Biolabs ST:LAST_NAME Tsai ST:FIRST_NAME Yueh-Lin ST:ADDRESS 44 Dunham Ridge, Beverly, Massachusetts, 01915, USA ST:EMAIL atsai@neb.com ST:PHONE 978-380-6587 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Thermococcus kodakarensis SU:TAXONOMY_ID 311400 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - TS559_rep1-r001 Genotype:Wild-type | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=Tk_total_RNAmods_01-r001.d SUBJECT_SAMPLE_FACTORS - TS559_rep1-r002 Genotype:Wild-type | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=Tk_total_RNAmods_01-r002.d SUBJECT_SAMPLE_FACTORS - TS559_rep1-40x Genotype:Wild-type | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=Tk_40x_total_RNAmods_01.d SUBJECT_SAMPLE_FACTORS - TK0008_rep1-r001 Genotype:TK0008 deletion | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=Tk_total_RNAmods_02-r001.d SUBJECT_SAMPLE_FACTORS - TK0008_rep1-r002 Genotype:TK0008 deletion | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=Tk_total_RNAmods_02-r002.d SUBJECT_SAMPLE_FACTORS - TK0008_rep1-40x Genotype:TK0008 deletion | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=Tk_40x_total_RNAmods_02.d SUBJECT_SAMPLE_FACTORS - TS559_rep2-r001 Genotype:Wild-type | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TK0008_Total_01.d SUBJECT_SAMPLE_FACTORS - TS559_rep2-20x Genotype:Wild-type | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TK0008_Total_20x_01.d SUBJECT_SAMPLE_FACTORS - TK0008_rep2-r001 Genotype:TK0008 deletion | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TK0008_Total_02.d SUBJECT_SAMPLE_FACTORS - TK0008_rep2-20x Genotype:TK0008 deletion | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TK0008_Total_20x_02.d SUBJECT_SAMPLE_FACTORS - TS559_total_RNA_rep1-r001 Genotype:Wild-type | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TS559_Expo_TotalRNA_2018-r001.d SUBJECT_SAMPLE_FACTORS - TS559_total_RNA_rep1-r002 Genotype:Wild-type | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TS559_Expo_TotalRNA_2018-r002.d SUBJECT_SAMPLE_FACTORS - TK0008_total_RNA_rep1-r001 Genotype:TK0008 deletion | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TK0008_Expo_TotalRNA_2018-r001.d SUBJECT_SAMPLE_FACTORS - TK0008_total_RNA_rep1-r002 Genotype:TK0008 deletion | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TK0008_Expo_TotalRNA_2018-r002.d SUBJECT_SAMPLE_FACTORS - TS559_total_RNA_rep2-r001 Genotype:Wild-type | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TK0008_total_m7G_quant_01-1-r001.d SUBJECT_SAMPLE_FACTORS - TS559_total_RNA_rep2-r002 Genotype:Wild-type | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TK0008_total_m7G_quant_01-1-r002.d SUBJECT_SAMPLE_FACTORS - TK0008_total_RNA_rep2-r001 Genotype:TK0008 deletion | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TK0008_total_m7G_quant_02-1-r001.d SUBJECT_SAMPLE_FACTORS - TK0008_total_RNA_rep2-r002 Genotype:TK0008 deletion | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TK0008_total_m7G_quant_02-1-r002.d SUBJECT_SAMPLE_FACTORS - TS559_23S_rep1-r001 Genotype:Wild-type | Sample source:23S_rRNA_Enrichment RAW_FILE_NAME(Raw_File_Names)=TS559_23S-Enriched-r001.d SUBJECT_SAMPLE_FACTORS - TS559_23S_rep1-r002 Genotype:Wild-type | Sample source:23S_rRNA_Enrichment RAW_FILE_NAME(Raw_File_Names)=TS559_23S-Enriched-r002.d SUBJECT_SAMPLE_FACTORS - TK0008_23S_rep1-r001 Genotype:TK0008 deletion | Sample source:23S_rRNA_Enrichment RAW_FILE_NAME(Raw_File_Names)=TK0008_23S-Enriched-r001.d SUBJECT_SAMPLE_FACTORS - TK0008_23S_rep1-r002 Genotype:TK0008 deletion | Sample source:23S_rRNA_Enrichment RAW_FILE_NAME(Raw_File_Names)=TK0008_23S-Enriched-r002.d SUBJECT_SAMPLE_FACTORS - TS559_23S_rep2-r001 Genotype:Wild-type | Sample source:23S_rRNA_Enrichment RAW_FILE_NAME(Raw_File_Names)=TK0008_23S_G_quant_01-r001.d SUBJECT_SAMPLE_FACTORS - TS559_23S_rep2-r002 Genotype:Wild-type | Sample source:23S_rRNA_Enrichment RAW_FILE_NAME(Raw_File_Names)=TK0008_23S_G_quant_01-r002.d SUBJECT_SAMPLE_FACTORS - TK0008_23S_rep2-r001 Genotype:TK0008 deletion | Sample source:23S_rRNA_Enrichment RAW_FILE_NAME(Raw_File_Names)=TK0008_23S_G_quant_02-r001.d SUBJECT_SAMPLE_FACTORS - TK0008_23S_rep2-r002 Genotype:TK0008 deletion | Sample source:23S_rRNA_Enrichment RAW_FILE_NAME(Raw_File_Names)=TK0008_23S_G_quant_02-r002.d SUBJECT_SAMPLE_FACTORS - IVT23S_NE_rep1-r001 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_01-r001.d SUBJECT_SAMPLE_FACTORS - IVT23S_NE_rep1-r002 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_01-r002.d SUBJECT_SAMPLE_FACTORS - IVT23S_rTK0008_0.1uM_rep1-r001 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_02-r001.d SUBJECT_SAMPLE_FACTORS - IVT23S_rTK0008_0.1uM_rep1-r002 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_02-r002.d SUBJECT_SAMPLE_FACTORS - IVT23S_rTK0008_0.2uM_rep1-r001 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_03-r001.d SUBJECT_SAMPLE_FACTORS - IVT23S_rTK0008_0.2uM_rep1-r002 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_03-r002.d SUBJECT_SAMPLE_FACTORS - IVT23S_rTK0008_1uM_rep1-r001 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_04-r001.d SUBJECT_SAMPLE_FACTORS - IVT23S_rTK0008_1uM_rep1-r002 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_04-r002.d SUBJECT_SAMPLE_FACTORS - IVT23S_rTK0008_4uM_rep1-r001 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_05-r001.d SUBJECT_SAMPLE_FACTORS - IVT23S_rTK0008_4uM_rep1-r002 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_05-r002.d SUBJECT_SAMPLE_FACTORS - IVT23S_NE_rep2-r001 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_01_rep2-r001.d SUBJECT_SAMPLE_FACTORS - IVT23S_NE_rep2-r002 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_01_rep2-r002.d SUBJECT_SAMPLE_FACTORS - IVT23S_rTK0008_0.1uM_rep2-r001 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_02_rep2-r001.d SUBJECT_SAMPLE_FACTORS - IVT23S_rTK0008_0.1uM_rep2-r002 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_02_rep2-r002.d SUBJECT_SAMPLE_FACTORS - IVT23S_rTK0008_0.2uM_rep2-r001 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_03_rep2-r001.d SUBJECT_SAMPLE_FACTORS - IVT23S_rTK0008_0.2uM_rep2-r002 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_03_rep2-r002.d SUBJECT_SAMPLE_FACTORS - IVT23S_rTK0008_1uM_rep2-r001 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_04_rep2-r001.d SUBJECT_SAMPLE_FACTORS - IVT23S_rTK0008_1uM_rep2-r002 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_04_rep2-r002.d SUBJECT_SAMPLE_FACTORS - IVT23S_rTK0008_4uM_rep2-r001 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_05_rep2-r001.d SUBJECT_SAMPLE_FACTORS - IVT23S_rTK0008_4uM_rep2-r002 Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_05_rep2-r002.d SUBJECT_SAMPLE_FACTORS - TS559_Total_RNA_01 Genotype:Wild-type | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TS559_total_RNA_rep1-1.d SUBJECT_SAMPLE_FACTORS - TS559_Total_RNA_02 Genotype:Wild-type | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TS559_total_RNA_rep2-1.d SUBJECT_SAMPLE_FACTORS - TS559_23S_m7G_Frag_Enrich_01 Genotype:Wild-type | Sample source:23S_Fragment_Enrichment RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Frag_Enrich_rep1.d SUBJECT_SAMPLE_FACTORS - TS559_23S_m7G_Frag_Enrich_02 Genotype:Wild-type | Sample source:23S_Fragment_Enrichment RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Frag_Enrich_rep2.d SUBJECT_SAMPLE_FACTORS - TS559_23S_m7G_Fragment_01_NoDepl-r001 Genotype:Wild-type | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Fragment_01-r001-1.d SUBJECT_SAMPLE_FACTORS - TS559_23S_m7G_Fragment_01_NoDepl-r002 Genotype:Wild-type | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Fragment_01-r002-1.d SUBJECT_SAMPLE_FACTORS - TS559_23S_m7G_Fragment_02_NoDepl-r001 Genotype:Wild-type | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Fragment_02-r001-1.d SUBJECT_SAMPLE_FACTORS - TS559_23S_m7G_Fragment_02_NoDepl-r002 Genotype:Wild-type | Sample source:Total RNA RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Fragment_02-r002-1.d SUBJECT_SAMPLE_FACTORS - TS559_23S_m7G_Fragment_01_Depl-r001 Genotype:Wild-type | Sample source:23S_Fragment_Depletion RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Fragment_03-r001-1.d SUBJECT_SAMPLE_FACTORS - TS559_23S_m7G_Fragment_01_Depl-r002 Genotype:Wild-type | Sample source:23S_Fragment_Depletion RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Fragment_03-r002-1.d SUBJECT_SAMPLE_FACTORS - TS559_23S_m7G_Fragment_02_Depl-r001 Genotype:Wild-type | Sample source:23S_Fragment_Depletion RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Fragment_04-r001-1.d SUBJECT_SAMPLE_FACTORS - TS559_23S_m7G_Fragment_02_Depl-r002 Genotype:Wild-type | Sample source:23S_Fragment_Depletion RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Fragment_04-r002-1.d #COLLECTION CO:COLLECTION_SUMMARY The TK0008 open reading frame (ORF) spans positions 7655 to 8755 on the T. CO:COLLECTION_SUMMARY kodakarensis genome. PCR was used to generate an amplicon including the TK0008 CO:COLLECTION_SUMMARY ORF flanked by ~700 bp both up- and down-stream that was cloned into the unique CO:COLLECTION_SUMMARY SwaI site of pTS700 (reference Hileman and Santangelo, PMID: 22701112) 82, CO:COLLECTION_SUMMARY resulting in pCSU-TK0008A. The coding sequence of TK0008 was excised from CO:COLLECTION_SUMMARY pCSU-TK0008A using site-directed QuikChange mutagenesis (Agilent Cat# 200516) to CO:COLLECTION_SUMMARY generate pCSU-TK0008B. All plasmid sequences were confirmed by Sanger CO:COLLECTION_SUMMARY sequencing. pCSU-TK0008B was transformed into T. kodakarensis strain TS559 and CO:COLLECTION_SUMMARY plated onto agmatine-free rich media plates as previously described (reference CO:COLLECTION_SUMMARY PMID: 34752288) 85. Transformants were allowed to grow for 4 days anaerobically CO:COLLECTION_SUMMARY at 85 °C and subsequent colonies were picked into rich liquid media lacking CO:COLLECTION_SUMMARY agmatine and grown overnight. Genomic DNA extracted from 1 mL of cells was used CO:COLLECTION_SUMMARY as a template for PCRs using two sets of primer pairs to determine the site and CO:COLLECTION_SUMMARY directionality of plasmid integration into the genome. One primer from each pair CO:COLLECTION_SUMMARY has homology to the genome with the other having homology to the plasmid to CO:COLLECTION_SUMMARY ensure PCR amplification involves a region flanking TK0008 on the genome. CO:COLLECTION_SUMMARY Colonies confirmed to contain proper integrations at the TK0008 locus were CO:COLLECTION_SUMMARY plated on minimal media containing 1 mM agmatine and 100 µM 6-methyl purine and CO:COLLECTION_SUMMARY grown anaerobically at 85 °C for 2-5 days. Genomic DNA from resultant CO:COLLECTION_SUMMARY colonies was used in diagnostic PCRs to identify strains deleted for TK0008 CO:COLLECTION_SUMMARY preliminarily. The deletion of TK0008 was ultimately confirmed by Sanger CO:COLLECTION_SUMMARY sequencing. In vitro transcribed (IVT) 23S rRNA was prepared using a linearized CO:COLLECTION_SUMMARY plasmid template (pBluescript II KS(-)) and HiScribe T7 High Yield RNA Synthesis CO:COLLECTION_SUMMARY Kit (NEB, Cat #E2040S). The synthesized transcripts were cleaned up using CO:COLLECTION_SUMMARY Monarch Spin RNA Cleanup Kit (NEB, Cat #T2050L). To perform in vitro methylation CO:COLLECTION_SUMMARY reactions, IVT 23S rRNA was heat-denatured at 95 C for 30 seconds and cooled to CO:COLLECTION_SUMMARY 22 C prior to enzyme treatment. Purified recombinant TK0008 (0.1 – 4 uM) was CO:COLLECTION_SUMMARY incubated with 1 uM of IVT 23S rRNA in 1x methyltransferase buffer (25 mM CO:COLLECTION_SUMMARY Tris-HCl pH 7.5, 100 mM NaCl, 1 mM DTT) supplemented with 160 uM of CO:COLLECTION_SUMMARY S-adenosyl-methionine (SAM) (NEB, Cat #B9003S) at 85 C for 30 minutes. The CO:COLLECTION_SUMMARY reactions were cleaned up using Monarch Spin RNA Cleanup Kit (NEB, Cat #T2040L). CO:SAMPLE_TYPE Ribonucleic acid, In Vitro Transcription #TREATMENT TR:TREATMENT_SUMMARY Harvested archaeal cell pellets were resuspended in 10 mL of TRI reagent TR:TREATMENT_SUMMARY (Molecular Research Center, Inc., Cat #TR118). The resuspended cells were TR:TREATMENT_SUMMARY homogenized using a beads beater at 4.0 m/s for 20 seconds x 2 cycles (MP TR:TREATMENT_SUMMARY Biomedicals, FastPrep-24TM). Subsequently, the mixture was centrifuged at 14000 TR:TREATMENT_SUMMARY g for 5 minutes to precipitate any cell debris. Supernatants were collected TR:TREATMENT_SUMMARY post-centrifugation and treated with 50 μL of BAN reagent (Molecular Research TR:TREATMENT_SUMMARY Center, Inc., Cat #BN191) per mL of supernatant for aqueous-organic phase TR:TREATMENT_SUMMARY separation. RNA from the aqueous phase was isolated by isopropanol precipitation TR:TREATMENT_SUMMARY and subjected to DNase I treatment (NEB, Cat #M0303S) to remove genomic DNA TR:TREATMENT_SUMMARY contamination. To further purify the DNase I-treated RNA, an equal volume of TR:TREATMENT_SUMMARY acid phenol-chloroform with isoamyl alcohol (125:24:1, Thermo Fisher Scientific, TR:TREATMENT_SUMMARY Cat #AM9722) was added to the reaction and centrifuged at 21300 g for 2 minutes TR:TREATMENT_SUMMARY to separate the aqueous phase from the organic phase. The aqueous phase TR:TREATMENT_SUMMARY containing RNA was then precipitated with 1.5 volumes of isopropanol and 0.1 TR:TREATMENT_SUMMARY volume of sodium acetate (Sigma Aldrich, Cat #S7899) at –20°C overnight. TR:TREATMENT_SUMMARY Finally, the precipitated RNA pellets were washed with 75% ethanol and dissolved TR:TREATMENT_SUMMARY in nuclease-free water. To enrich m7G-modified rRNA fragment, 50 ug of total RNA TR:TREATMENT_SUMMARY from T. kodakarensis TS559 wild-type strain was mixed with a biotinylated DNA TR:TREATMENT_SUMMARY probe (1 uM) fully complementary to the target sequence in 10 mM Tris buffer (pH TR:TREATMENT_SUMMARY 7.5) and heated to 95 C for 2 minutes followed by a ramp down to 22 C. TR:TREATMENT_SUMMARY Biotinylated DNA probe sequence: TR:TREATMENT_SUMMARY 5’-Biotin-ACCTTCGAGGTGTGCGCCAGCAACTGCCACT-3’. The unhybridized RNAs were TR:TREATMENT_SUMMARY digested with 5 uL of RNase 4 (NEB, Cat #M1284L) in 1x NEB buffer r1.1 at 37 TR:TREATMENT_SUMMARY C for 1 hour. The digestion was stopped by adding 10 L of human placental TR:TREATMENT_SUMMARY RNase inhibitor (NEB, Cat #M0307S) to the reaction mixture and incubated at room TR:TREATMENT_SUMMARY temperature for 10 minutes. Selective enrichment of the probe-hybridized rRNA TR:TREATMENT_SUMMARY region was performed using streptavidin magnetic beads (NEB, Cat #S1421S). TR:TREATMENT_SUMMARY Briefly, 250 uL of streptavidin magnetic beads were washed twice with equal TR:TREATMENT_SUMMARY volume of low-salt buffer (100 mM Tris pH 7.5, 10 mM EDTA, 50 mM NaCl) and TR:TREATMENT_SUMMARY resuspended in 50 uL of high-salt buffer (100 mM Tris pH 7.5, 10 mM EDTA, 250 mM TR:TREATMENT_SUMMARY NaCl). The high-salt buffer was removed, and the beads were incubated with the TR:TREATMENT_SUMMARY reaction mixture for 15 minutes at room temperature with occasional agitation. TR:TREATMENT_SUMMARY The bead-captured DNA-RNA hybrid was washed and eluted by heating to 80 C for 5 TR:TREATMENT_SUMMARY minutes in 30 uL of nuclease-free water. For m7G-modified rRNA fragment TR:TREATMENT_SUMMARY depletion, the same biotinylated DNA probe (1 uM) described above was mixed with TR:TREATMENT_SUMMARY 5 ug of total RNA from TS559 strain in the probe hybridization buffer provided TR:TREATMENT_SUMMARY in NEBNext rRNA Depletion Kit (NEB, Cat #E7850X). The probe-hybridized rRNA TR:TREATMENT_SUMMARY mixture was subjected to rRNA depletion according to the NEBNext rRNA Depletion TR:TREATMENT_SUMMARY Kit protocol. Following the enzymatic treatment, the reactions were cleaned up TR:TREATMENT_SUMMARY using RNA Clean and Concentrator Kit (Zymo Research, Cat #R1017). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Total or isolated RNAs were digested to nucleosides at 37°C overnight using a SP:SAMPLEPREP_SUMMARY Nucleoside Digestion Mix (NEB, Cat #M0649S). The digested RNAs were subsequently SP:SAMPLEPREP_SUMMARY injected without prior purification on an Agilent 1290 Infinity II UHPLC SP:SAMPLEPREP_SUMMARY equipped with a G7117 diode array detector and an Agilent 6495C SP:SAMPLEPREP_SUMMARY Triple-Quadrupole Mass Spectrometer operating in positive electrospray SP:SAMPLEPREP_SUMMARY ionization (+ESI) mode. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Solvent A pH is 4.5 CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Waters XSelect HSS T3 XP (100 × 2.1mm, 2.5um) CH:SOLVENT_A 100% water; 10mM ammonium acetate CH:SOLVENT_B 100% methanol CH:FLOW_GRADIENT 1%-23% Solvent B in 7.5 min CH:FLOW_RATE 0.6mL/min CH:COLUMN_TEMPERATURE 30 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6495 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Mass spectrometric data were acquired using dynamic multiple reaction monitoring MS:MS_COMMENTS (DMRM) mode. Identification of each nucleoside species was based on the MS:MS_COMMENTS associated retention time and mass transition in the extracted chromatogram. MS:MS_COMMENTS Quantification of nucleosides were based on external calibration curves. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS femtomole MS_METABOLITE_DATA_START Samples TS559_total_RNA_rep1-r001 TS559_total_RNA_rep1-r002 TK0008_total_RNA_rep1-r001 TK0008_total_RNA_rep1-r002 TS559_total_RNA_rep2-r001 TS559_total_RNA_rep2-r002 TK0008_total_RNA_rep2-r001 TK0008_total_RNA_rep2-r002 TS559_23S_rep1-r001 TS559_23S_rep1-r002 TK0008_23S_rep1-r001 TK0008_23S_rep1-r002 TS559_23S_rep2-r001 TS559_23S_rep2-r002 TK0008_23S_rep2-r001 TK0008_23S_rep2-r002 IVT23S_NE_rep1-r001 IVT23S_NE_rep1-r002 IVT23S_rTK0008_0.1uM_rep1-r001 IVT23S_rTK0008_0.1uM_rep1-r002 IVT23S_rTK0008_0.2uM_rep1-r001 IVT23S_rTK0008_0.2uM_rep1-r002 IVT23S_rTK0008_1uM_rep1-r001 IVT23S_rTK0008_1uM_rep1-r002 IVT23S_rTK0008_4uM_rep1-r001 IVT23S_rTK0008_4uM_rep1-r002 IVT23S_NE_rep2-r001 IVT23S_NE_rep2-r002 IVT23S_rTK0008_0.1uM_rep2-r001 IVT23S_rTK0008_0.1uM_rep2-r002 IVT23S_rTK0008_0.2uM_rep2-r001 IVT23S_rTK0008_0.2uM_rep2-r002 IVT23S_rTK0008_1uM_rep2-r001 IVT23S_rTK0008_1uM_rep2-r002 IVT23S_rTK0008_4uM_rep2-r001 IVT23S_rTK0008_4uM_rep2-r002 TS559_Total_RNA_01 TS559_Total_RNA_02 TS559_23S_m7G_Frag_Enrich_01 TS559_23S_m7G_Frag_Enrich_02 TS559_23S_m7G_Fragment_01_NoDepl-r001 TS559_23S_m7G_Fragment_01_NoDepl-r002 TS559_23S_m7G_Fragment_02_NoDepl-r001 TS559_23S_m7G_Fragment_02_NoDepl-r002 TS559_23S_m7G_Fragment_01_Depl-r001 TS559_23S_m7G_Fragment_01_Depl-r002 TS559_23S_m7G_Fragment_02_Depl-r001 TS559_23S_m7G_Fragment_02_Depl-r002 Factors Genotype:Wild-type | Sample source:Total RNA Genotype:Wild-type | Sample source:Total RNA Genotype:TK0008 deletion | Sample source:Total RNA Genotype:TK0008 deletion | Sample source:Total RNA Genotype:Wild-type | Sample source:Total RNA Genotype:Wild-type | Sample source:Total RNA Genotype:TK0008 deletion | Sample source:Total RNA Genotype:TK0008 deletion | Sample source:Total RNA Genotype:Wild-type | Sample source:23S_rRNA_Enrichment Genotype:Wild-type | Sample source:23S_rRNA_Enrichment Genotype:TK0008 deletion | Sample source:23S_rRNA_Enrichment Genotype:TK0008 deletion | Sample source:23S_rRNA_Enrichment Genotype:Wild-type | Sample source:23S_rRNA_Enrichment Genotype:Wild-type | Sample source:23S_rRNA_Enrichment Genotype:TK0008 deletion | Sample source:23S_rRNA_Enrichment Genotype:TK0008 deletion | Sample source:23S_rRNA_Enrichment Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription Genotype:Wild-type | Sample source:Total RNA Genotype:Wild-type | Sample source:Total RNA Genotype:Wild-type | Sample source:23S_Fragment_Enrichment Genotype:Wild-type | Sample source:23S_Fragment_Enrichment Genotype:Wild-type | Sample source:Total RNA Genotype:Wild-type | Sample source:Total RNA Genotype:Wild-type | Sample source:Total RNA Genotype:Wild-type | Sample source:Total RNA Genotype:Wild-type | Sample source:23S_Fragment_Depletion Genotype:Wild-type | Sample source:23S_Fragment_Depletion Genotype:Wild-type | Sample source:23S_Fragment_Depletion Genotype:Wild-type | Sample source:23S_Fragment_Depletion N7-methylguanosine 1.069404542 1.140290953 Below quantification threshold Below quantification threshold 2.067702764 2.045020596 Below quantification threshold Below quantification threshold 1.505198523 1.63098593 Below quantification threshold Below quantification threshold 1.379349924 1.39669465 Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold 4.82151824 4.823462046 4.845629111 4.86965539 9.04628025 9.189905549 14.80420995 14.58444555 Below quantification threshold Below quantification threshold 4.155099643 4.0735146 4.892938853 4.913719581 10.32433379 10.2407819 15.99932366 16.10461049 1.106687249 1.148090997 13.59509828 13.56042644 1.299136788 1.285427573 1.148648408 1.172997387 Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold 2'-O-methylguanosine 30.52495 33.9301667 34.11093342 34.59675167 26.98794189 26.87576099 26.01251787 26.56282953 24.52489833 26.34700572 27.02985679 28.26926751 11.86217857 11.76215863 11.15726267 11.29835347 20.11060709 23.52792285 19.95638948 20.04683372 23.78308821 24.02844311 21.3962676 22.14792481 22.53674769 21.17051488 19.23391715 19.72384225 1-methylguanosine 2.978652183 3.118239816 2.550808016 2.382940316 2.789260559 2.40467494 2.76343368 2.789047329 Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold N2-methylguanosine 3.026629282 3.455318358 3.262562398 3.497205849 2.64270022 2.43175043 2.610252437 2.656593673 Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold N2,N2-dimethylguanosine 6.36459465 6.733362361 6.386636246 6.921547598 3.74254043 3.841615761 3.733377895 3.808665616 Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold N2,2'-O-dimethylguanosine 0.634697195 0.670559138 0.68015444 0.711311892 0.612311413 0.624565794 0.58320341 0.61396113 Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold Below quantification threshold guanosine 2390.772788 2474.724114 2290.068802 2320.781198 1959.380457 1909.986201 1949.272623 1939.596462 1787.867023 1903.895681 1817.361054 1958.682289 882.9355854 897.8569827 824.0849548 872.8014019 731.7381594 728.9300989 723.1059321 723.3564415 731.087679 732.9107201 687.4400548 693.6221973 632.093779 618.0317143 642.2355634 630.4995318 621.0317433 619.7349263 568.9198595 566.9349309 631.3691144 622.4700873 527.7945467 532.1928914 1432.987348 1669.252991 1554.429032 1538.281698 1630.815742 1635.119504 1525.188201 1582.070006 1575.182478 1524.379369 1426.915016 1431.06202 N4-acetylcytidine 55.39391224 61.5039164 16.96382907 16.70524155 35.25070282 34.97489445 31.70272548 32.76867936 34.22930354 33.47028881 30.22507022 30.13655461 2'-O-methylcytidine 20.90136965 24.86298594 3.903562864 4.028678756 12.52409419 12.51940805 12.24142176 12.60057417 12.72499461 12.24043332 11.46499129 11.79870444 5-methylcytidine 23.39266526 27.97527197 4.845177298 4.655845153 14.53674113 14.82199235 14.30376686 14.60656303 15.34284149 14.13620289 14.08655142 14.45777179 cytidine 906.3217461 1063.98195 980.8034523 967.7749634 963.6829353 969.2126242 921.1383788 955.9028862 942.7007041 906.6480165 864.9813256 866.7020679 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Mass Transition N7-methylguanosine 298.1 -> 166.2 2'-O-methylguanosine 298.1 -> 152.1 1-methylguanosine 298.1 -> 166.2 N2-methylguanosine 298.1 -> 166.2 N2,N2-dimethylguanosine 312.1 -> 180.2 N2,2'-O-dimethylguanosine 312.1 -> 166.1 guanosine 284.1 -> 152.1 N4-acetylcytidine 286.1 -> 154.1 2'-O-methylcytidine 258.1 -> 112.1 5-methylcytidine 258.1 -> 126.1 cytidine 244.1 -> 112.1 METABOLITES_END #END