Summary of Study ST003728

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002314. The data can be accessed directly via it's Project DOI: 10.21228/M8HR7S This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003728
Study Title	Identification and quantification of a m7G modification in T. kodakarensis ribosomal RNA
Study Summary	To investigate the activity of the protein encoded by TK0008, we conducted a comprehensive nucleoside analysis comparing the total RNA from the TS559 wild-type strain with that of the TK0008-deletion strain.
Institute	New England Biolabs
Last Name	Tsai
First Name	Yueh-Lin
Address	44 Dunham Ridge, Beverly, Massachusetts, 01915, USA
Email	atsai@neb.com
Phone	978-380-6587
Submit Date	2025-02-07
Raw Data Available	Yes
Raw Data File Type(s)	mzML, d
Analysis Type Detail	LC-MS
Release Date	2025-02-14
Release Version	1

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Project:

Project ID:	PR002314
Project DOI:	doi: 10.21228/M8HR7S
Project Title:	Comprehensive Nucleoside Analysis of Archaeal RNA Modification Profiles Reveals a m7G in the Conserved P-loop of 23S rRNA
Project Summary:	Extremophilic archaea employ diverse chemical RNA modifications, providing a rich source of new enzymes for biotechnologically valuable RNA manipulations. Our understanding of the modified nucleoside profiles in Archaea, as well as the functions and dynamic regulation of specific RNA modifications is far from complete. Here, we established an extensive profile of nucleoside modifications in thermophilic and mesophilic Archaea through highly sensitive LC-MS/MS analysis and rigorous non-coding RNA depletion, identifying - with high confidence - at least four previously unannotated modifications in archaeal mRNAs. Nucleoside quantification analysis conducted on total, large, small, and mRNA-enriched subfractions of the model hyperthermophilic archaeon Thermococcus kodakarensis revealed a series of modifications whose abundance is dynamically responsive to growth temperatures, implying that specific RNA modifications are fitness relevant under specific growth conditions. To predict the RNA-modifying enzymes most likely to generate the new and dynamic RNA modifications, we leveraged a bioinformatics analysis of open-access databases to annotate likely functional domains of archaeal proteins. Putative enzyme activities were confirmed in vitro and in vivo by assessing the presence of the target RNA modification in genetic deletion strains of T. kodakarensis. Our approach led to the discovery of a methyltransferase-encoded gene responsible for m7G modification in the P-loop of 23S rRNA peptidyl transferase center and validates a novel and effective platform for discovering RNA-modifying enzymes through LC-MS/MS analysis that will accelerate efforts of the community towards uncovering the complex and dynamic roles of RNA modifications.
Institute:	New England Biolabs
Last Name:	Tsai
First Name:	Yueh-Lin
Address:	44 Dunham Ridge, Beverly, MA 01915
Email:	atsai@neb.com
Phone:	978-380-6587

Subject:

Subject ID:	SU003860
Subject Type:	Cultured cells
Subject Species:	Thermococcus kodakarensis
Taxonomy ID:	311400

Factors:

Subject type: Cultured cells; Subject species: Thermococcus kodakarensis (Factor headings shown in green)

mb_sample_id	local_sample_id	Genotype	Sample source
SA407232	IVT23S_NE_rep2-r001	Synthetic 23S_rRNA	In vitro transcription
SA407233	IVT23S_rTK0008_0.1uM_rep1-r001	Synthetic 23S_rRNA	In vitro transcription
SA407234	IVT23S_rTK0008_0.2uM_rep1-r001	Synthetic 23S_rRNA	In vitro transcription
SA407235	IVT23S_rTK0008_0.2uM_rep1-r002	Synthetic 23S_rRNA	In vitro transcription
SA407236	IVT23S_rTK0008_1uM_rep1-r001	Synthetic 23S_rRNA	In vitro transcription
SA407237	IVT23S_rTK0008_1uM_rep1-r002	Synthetic 23S_rRNA	In vitro transcription
SA407238	IVT23S_rTK0008_4uM_rep1-r001	Synthetic 23S_rRNA	In vitro transcription
SA407239	IVT23S_rTK0008_4uM_rep1-r002	Synthetic 23S_rRNA	In vitro transcription
SA407240	IVT23S_NE_rep2-r002	Synthetic 23S_rRNA	In vitro transcription
SA407241	IVT23S_NE_rep1-r001	Synthetic 23S_rRNA	In vitro transcription
SA407242	IVT23S_rTK0008_0.1uM_rep2-r001	Synthetic 23S_rRNA	In vitro transcription
SA407243	IVT23S_rTK0008_0.1uM_rep2-r002	Synthetic 23S_rRNA	In vitro transcription
SA407244	IVT23S_rTK0008_0.2uM_rep2-r001	Synthetic 23S_rRNA	In vitro transcription
SA407245	IVT23S_rTK0008_0.2uM_rep2-r002	Synthetic 23S_rRNA	In vitro transcription
SA407246	IVT23S_rTK0008_1uM_rep2-r001	Synthetic 23S_rRNA	In vitro transcription
SA407247	IVT23S_rTK0008_1uM_rep2-r002	Synthetic 23S_rRNA	In vitro transcription
SA407248	IVT23S_rTK0008_4uM_rep2-r001	Synthetic 23S_rRNA	In vitro transcription
SA407249	IVT23S_rTK0008_4uM_rep2-r002	Synthetic 23S_rRNA	In vitro transcription
SA407250	IVT23S_NE_rep1-r002	Synthetic 23S_rRNA	In vitro transcription
SA407251	IVT23S_rTK0008_0.1uM_rep1-r002	Synthetic 23S_rRNA	In vitro transcription
SA407252	TK0008_23S_rep1-r002	TK0008 deletion	23S_rRNA_Enrichment
SA407253	TK0008_23S_rep2-r001	TK0008 deletion	23S_rRNA_Enrichment
SA407254	TK0008_23S_rep2-r002	TK0008 deletion	23S_rRNA_Enrichment
SA407255	TK0008_23S_rep1-r001	TK0008 deletion	23S_rRNA_Enrichment
SA407256	TK0008_total_RNA_rep1-r001	TK0008 deletion	Total RNA
SA407257	TK0008_rep1-r001	TK0008 deletion	Total RNA
SA407258	TK0008_rep1-r002	TK0008 deletion	Total RNA
SA407259	TK0008_rep1-40x	TK0008 deletion	Total RNA
SA407260	TK0008_rep2-r001	TK0008 deletion	Total RNA
SA407261	TK0008_rep2-20x	TK0008 deletion	Total RNA
SA407262	TK0008_total_RNA_rep1-r002	TK0008 deletion	Total RNA
SA407263	TK0008_total_RNA_rep2-r002	TK0008 deletion	Total RNA
SA407264	TK0008_total_RNA_rep2-r001	TK0008 deletion	Total RNA
SA407265	TS559_23S_m7G_Fragment_02_Depl-r001	Wild-type	23S_Fragment_Depletion
SA407266	TS559_23S_m7G_Fragment_01_Depl-r002	Wild-type	23S_Fragment_Depletion
SA407267	TS559_23S_m7G_Fragment_01_Depl-r001	Wild-type	23S_Fragment_Depletion
SA407268	TS559_23S_m7G_Fragment_02_Depl-r002	Wild-type	23S_Fragment_Depletion
SA407269	TS559_23S_m7G_Frag_Enrich_01	Wild-type	23S_Fragment_Enrichment
SA407270	TS559_23S_m7G_Frag_Enrich_02	Wild-type	23S_Fragment_Enrichment
SA407271	TS559_23S_rep2-r001	Wild-type	23S_rRNA_Enrichment
SA407272	TS559_23S_rep1-r001	Wild-type	23S_rRNA_Enrichment
SA407273	TS559_23S_rep1-r002	Wild-type	23S_rRNA_Enrichment
SA407274	TS559_23S_rep2-r002	Wild-type	23S_rRNA_Enrichment
SA407275	TS559_23S_m7G_Fragment_02_NoDepl-r002	Wild-type	Total RNA
SA407276	TS559_23S_m7G_Fragment_02_NoDepl-r001	Wild-type	Total RNA
SA407277	TS559_23S_m7G_Fragment_01_NoDepl-r002	Wild-type	Total RNA
SA407278	TS559_23S_m7G_Fragment_01_NoDepl-r001	Wild-type	Total RNA
SA407279	TS559_rep2-20x	Wild-type	Total RNA
SA407280	TS559_Total_RNA_02	Wild-type	Total RNA
SA407281	TS559_Total_RNA_01	Wild-type	Total RNA
SA407282	TS559_rep1-40x	Wild-type	Total RNA
SA407283	TS559_rep2-r001	Wild-type	Total RNA
SA407284	TS559_total_RNA_rep1-r001	Wild-type	Total RNA
SA407285	TS559_total_RNA_rep1-r002	Wild-type	Total RNA
SA407286	TS559_total_RNA_rep2-r001	Wild-type	Total RNA
SA407287	TS559_total_RNA_rep2-r002	Wild-type	Total RNA
SA407288	TS559_rep1-r002	Wild-type	Total RNA
SA407289	TS559_rep1-r001	Wild-type	Total RNA

Showing results 1 to 58 of 58

Showing results 1 to 58 of 58

Collection:

Collection ID:	CO003853
Collection Summary:	The TK0008 open reading frame (ORF) spans positions 7655 to 8755 on the T. kodakarensis genome. PCR was used to generate an amplicon including the TK0008 ORF flanked by ~700 bp both up- and down-stream that was cloned into the unique SwaI site of pTS700 (reference Hileman and Santangelo, PMID: 22701112) 82, resulting in pCSU-TK0008A. The coding sequence of TK0008 was excised from pCSU-TK0008A using site-directed QuikChange mutagenesis (Agilent Cat# 200516) to generate pCSU-TK0008B. All plasmid sequences were confirmed by Sanger sequencing. pCSU-TK0008B was transformed into T. kodakarensis strain TS559 and plated onto agmatine-free rich media plates as previously described (reference PMID: 34752288) 85. Transformants were allowed to grow for 4 days anaerobically at 85 °C and subsequent colonies were picked into rich liquid media lacking agmatine and grown overnight. Genomic DNA extracted from 1 mL of cells was used as a template for PCRs using two sets of primer pairs to determine the site and directionality of plasmid integration into the genome. One primer from each pair has homology to the genome with the other having homology to the plasmid to ensure PCR amplification involves a region flanking TK0008 on the genome. Colonies confirmed to contain proper integrations at the TK0008 locus were plated on minimal media containing 1 mM agmatine and 100 µM 6-methyl purine and grown anaerobically at 85 °C for 2-5 days. Genomic DNA from resultant colonies was used in diagnostic PCRs to identify strains deleted for TK0008 preliminarily. The deletion of TK0008 was ultimately confirmed by Sanger sequencing. In vitro transcribed (IVT) 23S rRNA was prepared using a linearized plasmid template (pBluescript II KS(-)) and HiScribe T7 High Yield RNA Synthesis Kit (NEB, Cat #E2040S). The synthesized transcripts were cleaned up using Monarch Spin RNA Cleanup Kit (NEB, Cat #T2050L). To perform in vitro methylation reactions, IVT 23S rRNA was heat-denatured at 95 C for 30 seconds and cooled to 22 C prior to enzyme treatment. Purified recombinant TK0008 (0.1 – 4 uM) was incubated with 1 uM of IVT 23S rRNA in 1x methyltransferase buffer (25 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM DTT) supplemented with 160 uM of S-adenosyl-methionine (SAM) (NEB, Cat #B9003S) at 85 C for 30 minutes. The reactions were cleaned up using Monarch Spin RNA Cleanup Kit (NEB, Cat #T2040L).
Sample Type:	Ribonucleic acid, In Vitro Transcription

Treatment:

Treatment ID:	TR003869
Treatment Summary:	Harvested archaeal cell pellets were resuspended in 10 mL of TRI reagent (Molecular Research Center, Inc., Cat #TR118). The resuspended cells were homogenized using a beads beater at 4.0 m/s for 20 seconds x 2 cycles (MP Biomedicals, FastPrep-24TM). Subsequently, the mixture was centrifuged at 14000 g for 5 minutes to precipitate any cell debris. Supernatants were collected post-centrifugation and treated with 50 μL of BAN reagent (Molecular Research Center, Inc., Cat #BN191) per mL of supernatant for aqueous-organic phase separation. RNA from the aqueous phase was isolated by isopropanol precipitation and subjected to DNase I treatment (NEB, Cat #M0303S) to remove genomic DNA contamination. To further purify the DNase I-treated RNA, an equal volume of acid phenol-chloroform with isoamyl alcohol (125:24:1, Thermo Fisher Scientific, Cat #AM9722) was added to the reaction and centrifuged at 21300 g for 2 minutes to separate the aqueous phase from the organic phase. The aqueous phase containing RNA was then precipitated with 1.5 volumes of isopropanol and 0.1 volume of sodium acetate (Sigma Aldrich, Cat #S7899) at –20°C overnight. Finally, the precipitated RNA pellets were washed with 75% ethanol and dissolved in nuclease-free water. To enrich m7G-modified rRNA fragment, 50 ug of total RNA from T. kodakarensis TS559 wild-type strain was mixed with a biotinylated DNA probe (1 uM) fully complementary to the target sequence in 10 mM Tris buffer (pH 7.5) and heated to 95 C for 2 minutes followed by a ramp down to 22 C. Biotinylated DNA probe sequence: 5’-Biotin-ACCTTCGAGGTGTGCGCCAGCAACTGCCACT-3’. The unhybridized RNAs were digested with 5 uL of RNase 4 (NEB, Cat #M1284L) in 1x NEB buffer r1.1 at 37 C for 1 hour. The digestion was stopped by adding 10 L of human placental RNase inhibitor (NEB, Cat #M0307S) to the reaction mixture and incubated at room temperature for 10 minutes. Selective enrichment of the probe-hybridized rRNA region was performed using streptavidin magnetic beads (NEB, Cat #S1421S). Briefly, 250 uL of streptavidin magnetic beads were washed twice with equal volume of low-salt buffer (100 mM Tris pH 7.5, 10 mM EDTA, 50 mM NaCl) and resuspended in 50 uL of high-salt buffer (100 mM Tris pH 7.5, 10 mM EDTA, 250 mM NaCl). The high-salt buffer was removed, and the beads were incubated with the reaction mixture for 15 minutes at room temperature with occasional agitation. The bead-captured DNA-RNA hybrid was washed and eluted by heating to 80 C for 5 minutes in 30 uL of nuclease-free water. For m7G-modified rRNA fragment depletion, the same biotinylated DNA probe (1 uM) described above was mixed with 5 ug of total RNA from TS559 strain in the probe hybridization buffer provided in NEBNext rRNA Depletion Kit (NEB, Cat #E7850X). The probe-hybridized rRNA mixture was subjected to rRNA depletion according to the NEBNext rRNA Depletion Kit protocol. Following the enzymatic treatment, the reactions were cleaned up using RNA Clean and Concentrator Kit (Zymo Research, Cat #R1017).

Treatment ID:

TR003869

Treatment Summary:

Harvested archaeal cell pellets were resuspended in 10 mL of TRI reagent (Molecular Research Center, Inc., Cat #TR118). The resuspended cells were homogenized using a beads beater at 4.0 m/s for 20 seconds x 2 cycles (MP Biomedicals, FastPrep-24TM). Subsequently, the mixture was centrifuged at 14000 g for 5 minutes to precipitate any cell debris. Supernatants were collected post-centrifugation and treated with 50 μL of BAN reagent (Molecular Research Center, Inc., Cat #BN191) per mL of supernatant for aqueous-organic phase separation. RNA from the aqueous phase was isolated by isopropanol precipitation and subjected to DNase I treatment (NEB, Cat #M0303S) to remove genomic DNA contamination. To further purify the DNase I-treated RNA, an equal volume of acid phenol-chloroform with isoamyl alcohol (125:24:1, Thermo Fisher Scientific, Cat #AM9722) was added to the reaction and centrifuged at 21300 g for 2 minutes to separate the aqueous phase from the organic phase. The aqueous phase containing RNA was then precipitated with 1.5 volumes of isopropanol and 0.1 volume of sodium acetate (Sigma Aldrich, Cat #S7899) at –20°C overnight. Finally, the precipitated RNA pellets were washed with 75% ethanol and dissolved in nuclease-free water. To enrich m7G-modified rRNA fragment, 50 ug of total RNA from T. kodakarensis TS559 wild-type strain was mixed with a biotinylated DNA probe (1 uM) fully complementary to the target sequence in 10 mM Tris buffer (pH 7.5) and heated to 95 C for 2 minutes followed by a ramp down to 22 C. Biotinylated DNA probe sequence: 5’-Biotin-ACCTTCGAGGTGTGCGCCAGCAACTGCCACT-3’. The unhybridized RNAs were digested with 5 uL of RNase 4 (NEB, Cat #M1284L) in 1x NEB buffer r1.1 at 37 C for 1 hour. The digestion was stopped by adding 10 L of human placental RNase inhibitor (NEB, Cat #M0307S) to the reaction mixture and incubated at room temperature for 10 minutes. Selective enrichment of the probe-hybridized rRNA region was performed using streptavidin magnetic beads (NEB, Cat #S1421S). Briefly, 250 uL of streptavidin magnetic beads were washed twice with equal volume of low-salt buffer (100 mM Tris pH 7.5, 10 mM EDTA, 50 mM NaCl) and resuspended in 50 uL of high-salt buffer (100 mM Tris pH 7.5, 10 mM EDTA, 250 mM NaCl). The high-salt buffer was removed, and the beads were incubated with the reaction mixture for 15 minutes at room temperature with occasional agitation. The bead-captured DNA-RNA hybrid was washed and eluted by heating to 80 C for 5 minutes in 30 uL of nuclease-free water. For m7G-modified rRNA fragment depletion, the same biotinylated DNA probe (1 uM) described above was mixed with 5 ug of total RNA from TS559 strain in the probe hybridization buffer provided in NEBNext rRNA Depletion Kit (NEB, Cat #E7850X). The probe-hybridized rRNA mixture was subjected to rRNA depletion according to the NEBNext rRNA Depletion Kit protocol. Following the enzymatic treatment, the reactions were cleaned up using RNA Clean and Concentrator Kit (Zymo Research, Cat #R1017).

Sample Preparation:

Sampleprep ID:	SP003866
Sampleprep Summary:	Total or isolated RNAs were digested to nucleosides at 37°C overnight using a Nucleoside Digestion Mix (NEB, Cat #M0649S). The digested RNAs were subsequently injected without prior purification on an Agilent 1290 Infinity II UHPLC equipped with a G7117 diode array detector and an Agilent 6495C Triple-Quadrupole Mass Spectrometer operating in positive electrospray ionization (+ESI) mode.

Chromatography:

Chromatography ID:	CH004644
Chromatography Summary:	Solvent A pH is 4.5
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Waters XSelect HSS T3 XP (100 × 2.1mm, 2.5um)
Column Temperature:	30
Flow Gradient:	1%-40% Solvent B in 26.5 min
Flow Rate:	0.6 mL/min
Solvent A:	100% water; 10mM ammonium acetate
Solvent B:	100% methanol
Chromatography Type:	Reversed phase

Chromatography ID:	CH004645
Chromatography Summary:	Solvent A pH is 4.5
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Waters XSelect HSS T3 XP (100 × 2.1mm, 2.5um)
Column Temperature:	30
Flow Gradient:	1%-23% Solvent B in 7.5 min
Flow Rate:	0.6mL/min
Solvent A:	100% water; 10mM ammonium acetate
Solvent B:	100% methanol
Chromatography Type:	Reversed phase

Analysis:

Analysis ID:	AN006115
Analysis Type:	MS
Chromatography ID:	CH004644
Num Factors:	7
Num Metabolites:	98
Units:	femtomole

Analysis ID:	AN006116
Analysis Type:	MS
Chromatography ID:	CH004645
Num Factors:	7
Num Metabolites:	11
Units:	Mass Response Ratio