Summary of Study ST003726
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 | ST003726 |
| Study Title | RNA modification profiles in archaeal and non-archaeal species |
| Study Summary | Total RNA from five archaea, E. coli, S. cerevisiae and universal human reference RNA were digested to nucleosides and subjected to UHPLC-QqQ analysis. Each species was searched against a panel of 76 nucleoside standards. |
| Institute | New England Biolabs |
| Last Name | Tsai |
| First Name | Yueh-Lin |
| Address | 44 Dunham Ridge, Beverly, MA 01915 |
| atsai@neb.com | |
| Phone | 978-380-6587 |
| Submit Date | 2025-02-01 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML, d |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-02-14 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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: | SU003858 |
| Subject Type: | Bacteria |
| Subject Species: | Thermococcus kodakarensis; Thermococcus sp. AM4; Methanococcus maripaludis; Sulfolobus acidocaldarius; Sulfolobus islandicus; Saccharomyces cerevisiae; Escherichia coli; Homo sapiens |
| Taxonomy ID: | 311400; 246969; 39152; 2285; 43080; 4932; 562; 9606 |
Factors:
Subject type: Bacteria; Subject species: Thermococcus kodakarensis; Thermococcus sp. AM4; Methanococcus maripaludis; Sulfolobus acidocaldarius; Sulfolobus islandicus; Saccharomyces cerevisiae; Escherichia coli; Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Species | Sample source |
|---|---|---|---|
| SA407145 | E_coli_AT_01-r002 | Escherichia coli | Total RNA |
| SA407146 | E_coli_AT_01-r001 | Escherichia coli | Total RNA |
| SA407147 | Human_AT_02-r002 | Homo sapiens | Total RNA |
| SA407148 | Human_AT_02-r001 | Homo sapiens | Total RNA |
| SA407155 | M_mari_rRNA_depl_rep2_AT_03-r001 | Methanococcus maripaludis | rRNA_depletion |
| SA407156 | M_mari_rRNA_depl_rep1_AT_01-r001 | Methanococcus maripaludis | rRNA_depletion |
| SA407157 | M_mari_rRNA_depl_rep1_AT_01-r002 | Methanococcus maripaludis | rRNA_depletion |
| SA407158 | M_mari_rRNA_depl_rep2_AT_03-r002 | Methanococcus maripaludis | rRNA_depletion |
| SA407149 | M_mari_total_rep2_AT_04-r001 | Methanococcus maripaludis | Total RNA |
| SA407150 | Mmari_Total_RNA-r001 | Methanococcus maripaludis | Total RNA |
| SA407151 | M_mari_total_rep2_AT_04-r002 | Methanococcus maripaludis | Total RNA |
| SA407152 | Mmari_Total_RNA-r002 | Methanococcus maripaludis | Total RNA |
| SA407153 | M_mari_total_rep1_AT_02-r002 | Methanococcus maripaludis | Total RNA |
| SA407154 | M_mari_total_rep1_AT_02-r001 | Methanococcus maripaludis | Total RNA |
| SA407159 | Yeast_AT_03-r002 | Saccharomyces cerevisiae | Total RNA |
| SA407160 | Yeast_AT_03-r001 | Saccharomyces cerevisiae | Total RNA |
| SA407165 | S_acid_rRNA_depl_rep2_AT_01-r002 | Sulfolobus acidocaldarius | rRNA_depletion |
| SA407166 | S_acid_rRNA_depl_rep2_AT_01-r001 | Sulfolobus acidocaldarius | rRNA_depletion |
| SA407167 | S_acid_rRNA_depl_rep1_AT_03-r002 | Sulfolobus acidocaldarius | rRNA_depletion |
| SA407168 | S_acid_rRNA_depl_rep1_AT_03-r001 | Sulfolobus acidocaldarius | rRNA_depletion |
| SA407161 | S_acid_total_rep2_AT_02-r001 | Sulfolobus acidocaldarius | Total RNA |
| SA407162 | S_acid_total_rep2_AT_02-r002 | Sulfolobus acidocaldarius | Total RNA |
| SA407163 | S_acid_total_rep1_AT_04-r002 | Sulfolobus acidocaldarius | Total RNA |
| SA407164 | S_acid_total_rep1_AT_04-r001 | Sulfolobus acidocaldarius | Total RNA |
| SA407175 | S_island_rRNA_depl_rep1_AT_01_r002 | Sulfolobus islandicus | rRNA_depletion |
| SA407176 | S_island_rRNA_depl_rep1_AT_01_r001 | Sulfolobus islandicus | rRNA_depletion |
| SA407169 | S_island_total_rep1_AT_01-r001 | Sulfolobus islandicus | Total RNA |
| SA407170 | S_acid_total_rep1_AT_02-r002 | Sulfolobus islandicus | Total RNA |
| SA407171 | S_acid_total_rep1_AT_02-r001 | Sulfolobus islandicus | Total RNA |
| SA407172 | S_island_total_rep1_AT_01-r002 | Sulfolobus islandicus | Total RNA |
| SA407173 | S_island_total_rep2_AT_02-r002 | Sulfolobus islandicus | Total RNA |
| SA407174 | S_island_total_rep2_AT_02-r001 | Sulfolobus islandicus | Total RNA |
| SA407181 | Tk_rRNA_depl_rep1_AT_01-r001 | Thermococcus kodakarensis | rRNA_depletion |
| SA407182 | Tk_rRNA_depl_rep1_AT_01-r002 | Thermococcus kodakarensis | rRNA_depletion |
| SA407183 | Tk_rRNA_depl_rep2_AT_01-r001 | Thermococcus kodakarensis | rRNA_depletion |
| SA407184 | Tk_rRNA_depl_rep2_AT_01-r002 | Thermococcus kodakarensis | rRNA_depletion |
| SA407177 | Tk_total_rep1_AT_02-r001 | Thermococcus kodakarensis | Total RNA |
| SA407178 | Tk_total_rep1_AT_02-r002 | Thermococcus kodakarensis | Total RNA |
| SA407179 | Tk_total_rep2_AT_02-r001 | Thermococcus kodakarensis | Total RNA |
| SA407180 | Tk_total_rep2_AT_02-r002 | Thermococcus kodakarensis | Total RNA |
| SA407188 | TAM4_rRNA_depl_rep1-r002 | Thermococcus sp. AM4 | rRNA_depletion |
| SA407189 | TAM4_rRNA_depl_rep1-r001 | Thermococcus sp. AM4 | rRNA_depletion |
| SA407185 | TAM4_total_rep2_AT_02 | Thermococcus sp. AM4 | Total RNA |
| SA407186 | TAM4_total_rep1_AT_03-r001 | Thermococcus sp. AM4 | Total RNA |
| SA407187 | TAM4_total_rep1_AT_03-r002 | Thermococcus sp. AM4 | Total RNA |
| Showing results 1 to 45 of 45 |
Collection:
| Collection ID: | CO003851 |
| Collection Summary: | Universal human reference RNA (Agilent Technologies, Cat #740000) and Escherichia coli total RNAs (Thermo Fisher Scientific, Cat #AM7940) were purchased from commercially available sources. Saccharomyces cerevisiae (strain S288C) total RNA was obtained as described in Mulroney, L., Wulf, M.G., Schildkraut, I., Tzertzinis, G., Buswell, J., Jain, M., Olsen, H., Diekhans, M., Correa, I.R., Jr., Akeson, M., and Ettwiller, L. (2022). Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing. RNA 28, 162-176. 10.1261/rna.078703.121. 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. |
| Sample Type: | Ribonucleic acid |
Treatment:
| Treatment ID: | TR003867 |
| Treatment Summary: | To remove rRNA and tRNA, total RNA was separated into large (> 200 nt) and small RNA (< 200 nt) fractions using the RNA Clean and Concentrator Kit (Zymo Research, Cat #R1017). Subsequently, 50 μg of the large RNA fraction were subjected to rRNA depletion using the NEBNext rRNA Depletion Kit (NEB, Cat #E7850X) with the following changes: The NEBNext rRNA depletion solutions provided in the kit were substituted for customized DNA probe mixtures (at 1 uM for each probe) fully complementary to rRNA sequences corresponding to each archaeal species; All volumes for the probe hybridization, RNase H and DNase I digestion reactions were scaled up by fivefold in 10 parallel reactions. Following the enzymatic treatment, the reactions were cleaned up using RNA Clean and Concentrator Kit (Zymo Research, Cat #R1017), the mRNA-enriched fractions were eluted in 10 uL water and combined. |
Sample Preparation:
| Sampleprep ID: | SP003864 |
| Sampleprep Summary: | Total RNA, RNA subfractions, and mRNA-enriched samples were digested to nucleosides at 37°C overnight using a Nucleoside Digestion Mix (NEB, Cat #M0649S). |
Chromatography:
| Chromatography ID: | CH004641 |
| Chromatography Summary: | Solvent A was at pH 4.5 |
| Instrument Name: | Agilent 1290 Infinity II UHPLC |
| Column Name: | Waters XSelect HSS T3 XP (100 x 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 |
Analysis:
| Analysis ID: | AN006112 |
| Analysis Type: | MS |
| Chromatography ID: | CH004641 |
| Num Factors: | 13 |
| Num Metabolites: | 100 |
| Rt Units: | Minutes |
| Units: | Integrative area under chromatographic peak |
