Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC–UV–MS

Russell, Susan P.; Limbach, Patrick A.

doi:10.1016/j.jchromb.2013.02.010

Cited by 43 publications

(46 citation statements)

References 54 publications

(72 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Y. C. Chan et al, 2015). While descriptive analysis of ribonucleosides using MS was established two decades ago (Pomerantz & McCloskey, 1990; Suzuki, Ikeuchi, Noma, & Sakaguchi, 2007), quantitative MS analysis of ribonucleosides presents significantly greater challenges (Russell & Limbach, 2013; Su et al, 2014). Both relative and absolute quantification are best achieved using HPLC-coupled tandem or triple quadrupole (QQQ) mass spectrometry (LC-MS/MS) with electrospray ionization (ESI), given the increased sensitivity over other instrument types.…”

Section: Methodsmentioning

confidence: 99%

A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA

Cai

Chionh

Hia

et al. 2015

Methods in Enzymology

View full text Add to dashboard Cite

Here we describe an analytical platform for systems-level quantitative analysis of modified ribonucleosides in any RNA species, with a focus on stress-induced reprogramming of tRNA as part of a system of translational control of cell stress response. The chapter emphasizes strategies and caveats for each of the seven steps of the platform workflow: 1) RNA isolation, 2) RNA purification, 3) RNA hydrolysis to individual ribonucleosides, 4) chromatographic resolution of ribonucleosides, 5) identification of the full set of modified ribonucleosides, 6) mass spectrometric quantification of ribonucleosides, 6) interrogation of ribonucleoside datasets, and 7) mapping the location of stress-sensitive modifications in individual tRNA molecules. We have focused on the critical determinants of analytical sensitivity, specificity, precision and accuracy in an effort to ensure the most biologically meaningful data on mechanisms of translational control of cell stress response. The methods described here should find wide use in virtually any analysis involving RNA modifications.

show abstract

Section: Methodsmentioning

confidence: 99%

A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA

Cai

Chionh

Hia

et al. 2015

Methods in Enzymology

View full text Add to dashboard Cite

show abstract

“…The fractionated oligomer was digested with snake venom phosphodiesterase and Antarctic phosphatase before subjecting it to subsequent LC-MS (56,58) with detection in positive polarity.…”

Section: Methodsmentioning

confidence: 99%

Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF

Addepalli

Limbach

2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Pseudouridine is found in almost all cellular ribonucleic acids (RNAs). Of the multiple characteristics attributed to pseudouridine, making messenger RNAs (mRNAs) highly translatable and non-immunogenic is one such feature that directly implicates this modification in protein synthesis. We report the existence of pseudouridine in the anticodon of Escherichia coli tyrosine transfer RNAs (tRNAs) at position 35. Pseudouridine was verified by multiple detection methods, which include pseudouridine-specific chemical derivatization and gas phase dissociation of RNA during liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of total tRNA isolated from E. coli pseudouridine synthase knock-out mutants identified RluF as the enzyme responsible for this modification. Furthermore, the absence of this modification compromises the translational ability of a luciferase reporter gene coding sequence when it is preceded by multiple tyrosine codons. This effect has implications for the translation of mRNAs that are rich in tyrosine codons in bacterial expression systems.Of 150-plus chemical modifications (1, 2) found in cellular RNA, 5-ribosyluridine or pseudouridine (⌿) 3 (3) is the most abundant but still poorly understood (4) modification. Pseudouridine, which has been found in the functionally important regions of RNAs (5), is known to modulate codon-anticodon interactions between mRNA and tRNA (6) and assist assembly of the ribosome (7) and spliceosome (8). The presence of pseudouridine in mRNA makes the message non-immunogenic and highly translatable, thus offering therapeutic opportunities in medical applications (9).Although the exact mechanism of isomerization is still not clear (4), the incorporation of C5 of the uracil base into the glycosidic bond is catalyzed by either standalone proteins (referred to as ⌿ synthases) (10, 11) or RNA-protein complexes (referred to as H/ACA box ribonucleoproteins) (12). Following site-specific recognition, the nitrogen-carbon glycosidic bond is cleaved, and uracil is rotated and then reattached to the sugar, forming a carbon-carbon glycosidic bond on the polyribonucleotide (Fig. 1) by Michael addition-like or glycal mechanisms (13).The ⌿ synthases have been initially classified based on the Escherichia coli enzymes RluA, RsuA, TruA, TruB, and TruD (10, 11). A sixth family with members from archaea and eukaryotes but not from bacteria was added after discovery of Pus10 (14). The enzyme active site carries a catalytically essential aspartate, which is the only absolutely conserved residue in all ⌿ synthases (15, 16). Substrate recognition by the ⌿ synthase is usually in the context of the sequence or structure of the target site in RNA. Site specificity also varies depending on the ⌿ synthase. For example, RsuA isomerizes U516 of 16S rRNA (17) exclusively, whereas the universally conserved ⌿55 of the T⌿C loop in all elongator tRNAs is catalyzed by TruB (18). RluA catalyzes modification of two distinct RNAs, 23S rRNA (position 746) and some tRNAs (position 32) (19). Member...

show abstract

“…Recent developments, especially the application of sensitive mass spectrometry tools to nucleic acid chemistry (i.e., liquid chromatography combined with mass spectrometry, LC-MS) has considerably increased analytical sensitivity, opening up possibilities for quantifying also the modifications status of low-abundance RNAs (Kellner et al, 2014;Li et al, 2008;Russell & Limbach, 2013). Using LC-MS has provided unprecedented insights into the dynamic landscape of RNA modifications (including m 5 C) in abundant RNAs, especially during stress conditions (Chan et al, 2010(Chan et al, , 2012.…”

Section: Methods For Identifying M 5 C-modified Rna In Sequence Contextmentioning

confidence: 99%

RNA 5-Methylcytosine Analysis by Bisulfite Sequencing

Schaefer

2015

Methods in Enzymology

View full text Add to dashboard Cite

Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC–UV–MS

Cited by 43 publications

References 54 publications

A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA

A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA

Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF

RNA 5-Methylcytosine Analysis by Bisulfite Sequencing

Contact Info

Product

Resources

About