Number, position, and significance of the pseudouridines in the large subunit ribosomal RNA of <i>Haloarcula marismortui</i> and <i>Deinococcus radiodurans</i>

Campo, Mark Del; Recinos, Claudia C; Yanez, Giscard; Pomerantz, Steven C.; Guymon, Rebecca; Crain, Pamela F.; McCloskey, James A.; Ofengand, James

doi:10.1261/rna.7209905

Cited by 43 publications

(45 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Figure 2b is a representative mass spectrum arising from the RNase T1 digestion of tRNA TyrII after reaction with CMC and alkaline treatment. Optimal reaction conditions utilized for the CMC modification of RNase T1 fragments of tRNA TyrII were found to be very similar to those conditions used for the CMC reactions with dT 10 and r(AUGCAUGC). Table 1 includes those ions appearing in Figure 2b which are integer multiples of 252 Da from any ions detected in Figure 2a as those represent CMC-derivatized digestion products stable to alkaline treatment.…”

Section: Analysis Of Cmc-derivatized Rnase T1 Digestion Products Of Ementioning

confidence: 83%

“…The solution was vortexed and incubated in a water bath. Reaction mixtures containing 0.5 μg to 2 μg of dT 10 or r(AUGCAUGC) or 5 μg of RNase digested tRNA were incubated with the stock CMC solution. Incubation time, temperature, pH and the ratio of amounts of CMC to sample were varied for optimization.…”

Section: Cmc Derivatization and Alkaline Treatmentmentioning

confidence: 99%

“…Initial optimization studies were conducted using the oligodeoxynucleotide dT 10 (data not shown) and the oligoribonucleotide r (AUGCAUGC) (Figure 1). The homopolymer was selected to identify the maximum number of CMC units that can bond to an oligonucleotide before steric crowding becomes limiting.…”

Section: Cmc Derivatization and Mass Spectrometric Analysis Of Model mentioning

confidence: 99%

“…CMC-reacted DNA has also been shown to be inhibited in its ability to support transcription by RNA polymerase [5]. One of the most powerful applications of CMC derivatization has been its use as a sequencing tool for the detection of the posttranscriptionally modified nucleoside, pseudouridine (Ψ), in RNA [6][7][8][9][10]. Reaction with CMC at a pH of 8.5 modifies guanosine, uridines and the N-1 and N-3 positions of pseudouridine, but the CMC unit is retained selectively at higher pH (10.4) on the N-3 position of pseudouridine (N 3 -CMC-Ψ).…”

Section: Introductionmentioning

confidence: 99%

“…Most significantly, the presence of unreacted oligonucleotides in the reaction mixture can hinder the identification of undermodified pseudouridines, and a failure to release CMC from guanosine and uridine residues during alkaline treatment can lead to a misinterpretation of those locations as pseudouridine containing sites. Ofengand and co-workers also advise this to be a major source of their error when they inadvertently reported an incorrect number of pseudouridines in Haloarcula marismortui RNA [10].…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Improving CMC-derivatization of pseudouridine in RNA for mass spectrometric detection

Durairaj

Limbach

2008

Analytica Chimica Acta

View full text Add to dashboard Cite

A protocol that utilizes matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and N-cyclohexyl-N′-β-(4-methylmorpholinium)ethylcarbodiimide (CMC) derivatization to detect the post-transcriptionally modified nucleoside, pseudouridine, in RNA has been optimized for RNase digests. Because pseudouridine is mass-silent (i.e. the mass of pseudouridine is the same as the mass of uridine), after CMC derivatization and alkaline treatment, all pseudouridine residues exhibit a mass shift of 252 Da that allows its presence to be easily detected by mass spectrometry. This protocol is illustrated by the direct MALDI-MS identification of pseudouridines within Escherichia coli tRNA TyrII starting from microgram amounts of sample. During this optimization study, it was discovered that the post-transcriptionally modified nucleoside 2-methylthio-N 6 -isopentenyladenosine, which is present in bacterial tRNAs, also retains a CMC unit after derivatization and incubation with base. Thus, care must be exercised when applying this MALDIbased CMC-derivatization approach for pseudouridine detection to samples containing transfer RNAs to minimize the misidentification of pseudouridine.

show abstract

Section: Analysis Of Cmc-derivatized Rnase T1 Digestion Products Of Ementioning

confidence: 83%

Section: Cmc Derivatization and Alkaline Treatmentmentioning

confidence: 99%

Section: Cmc Derivatization and Mass Spectrometric Analysis Of Model mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Improving CMC-derivatization of pseudouridine in RNA for mass spectrometric detection

Durairaj

Limbach

2008

Analytica Chimica Acta

View full text Add to dashboard Cite

show abstract

Removal of 3'‐phosphate group by bacterial alkaline phosphatase improves oligonucleotide sequence coverage of RNase digestion products analyzed by collision‐induced dissociation mass spectrometry

Krivos

Addepalli

Limbach

2011

Rapid Comm Mass Spectrometry

View full text Add to dashboard Cite

RNase mapping by nucleobase-specific endonucleases combined with liquid chromatography/tandem mass spectrometry (LC/MS/MS) is a powerful analytical method for characterizing ribonucleic acids (RNAs). Endonuclease digestion of RNA yields products that contain a 3'-terminal phosphate group. MS/MS via collision-induced dissociation (CID) of these digestion products on a linear ion trap generates fragmentation pathways that include the loss of phosphoric acid (-H(3)PO(4); -98 u), which does not provide information about the sequence of the digestion products and can reduce ion abundance from other pathways that provide sequence information. Here we investigate the use of bacterial alkaline phosphatase (BAP) after RNase digestion to remove the 3'-terminal phosphate from all RNase digestion products prior to LC/MS/MS analysis. RNase digestion products lacking the 3'-phosphate were found to produce CID spectra with more consistent, high-abundance c- and y-type fragment ions as well as significantly more a-Base and w-type ions than digestion products retaining the 3'-phosphate. In this manner, RNase mapping with LC/MS/MS can provide more complete RNA sequence information from fragment ions of higher abundance that are easier to interpret and identify.

show abstract

Naturally occurring modified ribonucleosides

et al. 2020

View full text Add to dashboard Cite

The chemical identity of RNA molecules beyond the four standard ribonucleosides has fascinated scientists since pseudouridine was characterized as the "fifth" ribonucleotide in 1951. Since then, the ever-increasing number and complexity of modified ribonucleosides have been found in viruses and throughout all three domains of life. Such modifications can be as simple as methylations, hydroxylations, or thiolations, complex as ring closures, glycosylations, acylations, or aminoacylations, or unusual as the incorporation of selenium. While initially found in transfer and ribosomal RNAs, modifications also exist in messenger RNAs and noncoding RNAs. Modifications have profound cellular outcomes at various levels, such as altering RNA structure or being essential for cell survival or organism viability. The aberrant presence or absence of RNA modifications can lead to human disease, ranging from cancer to various metabolic and developmental illnesses such as Hoyeraal-Hreidarsson syndrome, Bowen-Conradi syndrome, or Williams-Beuren syndrome. In this review article, we summarize the characterization of all 143 currently known modified ribonucleosides by describing their taxonomic distributions, the enzymes that generate the modifications, and any implications in cellular processes, RNA structure, and disease. We also highlight areas of active research, such as specific RNAs that contain a particular type of modification as well as methodologies used to identify novel RNA modifications.

show abstract

Number, position, and significance of the pseudouridines in the large subunit ribosomal RNA of Haloarcula marismortui and Deinococcus radiodurans

Abstract: ABSTRACT

Cited by 43 publications

References 35 publications

Improving CMC-derivatization of pseudouridine in RNA for mass spectrometric detection

Improving CMC-derivatization of pseudouridine in RNA for mass spectrometric detection

Removal of 3'‐phosphate group by bacterial alkaline phosphatase improves oligonucleotide sequence coverage of RNase digestion products analyzed by collision‐induced dissociation mass spectrometry

Naturally occurring modified ribonucleosides

Contact Info

Product

Resources

About