Improved RNA modification mapping of cellular non-coding RNAs using C- and U-specific RNases

Thakur, Priti; Estevez, Mariana; Lobue, Peter A.; Limbach, Patrick A.; Addepalli, Balasubrahmanyam

doi:10.1039/c9an02111f

Cited by 32 publications

(36 citation statements)

References 92 publications

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“…It is interesting to note that although RlmKL installs methyl groups at two different positions (2069 and 2,445) of 23S rRNA compared to RlmB (at position 2,251) ( Popova and Williamson, 2014 ; Sergiev et al, 2018 ), the latter strain seems to be more susceptible to oxidative stress ( Figure 4 ). Further studies involving modification mapping experiments ( Sun et al, 2020 ; Thakur et al, 2020 ) could identify the exact locations in the sequence that might serve as hotspots for oxidative damage. Nevertheless, these studies indicate that although some post-transcriptional methylations are not essential for normal growth, their absence could aggravate the vulnerability of rRNA to oxidative damage.…”

Section: Discussionmentioning

confidence: 99%

Oxidative Damage to RNA is Altered by the Presence of Interacting Proteins or Modified Nucleosides

Estevez

Valesyan

Jora

et al. 2021

Front. Mol. Biosci.

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Oxidative stress triggered by the Fenton reaction (chemical) or UVR exposure (photo) can damage cellular biomolecules including RNA through oxidation of nucleotides. Besides such xenobiotic chemical modifications, RNA also contains several post-transcriptional nucleoside modifications that are installed by enzymes to modulate structure, RNA-protein interactions, and biochemical functions. We examined the extent of oxidative damage to naturally modified RNA which is required for cellular protein synthesis under two different contexts. The extent of oxidative damage is higher when RNA is not associated with proteins, but the degree of damage is lower when the RNA is presented in the form of a ribonucleoprotein complex, such as an intact ribosome. Our studies also indicate that absence of methylations in ribosomal RNA at specific positions could make it more susceptible to photooxidative stress. However, the extent of guanosine oxidation varied with the position at which the modification is deficient, indicating position-dependent structural effects. Further, an E. coli strain deficient in 5-methylaminomethyl-2-thiouridine (mnm5s2U) (found in lysine and glutamate tRNA anticodon) is more vulnerable to oxidative RNA damage compared to its wildtype strain suggesting an auxiliary function for the mnm5s2U modification. These studies indicate that oxidative damage to RNA is altered by the presence of enzymatic modified nucleosides or protein association inside the cell.

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Section: Discussionmentioning

confidence: 99%

Oxidative Damage to RNA is Altered by the Presence of Interacting Proteins or Modified Nucleosides

Estevez

Valesyan

Jora

et al. 2021

Front. Mol. Biosci.

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“…Zhang, Shi, et al, 2019). Recently, multiple RNases were used to generate unique, overlapping digestion products, which has bypassed the need for the RNA to be highly purified (Thakur, Estevez, Lobue, Limbach, & Addepalli, 2020). Because LC-MS can be limited by the RNase cleavage site, novel RNases that have different substrate specificities can be helpful in sequence-specific detection of modifications (Addepalli et al, 2017).…”

Section: Mass Spectrometry-based Methods Of Detecting Modified Ribonucleosidesmentioning

confidence: 99%

Naturally occurring modified ribonucleosides

et al. 2020

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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.

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“…5-aminomethyl-2-geranylthiouridine, 425.1984 Da from epoxyqueuosine, 425.1547 Da); when no standard is available. Nucleoside analysis is then followed by the generation of oligomers of manageable size (5-mers to 20-mers) via the use of base-specific nucleases prior to chromatographic separation and mass spectrometric analysis (Thakur et al 2020). These approaches have been used successfully to sequence tRNAs but can easily be used with other types of RNAs.…”

Section: Modification Diversity and The Limits Of Detectionmentioning

confidence: 99%

“…Clearly the drawback with LC MS/MS has always been the requirement for relatively large amounts of pure sample, however, as chromatographic technology continues to improve, sample consumption continues to decrease. Recently the implementation of new base-specific nucleases has permitted the analysis of modification in their natural sequence context in complex mixtures (Thakur et al 2020). So the future for mass spectrometry based approaches is bright and in our opinion is truly the way of the future.…”

Section: Modification Diversity and The Limits Of Detectionmentioning

confidence: 99%

From canonical to modified nucleotides: balancing translation and metabolism

Accornero

Ross

Alfonzo

2020

Critical Reviews in Biochemistry and Molecular Biology

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Every type of nucleic acid in cells may undergo some kind of post-replicative or post-transcriptional chemical modification. Recent evidence has highlighted their importance in biology and their chemical complexity. In the following pages, we will describe new discoveries of modifications, with a focus on tRNA and mRNA. We will highlight current challenges and advances in modification detection and we will discuss how changes in nucleotide post-transcriptional modifications may affect cell homeostasis leading to malfunction. Although, RNA modifications prevail in all forms of life, the present review will focus on eukaryotic systems, where the great degree of intracellular compartmentalization provides barriers and filters for the level at which a given RNA is modified and will of course affect its fate and function. Additionally, although we will mention rRNA modification and modifications of the mRNA 5'-CAP structure, this will only be discussed in passing, as many substantive reviews have been written on these subjects. Here we will not spend much time describing all the possible modifications that have been observed; truly a daunting task. For reference, Bujnicki and coworkers have created MODOMICS, a useful repository for all types of modifications and their associated enzymes. Instead we will discuss a few examples, which illustrate our arguments on the connection of modifications, metabolism and ultimately translation. The fact remains, a full understanding of the long reach of nucleic acid modifications in cells requires both a global and targeted study of unprecedented scale, which at the moment may well be limited only by technology.

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Improved RNA modification mapping of cellular non-coding RNAs using C- and U-specific RNases

Abstract: Improved RNA modification mapping through complementary nucleobase-specific ribonucleases.

Cited by 32 publications

References 92 publications

Oxidative Damage to RNA is Altered by the Presence of Interacting Proteins or Modified Nucleosides

Oxidative Damage to RNA is Altered by the Presence of Interacting Proteins or Modified Nucleosides

Naturally occurring modified ribonucleosides

From canonical to modified nucleotides: balancing translation and metabolism

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