Kok Seong Lim

Quantitative analysis of ribonucleoside modifications in tRNA by HPLC-coupled mass spectrometry

Post-transcriptional modification of RNA is an important determinant of RNA quality control, translational efficiency, RNA-protein interactions and stress response. This is illustrated by the observation of toxicant-specific changes in the spectrum of tRNA modifications in a stress-response mechanism involving selective translation of codon-biased mRNA for crucial proteins. To facilitate systems-level studies of RNA modifications, we developed a liquid chromatography–mass spectrometry (LC-MS) technique for the quantitative analysis of modified ribonucleosides in tRNA. The protocol includes tRNA purification by HPLC, enzymatic hydrolysis, reversed-phase HPLC resolution of the ribonucleosides, and identification and quantification of individual ribonucleosides by LC-MS via dynamic multiple reaction monitoring (DMRM). In this approach, the relative proportions of modified ribonucleosides are quantified in several micrograms of tRNA in a 15-min LC-MS run. This protocol can be modified to analyze other types of RNA by modifying the steps for RNA purification as appropriate. By comparison, traditional methods for detecting modified ribonucleosides are labor- and time-intensive, they require larger RNA quantities, they are modification-specific or require radioactive labeling.

m6A-LAIC-seq reveals the census and complexity of the m6A epitranscriptome

N6-Methyladenosine (m6A) is a widespread, reversible chemical modification of RNA molecules, implicated in many aspects of RNA metabolism. Little quantitative information exists as to either how many transcript copies of particular genes are m6A modified (m6A levels) or the relationship of m6A modification(s) to alternative RNA isoforms. To deconvolute the m6A epitranscriptome, we developed m6A-level and isoform-characterization sequencing (m6A-LAIC-seq). We found that cells exhibit a broad range of nonstoichiometric m6A levels with cell-type specificity. At the level of isoform characterization, we discovered widespread differences in the use of tandem alternative polyadenylation (APA) sites by methylated and nonmethylated transcript isoforms of individual genes. Strikingly, there is a strong bias for methylated transcripts to be coupled with proximal APA sites, resulting in shortened 3′ untranslated regions, while nonmethylated transcript isoforms tend to use distal APA sites. m6A-LAIC-seq yields a new perspective on transcriptome complexity and links APA usage to m6A modifications.

Do Mitochondria make Nitric Oxide? No?

Several papers have claimed that mitochondria contain nitric oxide synthase (NOS) and make nitric oxide (NO•) in amounts sufficient to affect mitochondrial respiration. However, we found that the addition of l-arginine or the NOS inhibitor l-NMMA to intact rat liver mitochondria did not have any effect on the respiratory rate in both State 3 and State 4. We did not detect mitochondrial NO• production by the oxymyoglobin oxidation assay, or electrochemically using an NO• electrode. An apparent NO• production detected by the Griess assay was identified as an artifact. NO• generated by eNOS added to the mitochondria could easily be detected, although succinate-supplemented mitochondria appeared to consume NO•. Our data show that NO• production by normal rat liver mitochondria cannot be detected in our laboratory, even though the levels of production claimed in the literature should easily have been measured by the techniques used. The implications for the putative mitochondrial NOS are discussed.

Oxidative damage in mitochondrial DNA is not extensive

Abstract: Since 1988 several research groups have reported greater levels of oxidative damage in mitochondrial DNA than in nuclear DNA, while others have suggested that the greater damage in mtDNA might be due to artifactual oxidation. The popular theory that mtDNA is more heavily damaged in vivo than nDNA does not stand on firm ground. Using an improved GC‐MS method and pure mtDNA, our analyses revealed that the damage level in mtDNA is not higher, and may be somewhat lower, than that in nDNA.

A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA: Application to Stress-Induced Reprogramming of tRNA Modifications.

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. This 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.

Sequence-Dependent Variation in the Reactivity of 8-Oxo-7,8-dihydro-2′-deoxyguanosine toward Oxidation

The goal of this study was to define the effect of DNA sequence on the reactivity of 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) toward oxidation. To this end, we developed a quadrupole/time-of-flight (QTOF) mass spectrometric method to quantify the reactivity of site specifically modified oligodeoxyribonucleotides with two model oxidants: nitrosoperoxycarbonate (ONOOCO(2)(-)), a chemical mediator of inflammation, and photoactivated riboflavin, a classical one-electron oxidant widely studied in mutagenesis and charge transport in DNA. In contrast to previous observations with guanine [ Margolin , Y. , ( 2006 ) Nat. Chem. Biol. 2 , 365 ], sequence context did not affect the reactivity of ONOOCO(2)(-) with 8-oxodG, but photosensitized riboflavin showed a strong sequence preference in its reactivity with the following order (8-oxodG = O): COA ≈ AOG > GOG ≥ COT > TOC > AOC. That the COA context was the most reactive was unexpected and suggests a new sequence context where mutation hotspots might occur. These results point to both sequence- and agent-specific effects on 8-oxodG oxidation.

Identification of N6,N6-Dimethyladenosine in Transfer RNA from Mycobacterium bovis Bacille Calmette-Guérin

There are more than 100 different ribonucleoside structures incorporated as post-transcriptional modifications, mainly in tRNA and rRNA of both prokaryotes and eukaryotes, and emerging evidence suggests that these modifications function as a system in the translational control of cellular responses. However, our understanding of this system is hampered by the paucity of information about the complete set of RNA modifications present in individual organisms. To this end, we have employed a chromatography-coupled mass spectrometric approach to define the spectrum of modified ribonucleosides in microbial species, starting with Mycobacterium bovis BCG. This approach revealed a variety of ribonucleoside candidates in tRNA from BCG, of which 12 were definitively identified based on comparisons to synthetic standards and 5 were tentatively identified by exact mass comparisons to RNA modification databases. Among the ribonucleosides observed in BCG tRNA was one not previously described in tRNA, which we have now characterized as N6,N6-dimethyladenosine.

Pitfalls in the Denaturing High-Performance Liquid Chromatography Analysis of Mitochondrial DNA Mutation

Denaturing high-performance liquid chromatography (DHPLC) purification of heteroduplexes has been reported as a method to increase sensitivity of the detection of low-level heteroplasmy by DNA sequencing, and DHPLC profiling has been suggested as a method to allow the correlation of a characteristic chromatographic profile with a specific sequence alteration. Herein we report pitfalls associated with the use of DHPLC for these purposes. We show that the purified heteroduplex fraction does not contain a 50:50 mix of wild-type and mutant DNA in DNA samples containing low-level mutations, and that with a commonly used protocol, DNA sequencing gave false negative results at the 1% mutation level, potentially leading to misdiagnosis. We improved the protocol to detect low levels of mutations and evaluated the sensitivity of DNA sequencing in the detection of mutation in these fractions. We also studied the DHPLC profiles of several mutations in the tRNALeu(UUR) region of mitochondrial DNA and found a characteristic profile in only one of five mutants tested, whereas four other mutants showed identical chromatographic profiles.

Accumulation of CD11c+CD163+ Adipose Tissue Macrophages through Upregulation of Intracellular 11β-HSD1 in Human Obesity.

Adipose tissue (AT) macrophages (ATMs) are key players for regulation of AT homeostasis and obesity-related metabolic disorders. However, the phenotypes of human ATMs and regulatory mechanisms of their polarization have not been clearly described. In this study, we investigated human ATMs in both abdominal visceral AT and s.c. AT and proposed an 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1)–glucocorticoid receptor regulatory axis that might dictate M1/M2 polarization in ATMs. The accumulation of CD11c+CD163+ ATMs in both visceral AT and s.c. AT of obese individuals was confirmed at the cellular level and was found to be clearly correlated with body mass index and production of reactive oxygen species. Using our in vitro system where human peripheral blood monocytes (hPBMs) were cocultured with Simpson–Golabi–Behmel syndrome adipocytes, M1/M2 polarization was found to be dependent on 11β-HSD1, an intracellular glucocorticoid reactivating enzyme. Exposure of hPBMs to cortisol-induced expression of CD163 and RU-486, a glucocorticoid receptor antagonist, significantly abrogated CD163 expression through coculture of mature adipocytes with hPBMs. Moreover, 11β-HSD1 was expressed in crown ATMs in obese AT. Importantly, conditioned medium from coculture of adipocytes with hPBMs enhanced proliferation of human breast cancer MCF7 and MDA-MB-231 cells. In summary, the phenotypic switch of ATMs from M2 to mixed M1/M2 phenotype occurred through differentiation of adipocytes in obese individuals, and upregulation of intracellular 11β-HSD1 might play a role in the process.

In situ analysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine oxidation reveals sequence- and agent-specific damage spectra

Guanine is a major target for oxidation in DNA, with 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) as a major product. 8-oxodG is itself significantly more susceptible to oxidation than guanine, with the resulting damage consisting of more than 10 different products. This complexity has hampered efforts to understand the determinants of biologically relevant DNA oxidation chemistry. To address this problem, we have developed a high mass accuracy mass spectrometric method to quantify oxidation products arising site specifically in DNA. We applied this method to quantify the role of sequence context in defining the spectrum of damage products arising from oxidation of 8-oxodG by two oxidants: nitrosoperoxycarbonate (ONOOCO(2)(-)), a macrophage-derived chemical mediator of inflammation, and the classical one-electron oxidant, riboflavin-mediated photooxidation. The results reveal the predominance of dehydroguanidinohydantoin (DGh) in 8-oxodG oxidation by both oxidants. While the relative quantities of 8-oxodG oxidation products arising from ONOOCO(2)(-) did not vary as a function of sequence context, products of riboflavin-mediated photooxidation of 8-oxodG were highly sequence dependent. Several of the 8-oxodG oxidation products underwent hydrolytic conversion to new products with half-lives of 2-7 h. The results have implications for understanding the chemistry of DNA oxidation and the biological response to the damage, with DNA damage recognition and repair systems faced with a complex and dynamic set of damage targets.