The ART of RNAylation: covalent RNA-protein linkage in bacteriophage infection.

Bacteriophages have been a treasure trove for the discovery of fundamental biological principles and the expansion of our enzymatic toolkit since the dawn of molecular biology. In a recent study by Wolfram-Schauerte et al. these ubiquitous bacteria-infecting viruses reveal yet another new biological concept: post-translational modification through covalent RNA-protein linkages.

Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping.

N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.

Systematic mapping of rRNA 2'-O methylation during frog development and involvement of the methyltransferase Fibrillarin in eye and craniofacial development in Xenopus laevis.

Ribosomes are essential nanomachines responsible for protein production. Although ribosomes are present in every living cell, ribosome biogenesis dysfunction diseases, called ribosomopathies, impact particular tissues specifically. Here, we evaluate the importance of the box C/D snoRNA-associated ribosomal RNA methyltransferase fibrillarin (Fbl) in the early embryonic development of Xenopus laevis. We report that in developing embryos, the neural plate, neural crest cells (NCCs), and NCC derivatives are rich in fbl transcripts. Fbl knockdown leads to striking morphological defects affecting the eyes and craniofacial skeleton, due to lack of NCC survival caused by massive p53-dependent apoptosis. Fbl is required for efficient pre-rRNA processing and 18S rRNA production, which explains the early developmental defects. Using RiboMethSeq, we systematically reinvestigated ribosomal RNA 2'-O methylation in X. laevis, confirming all 89 previously mapped sites and identifying 15 novel putative positions in 18S and 28S rRNA. Twenty-three positions, including 10 of the new ones, were validated orthogonally by low dNTP primer extension. Bioinformatic screening of the X. laevis transcriptome revealed candidate box C/D snoRNAs for all methylated positions. Mapping of 2'-O methylation at six developmental stages in individual embryos indicated a trend towards reduced methylation at specific positions during development. We conclude that fibrillarin knockdown in early Xenopus embryos causes reduced production of functional ribosomal subunits, thus impairing NCC formation and migration.

Identification of a novel deFADding activity in human, yeast and bacterial 5' to 3' exoribonucleases.

Identification of metabolite caps including FAD on the 5' end of RNA has uncovered a previously unforeseen intersection between cellular metabolism and gene expression. To understand the function of FAD caps in cellular physiology, we characterised the proteins interacting with FAD caps in budding yeast. Here we demonstrate that highly conserved 5'-3' exoribonucleases, Xrn1 and Rat1, physically interact with the RNA 5' FAD cap and both possess FAD cap decapping (deFADding) activity and subsequently degrade the resulting RNA. Xrn1 deFADding activity was also evident in human cells indicating its evolutionary conservation. Furthermore, we report that the recently identified bacterial 5'-3' exoribonuclease RNase AM also possesses deFADding activity that can degrade FAD-capped RNAs in vitro and in Escherichia coli cells. To gain a molecular understanding of the deFADding reaction, an RNase AM crystal structure with three manganese ions coordinated by a sulfate molecule and the active site amino acids was generated that provided details underlying hydrolysis of the FAD cap. Our findings reveal a general propensity for 5'-3' exoribonucleases to hydrolyse and degrade RNAs with 5' end noncanonical caps in addition to their well characterized 5' monophosphate RNA substrates indicating an intrinsic property of 5'-3' exoribonucleases.

Probing small ribosomal subunit RNA helix 45 acetylation across eukaryotic evolution.

NAT10 is an essential enzyme that catalyzes N4-acetylcytidine (ac4C) in eukaryotic transfer RNA and 18S ribosomal RNA. Recent studies suggested that rRNA acetylation is dependent on SNORD13, a box C/D small nucleolar RNA predicted to base-pair with 18S rRNA via two antisense elements. However, the selectivity of SNORD13-dependent cytidine acetylation and its relationship to NAT10's essential function remain to be defined. Here, we demonstrate that SNORD13 is required for acetylation of a single cytidine of human and zebrafish 18S rRNA. In-depth characterization revealed that SNORD13-dependent ac4C is dispensable for human cell growth, ribosome biogenesis, translation and development. This loss of function analysis inspired a cross-evolutionary survey of the eukaryotic rRNA acetylation 'machinery' that led to the characterization of many novel metazoan SNORD13 genes. This includes an atypical SNORD13-like RNA in Drosophila melanogaster which guides ac4C to 18S rRNA helix 45 despite lacking one of the two rRNA antisense elements. Finally, we discover that Caenorhabditis elegans 18S rRNA is not acetylated despite the presence of an essential NAT10 homolog. Our findings shed light on the molecular mechanisms underlying SNORD13-mediated rRNA acetylation across eukaryotic evolution and raise new questions regarding the biological and evolutionary relevance of this highly conserved rRNA modification.

Evaluation of Proximate Composition, Cooking Quality, and Texture Profile Analysis in Himalayan Landraces of Black Gram (Vigna mungo).

The black gram is a traditional pulse crop and is a source of different nutritional components. Due to the scientific community's preference for yield and its components, the area of nutritional composition remains unexplored. Therefore, the evaluation of various quality traits such as proximate composition, cooking quality, texture profile, and association between them is keen important for the identification of the traits influencing the selection of the genotypes. This research aimed at the evaluation of the 25 black gram genotypes for their proximate composition, the effect of different cooking methods (conventional and microwave) on cooking quality parameters, and texture profile analysis. The genotypes were screened on 17 parameters, mean and replicated value of each variable were subjected to statistical analysis. The results for proximate composition showed the range from 11.2-11.7%, 24.24-28.22%, 1.25-1.85%, 3.10-4.45%, 5.35-6.60%, 60.23-64.86% and 368.35-372.75 Kcal/100 g for moisture, protein, fat, dietary fiber, ash, utilizable carbohydrate, and gross energy respectively. Cooking time ranged from 33 to 55.5 min (traditional) to 29.5-48.5 min (microwave), L: B ratio from 1.35 to 1.85, WUR from 1.85 to 2.60, and GSL from 0.25 to 11.30%. TPA's cohesiveness, gumminess, and chewiness ranged from 0.19 to 1.44 N, 0.14-1.30 N, 0.58-3.67 N, 1.14-10.81 N, and 0.58-5.29; 1.16-10.50 N in traditional and microwave cooking. Chewiness, gumminess, protein, ash, and cooking time were positively correlated. The first seven PCs have ≥ 1 eigenvalues, accounting for 23.30, 18.00, 13.50, 9.50, 7.40, 6.70%, and 6.40% of total variability. Mandi-2, Kinnour-1, Kirmour-1, Kangra-2, Bilaspur-1, Kangra-3, Kullu-1, Kullu-4, Chamba-3, and Chamba-7 to PCs 1-2 contributed the most to diversity, indicating good selection for subsequent upgrading initiatives.

Recent insights into noncanonical 5' capping and decapping of RNA.

The 5' N7-methylguanosine cap is a critical modification for mRNAs and many other RNAs in eukaryotic cells. Recent studies have uncovered an RNA 5' capping quality surveillance mechanism, with DXO/Rai1 decapping enzymes removing incomplete caps and enabling the degradation of the RNAs, in a process we also refer to as "no-cap decay." It has also been discovered recently that RNAs in eukaryotes, bacteria, and archaea can have noncanonical caps (NCCs), which are mostly derived from metabolites and cofactors such as NAD, FAD, dephospho-CoA, UDP-glucose, UDP-N-acetylglucosamine, and dinucleotide polyphosphates. These NCCs can affect RNA stability, mitochondrial functions, and possibly mRNA translation. The DXO/Rai1 enzymes and selected Nudix (nucleotide diphosphate linked to X) hydrolases have been shown to remove NCCs from RNAs through their deNADding, deFADding, deCoAping, and related activities, permitting the degradation of the RNAs. In this review, we summarize the recent discoveries made in this exciting new area of RNA biology.

Mammalian Nudix proteins cleave nucleotide metabolite caps on RNAs.

We recently reported the presence of nicotinamide adenine dinucleotide (NAD)-capped RNAs in mammalian cells and a role for DXO and the Nudix hydrolase Nudt12 in decapping NAD-capped RNAs (deNADding) in cells. Analysis of 5'caps has revealed that in addition to NAD, mammalian RNAs also contain other metabolite caps including flavin adenine dinucleotide (FAD) and dephosphoCoA (dpCoA). In the present study we systematically screened all mammalian Nudix proteins for their potential deNADing, FAD cap decapping (deFADding) and dpCoA cap decapping (deCoAping) activity. We demonstrate that Nudt16 is a novel deNADding enzyme in mammalian cells. Additionally, we identified seven Nudix proteins-Nudt2, Nudt7, Nudt8, Nudt12, Nudt15, Nudt16 and Nudt19, to possess deCoAping activity in vitro. Moreover, our screening revealed that both mammalian Nudt2 and Nudt16 hydrolyze FAD-capped RNAs in vitro with Nudt16 regulating levels of FAD-capped RNAs in cells. All decapping activities identified hydrolyze the metabolite cap substrate within the diphosphate linkage. Crystal structure of human Nudt16 in complex with FAD at 2.7 Å resolution provide molecular insights into the binding and metal-coordinated hydrolysis of FAD by Nudt16. In summary, our study identifies novel cellular deNADding and deFADding enzymes and establishes a foundation for the selective functionality of the Nudix decapping enzymes on non-canonical metabolite caps.

Identification of the 3-amino-3-carboxypropyl (acp) transferase enzyme responsible for acp3U formation at position 47 in Escherichia coli tRNAs.

tRNAs from all domains of life contain modified nucleotides. However, even for the experimentally most thoroughly characterized model organism Escherichia coli not all tRNA modification enzymes are known. In particular, no enzyme has been found yet for introducing the acp3U modification at position 47 in the variable loop of eight E. coli tRNAs. Here we identify the so far functionally uncharacterized YfiP protein as the SAM-dependent 3-amino-3-carboxypropyl transferase catalyzing this modification and thereby extend the list of known tRNA modification enzymes in E. coli. Similar to the Tsr3 enzymes that introduce acp modifications at U or m1Ψ nucleotides in rRNAs this protein contains a DTW domain suggesting that acp transfer reactions to RNA nucleotides are a general function of DTW domain containing proteins. The introduction of the acp3U-47 modification in E. coli tRNAs is promoted by the presence of the m7G-46 modification as well as by growth in rich medium. However, a deletion of the enzymes responsible for the modifications at position 46 and 47 in the variable loop of E. coli tRNAs did not lead to a clearly discernible phenotype suggesting that these two modifications play only a minor role in ensuring the proper function of tRNAs in E. coli.

Insulin action in the brain regulates both central and peripheral functions.

The brain has been traditionally thought to be insensitive to insulin, primarily because insulin does not stimulate glucose uptake/metabolism in the brain (as it does in classic insulin-sensitive tissues such as muscle, liver, and fat). However, over the past 20 years, research in this field has identified unique actions of insulin in the brain. There is accumulating evidence that insulin crosses into the brain and regulates central nervous system functions such as feeding, depression, and cognitive behavior. In addition, insulin acts in the brain to regulate systemic functions such as hepatic glucose production, lipolysis, lipogenesis, reproductive competence, and the sympathoadrenal response to hypoglycemia. Decrements in brain insulin action (or brain insulin resistance) can be observed in obesity, type 2 diabetes (T2DM), aging, and Alzheimer's disease (AD), indicating a possible link between metabolic and cognitive health. Here, we describe recent findings on the pleiotropic actions of insulin in the brain and highlight the precise sites, specific neuronal population, and roles for supportive astrocytic cells through which insulin acts in the brain. In addition, we also discuss how boosting brain insulin action could be a therapeutic option for people at an increased risk of developing metabolic and cognitive diseases such as AD and T2DM. Overall, this perspective article serves to highlight some of these key scientific findings, identify unresolved issues, and indicate future directions of research in this field that would serve to improve the lives of people with metabolic and cognitive dysfunctions.

Specialized box C/D snoRNPs act as antisense guides to target RNA base acetylation.

Box C/D snoRNAs are known to guide site-specific ribose methylation of ribosomal RNA. Here, we demonstrate a novel and unexpected role for box C/D snoRNAs in guiding 18S rRNA acetylation in yeast. Our results demonstrate, for the first time, that the acetylation of two cytosine residues in 18S rRNA catalyzed by Kre33 is guided by two orphan box C/D snoRNAs-snR4 and snR45 -not known to be involved in methylation in yeast. We identified Kre33 binding sites on these snoRNAs as well as on the 18S rRNA, and demonstrate that both snR4 and snR45 establish extended bipartite complementarity around the cytosines targeted for acetylation, similar to pseudouridylation pocket formation by the H/ACA snoRNPs. We show that base pairing between these snoRNAs and 18S rRNA requires the putative helicase activity of Kre33, which is also needed to aid early pre-rRNA processing. Compared to yeast, the number of orphan box C/D snoRNAs in higher eukaryotes is much larger and we hypothesize that several of these may be involved in base-modifications.

Cone-beam Computed Tomographic Evaluation of the Anatomical Changes of Temporomandibular Joint Use of Pre-post Dentures: A Time-control Study.

AIM AND OBJECTIVE: The growth of the temporomandibular joint (TMJ) gets affected by multiple factors like aging, occlusion state, and by the movement of the jaw while masticating and swallowing. Radiographic imaging is often utilized as a vital diagnostic adjunct in the evaluation of certain examinations of the TMJ. MATERIALS AND METHODS: In this in vivo study, 30 male participants with mean age 55 years, having edentulous maxillary and mandibular arches from the Outpatient Department of Prosthodontics, were randomly selected. Group I (n = 30) patients who were edentulous for the last 4-5 years but without wearing dentures. Whereas group II (n = 30) patients who were edentulous for the last 4-5 years but were wearing dentures for this period. Maxillary and mandibular dentures were fabricated and delivered to subjects. Subjects were subjected to the TMJ analysis with the help of CBCT. Radiological images of dentomaxillofacial structures were analyzed by a specialist with a dual monitor inside a darkened silent room. On the monitor, three times measurements were recorded followed by calculation of mean value. The recordings were taken on both sides and thus, 210 sites were analyzed altogether, followed by the statistical analysis using SPSS software version 15.0. RESULTS: The mean ages of group I and II were 59.00 ± 6.74 and 58.27 ± 6.75 years, respectively. The intra- and intergroup comparisons were done using a one-sample t-test. Differences in mean intercondylar width in groups I and II were not found to be statistically significant. The difference in mean length of glenoid fossa was not statistically significant at any of the above observation periods. A continuous decline in mean length of glenoid fossa was observed with time in both groups. The range of change in articular eminence length was found to be statistically significant for both the groups (p < 0.05). CONCLUSION: This study shows that the articular eminence flattening is correlated with age; on the other hand, the rate of deformation was found significantly more in total edentulous subjects as compared to subjects having normally maintained occlusion. The anatomical changes inside the TMJ have been much greater expressed within the completely edentulous subjects in whom the angle of sagittal condyle path declines and so does the articular eminence height. CLINICAL SIGNIFICANCE: It is essential to provide the edentulous patient with early prosthetic and occlusal rehabilitation after extractions to prevent the anatomical changes in TMJ.

'View From A Bridge': A New Perspective on Eukaryotic rRNA Base Modification.

Eukaryotic rRNA are modified frequently, although the diversity of modifications is low: in yeast rRNA, there are only 12 different types out of a possible natural repertoire exceeding 112. All nine rRNA base methyltransferases (MTases) and one acetyltransferase have recently been identified in budding yeast, and several instances of crosstalk between rRNA, tRNA, and mRNA modifications are emerging. Although the machinery has largely been identified, the functions of most rRNA modifications remain to be established. Remarkably, a eukaryote-specific bridge, comprising a single ribosomal protein (RP) from the large subunit (LSU), contacts four rRNA base modifications across the ribosomal subunit interface, potentially probing for their presence. We hypothesize in this article that long-range allosteric communication involving rRNA modifications is taking place between the two subunits during translation or, perhaps, the late stages of ribosome assembly.

Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans.

The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m(1)acp(3)Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells. In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes.

Tuning the ribosome: The influence of rRNA modification on eukaryotic ribosome biogenesis and function.

rRNAs are extensively modified during their transcription and subsequent maturation in the nucleolus, nucleus and cytoplasm. RNA modifications, which are installed either by snoRNA-guided or by stand-alone enzymes, generally stabilize the structure of the ribosome. However, they also cluster at functionally important sites of the ribosome, such as the peptidyltransferase center and the decoding site, where they facilitate efficient and accurate protein synthesis. The recent identification of sites of substoichiometric 2'-O-methylation and pseudouridylation has overturned the notion that all rRNA modifications are constitutively present on ribosomes, highlighting nucleotide modifications as an important source of ribosomal heterogeneity. While the mechanisms regulating partial modification and the functions of specialized ribosomes are largely unknown, changes in the rRNA modification pattern have been observed in response to environmental changes, during development, and in disease. This suggests that rRNA modifications may contribute to the translational control of gene expression.

Identification of a new ribose methylation in the 18S rRNA of S. cerevisiae.

Methylation of ribose sugars at the 2'-OH group is one of the major chemical modifications in rRNA, and is catalyzed by snoRNA directed C/D box snoRNPs. Previous biochemical and computational analyses of the C/D box snoRNAs have identified and mapped a large number of 2'-OH ribose methylations in rRNAs. In the present study, we systematically analyzed ribose methylations of 18S rRNA in Saccharomyces cerevisiae, using mung bean nuclease protection assay and RP-HPLC. Unexpectedly, we identified a hitherto unknown ribose methylation at position G562 in the helix 18 of 5' central domain of yeast 18S rRNA. Furthermore, we identified snR40 as being responsible to guide snoRNP complex to catalyze G562 ribose methylation, which makes it only second snoRNA known so far to target three ribose methylation sites: Gm562, Gm1271 in 18S rRNA, and Um898 in 25S rRNA. Our sequence and mutational analysis of snR40 revealed that snR40 uses the same D' box and methylation guide sequence for both Gm562 and Gm1271 methylation. With the identification of Gm562 and its corresponding snoRNA, complete set of ribose methylations of 18S rRNA and their corresponding snoRNAs have finally been established opening great prospects to understand the physiological function of these modifications.

Yeast Kre33 and human NAT10 are conserved 18S rRNA cytosine acetyltransferases that modify tRNAs assisted by the adaptor Tan1/THUMPD1.

The function of RNA is subtly modulated by post-transcriptional modifications. Here, we report an important crosstalk in the covalent modification of two classes of RNAs. We demonstrate that yeast Kre33 and human NAT10 are RNA cytosine acetyltransferases with, surprisingly, specificity toward both 18S rRNA and tRNAs. tRNA acetylation requires the intervention of a specific and conserved adaptor: yeast Tan1/human THUMPD1. In budding and fission yeasts, and in human cells, we found two acetylated cytosines on 18S rRNA, one in helix 34 important for translation accuracy and another in helix 45 near the decoding site. Efficient 18S rRNA acetylation in helix 45 involves, in human cells, the vertebrate-specific box C/D snoRNA U13, which, we suggest, exposes the substrate cytosine to modification through Watson-Crick base pairing with 18S rRNA precursors during small subunit biogenesis. Finally, while Kre33 and NAT10 are essential for pre-rRNA processing reactions leading to 18S rRNA synthesis, we demonstrate that rRNA acetylation is dispensable to yeast cells growth. The inactivation of NAT10 was suggested to suppress nuclear morphological defects observed in laminopathic patient cells through loss of microtubules modification and cytoskeleton reorganization. We rather propose the effects of NAT10 on laminopathic cells are due to reduced ribosome biogenesis or function.

The reverse transcription signature of N-1-methyladenosine in RNA-Seq is sequence dependent.

The combination of Reverse Transcription (RT) and high-throughput sequencing has emerged as a powerful combination to detect modified nucleotides in RNA via analysis of either abortive RT-products or of the incorporation of mismatched dNTPs into cDNA. Here we simultaneously analyze both parameters in detail with respect to the occurrence of N-1-methyladenosine (m(1)A) in the template RNA. This naturally occurring modification is associated with structural effects, but it is also known as a mediator of antibiotic resistance in ribosomal RNA. In structural probing experiments with dimethylsulfate, m(1)A is routinely detected by RT-arrest. A specifically developed RNA-Seq protocol was tailored to the simultaneous analysis of RT-arrest and misincorporation patterns. By application to a variety of native and synthetic RNA preparations, we found a characteristic signature of m(1)A, which, in addition to an arrest rate, features misincorporation as a significant component. Detailed analysis suggests that the signature depends on RNA structure and on the nature of the nucleotide 3' of m(1)A in the template RNA, meaning it is sequence dependent. The RT-signature of m(1)A was used for inspection and confirmation of suspected modification sites and resulted in the identification of hitherto unknown m(1)A residues in trypanosomal tRNA.

Identification of novel methyltransferases, Bmt5 and Bmt6, responsible for the m3U methylations of 25S rRNA in Saccharomyces cerevisiae.

RNA contains various chemical modifications that expand its otherwise limited repertoire to mediate complex processes like translation and gene regulation. 25S rRNA of the large subunit of ribosome contains eight base methylations. Except for the methylation of uridine residues, methyltransferases for all other known base methylations have been recently identified. Here we report the identification of BMT5 (YIL096C) and BMT6 (YLR063W), two previously uncharacterized genes, to be responsible for m3U2634 and m3U2843 methylation of the 25S rRNA, respectively. These genes were identified by RP-HPLC screening of all deletion mutants of putative RNA methyltransferases and were confirmed by gene complementation and phenotypic characterization. Both proteins belong to Rossmann-fold-like methyltransferases and the point mutations in the S-adenosyl-L-methionine binding pocket abolish the methylation reaction. Bmt5 localizes in the nucleolus, whereas Bmt6 is localized predominantly in the cytoplasm. Furthermore, we showed that 25S rRNA of yeast does not contain any m5U residues as previously predicted. With Bmt5 and Bmt6, all base methyltransferases of the 25S rRNA have been identified. This will facilitate the analyses of the significance of these modifications in ribosome function and cellular physiology.

Absolute and relative quantification of RNA modifications via biosynthetic isotopomers.

In the resurging field of RNA modifications, quantification is a bottleneck blocking many exciting avenues. With currently over 150 known nucleoside alterations, detection and quantification methods must encompass multiple modifications for a comprehensive profile. LC-MS/MS approaches offer a perspective for comprehensive parallel quantification of all the various modifications found in total RNA of a given organism. By feeding (13)C-glucose as sole carbon source, we have generated a stable isotope-labeled internal standard (SIL-IS) for bacterial RNA, which facilitates relative comparison of all modifications. While conventional SIL-IS approaches require the chemical synthesis of single modifications in weighable quantities, this SIL-IS consists of a nucleoside mixture covering all detectable RNA modifications of Escherichia coli, yet in small and initially unknown quantities. For absolute in addition to relative quantification, those quantities were determined by a combination of external calibration and sample spiking of the biosynthetic SIL-IS. For each nucleoside, we thus obtained a very robust relative response factor, which permits direct conversion of the MS signal to absolute amounts of substance. The application of the validated SIL-IS allowed highly precise quantification with standard deviations<2% during a 12-week period, and a linear dynamic range that was extended by two orders of magnitude.