Kathrin Thüring

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 (m1A) 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, m1A 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 m1A, 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 m1A in the template RNA, meaning it is sequence dependent. The RT-signature of m1A was used for inspection and confirmation of suspected modification sites and resulted in the identification of hitherto unknown m1A residues in trypanosomal tRNA.

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 13C-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.

Loss of Anticodon Wobble Uridine Modifications Affects tRNALys Function and Protein Levels in Saccharomyces cerevisiae

In eukaryotes, wobble uridines in the anticodons of tRNALys UUU, tRNAGlu UUC and tRNAGln UUG are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm5 side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm5s2U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm5U and s2U) without a lethal effect. However, an elp3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm5s2U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNALys UUU. These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm5 and s2 wobble uridine modification that could account for growth impairment and mainly originates from tRNALys UUU hypomodification and malfunction.

Phosphorylation of Elp1 by Hrr25 is required for elongator-dependent tRNA modification in yeast.

Elongator is a conserved protein complex comprising six different polypeptides that has been ascribed a wide range of functions, but which is now known to be required for modification of uridine residues in the wobble position of a subset of tRNAs in yeast, plants, worms and mammals. In previous work, we showed that Elongators largest subunit (Elp1; also known as Iki3) was phosphorylated and implicated the yeast casein kinase I Hrr25 in Elongator function. Here we report identification of nine in vivo phosphorylation sites within Elp1 and show that four of these, clustered close to the Elp1 C-terminus and adjacent to a region that binds tRNA, are important for Elongators tRNA modification function. Hrr25 protein kinase directly modifies Elp1 on two sites (Ser-1198 and Ser-1202) and through analyzing non-phosphorylatable (alanine) and acidic, phosphomimic substitutions at Ser-1198, Ser-1202 and Ser-1209, we provide evidence that phosphorylation plays a positive role in the tRNA modification function of Elongator and may regulate the interaction of Elongator both with its accessory protein Kti12 and with Hrr25 kinase.

Analysis of RNA modifications by liquid chromatography-tandem mass spectrometry.

The analysis of RNA modifications is of high importance in order to address a wide range of biological questions. Therefore, a highly sensitive and accurate method such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) has to be available. By using different LC-MS/MS procedures, it is not only possible to quantify very low amounts of RNA modifications, but also to detect probably unknown modified nucleosides. For these cases the dynamic multiple reaction monitoring and the neutral loss scan are the most common techniques. Here, we provide the whole workflow for analyzing RNA samples regarding their modification content. This includes an equipment list, the preparation of required solutions/enzymes and the creation of an internal standard or nucleoside stocks for internal or external calibration. Furthermore, we describe the preparation of RNA samples for the subsequent LC-MS/MS analysis and the corresponding analysis process.

Dynamic modulation of Dnmt2-dependent tRNA methylation by the micronutrient queuine

Dnmt2 enzymes are cytosine-5 methyltransferases that methylate C38 of several tRNAs. We report here that the activities of two Dnmt2 homologs, Pmt1 from Schizosaccharomyces pombe and DnmA from Dictyostelium discoideum, are strongly stimulated by prior queuosine (Q) modification of the substrate tRNA. In vivo tRNA methylation levels were stimulated by growth of cells in queuine-containing medium; in vitro Pmt1 activity was enhanced on Q-containing RNA; and queuine-stimulated in vivo methylation was abrogated by the absence of the enzyme that inserts queuine into tRNA, eukaryotic tRNA-guanine transglycosylase. Global analysis of tRNA methylation in S. pombe showed a striking selectivity of Pmt1 for tRNAAsp methylation, which distinguishes Pmt1 from other Dnmt2 homologs. The present analysis also revealed a novel Pmt1- and Q-independent tRNA methylation site in S. pombe, C34 of tRNAPro. Notably, queuine is a micronutrient that is scavenged by higher eukaryotes from the diet and gut microflora. This work therefore reveals an unanticipated route by which the environment can modulate tRNA modification in an organism.

Urmylation and tRNA thiolation functions of ubiquitin-like Uba4·Urm1 systems are conserved from yeast to man

The ubiquitin‐like protein Urm1 from budding yeast and its E1‐like activator Uba4 have dual roles in protein urmylation and tRNA thiolation pathways. To study whether these are conserved among eukaryotes, we used gene shuffles to replace the yeast proteins by their human counterparts, hURM1 and hUBA4/MOCS3. As judged from biochemical and genetical assays, hURM1 and hUBA4 are functional in yeast, albeit at reduced efficiencies. They mediate urmylation of the peroxiredoxin Ahp1, a known urmylation target in yeast, and support tRNA thiolation. Similar to hUBA4, yeast Uba4 itself is modified by Urm1 and hURM1 suggesting target overlap between eukaryal urmylation pathways. In sum, our study shows that dual‐function ubiquitin‐like Urm1·Uba4 systems are conserved and exchangeable between human and yeast cells.

Variable presence of 5-methylcytosine in commercial RNA and DNA

Nucleoside methylations and other nucleic acid modifications have recently encountered a surge in interest, prompted, among other things, by the detection of methylation and active demethylation of DNA and mRNA by similar mechanisms. In DNA, deoxycytidine methylation by Dnmt enzymes generates 5-methyldeoxycytidine,1 an important epigenetic mark that typically causes inactivation of transcription of the methylated promoter region. Recent exciting developments have shown that these marks are not concrete-cast, but can be actively removed by the oxidative action of TET enzymes,2 which generate, through a series of 2-electron oxidations, first hydroxymethylcytidine (hm5C), then formyldeoxycytidine (f5C),3 and finally carboxydeoxycytidine (ca5C), which may eventually regenerate deoxycytidine by decarboxylation. The apparent functional homolog in mRNA is m6A, which appears to reduce translation efficiency. Here, too, the methylation can be removed by TET-related enzymes generating first hydroxymethyladenosine (hm6A), then formyladenosine (f6A).4,5,6 Also, the presence of 5-methylcytidine in mRNA has been reported early on, and has recently raised renewed interest, although its function is as yet unclear and putative conversion into hydroxymethylcytidine is yet to be demonstrated.32 These developments are enhanced by the development of highly sensitive detection methods7,8 including the adaptation of the so-called bisulfite sequencing from DNA, where it is well established,9 to RNA, where its application has significantly contributed to the present high level of interest.10,11,12 However, bisulfite sequencing alone does not yield unassailable results13,14 and we have thus looked to expand the limits of detection of m5C in both DNA and RNA by LC-MS/MS. Here we report a straightforward regimen that provides values for the limit of quantification (LOQ) in the triple digit attomol range. Its application to presumed negative controls, namely synthetic oligonucleotides, surprisingly detected significant amounts of m5C in both types of synthetic nucleic acids.

Next‐Generation Sequencing‐Based RiboMethSeq Protocol for Analysis of tRNA 2′‐O‐Methylation

Analysis of RNA modifications by traditional physico-chemical approaches is labor intensive, requires substantial amounts of input material and only allows site-by-site measurements. The recent development of qualitative and quantitative approaches based on next-generation sequencing (NGS) opens new perspectives for the analysis of various cellular RNA species. The Illumina sequencing-based RiboMethSeq protocol was initially developed and successfully applied for mapping of ribosomal RNA (rRNA) 2′-O-methylations. This method also gives excellent results in the quantitative analysis of rRNA modifications in different species and under varying growth conditions. However, until now, RiboMethSeq was only employed for rRNA, and the whole sequencing and analysis pipeline was only adapted to this long and rather conserved RNA species. A deep understanding of RNA modification functions requires large and global analysis datasets for other important RNA species, namely for transfer RNAs (tRNAs), which are well known to contain a great variety of functionally-important modified residues. Here, we evaluated the application of the RiboMethSeq protocol for the analysis of tRNA 2′-O-methylation in Escherichia coli and in Saccharomyces cerevisiae. After a careful optimization of the bioinformatic pipeline, RiboMethSeq proved to be suitable for relative quantification of methylation rates for known modified positions in different tRNA species.

Bioconjugation of small Molecules to rna impedes its recognition by Toll- like receptor 7

A fundamental mechanism of the innate immune system is the recognition, via extra- and intracellular pattern-recognition receptors, of pathogen-associated molecular patterns. A prominent example is represented by foreign nucleic acids, triggering the activation of several signaling pathways. Among these, the endosomal toll-like receptor 7 (TLR7) is known to be activated by single-stranded RNA (ssRNA), which can be specifically influenced through elements of sequence structure and posttranscriptional modifications. Furthermore, small molecules TLR7 agonists (smTLRa) are applied as boosting adjuvants in vaccination processes. In this context, covalent conjugations between adjuvant and vaccines have been reported to exhibit synergistic effects. Here, we describe a concept to chemically combine three therapeutic functions in one RNA bioconjugate. This consists in the simultaneous TLR7 stimulation by ssRNA and smTLRa as well as the therapeutic function of the RNA itself, e.g., as a vaccinating or knockdown agent. We have hence synthesized bioconjugates of mRNA and siRNA containing covalently attached smTLRa and tested their function in TLR7 stimulation. Strikingly, the bioconjugates displayed decreased rather than synergistically increased stimulation. The decrease was distinct from the antagonistic action of an siRNA bearing a Gm motive, as observed by direct comparison of the effects in the presence of otherwise stimulatory RNA. In summary, these investigations showed that TRL7 activation can be impeded by bioconjugation of small molecules to RNA.