Abstract
The highly abundant N6-methyladenosine (m6A) RNA modification affects most aspects of mRNA function, yet the precise function of the rarer 5-methylcytidine (m5C) remains largely unknown. ...Here, we map m5C in the human transcriptome using methylation-dependent individual-nucleotide resolution cross-linking and immunoprecipitation (miCLIP) combined with RNA bisulfite sequencing. We identify NSUN6 as a methyltransferase with strong substrate specificity towards mRNA. NSUN6 primarily targeted three prime untranslated regions (3′UTR) at the consensus sequence motif CTCCA, located in loops of hairpin structures. Knockout and rescue experiments revealed enhanced mRNA and translation levels when NSUN6-targeted mRNAs were methylated. Ribosome profiling further demonstrated that NSUN6-specific methylation correlated with translation termination. While NSUN6 was dispensable for mouse embryonic development, it was down-regulated in human tumours and high expression of NSUN6 indicated better patient outcome of certain cancer types. In summary, our study identifies NSUN6 as a methyltransferase targeting mRNA, potentially as part of a quality control mechanism involved in translation termination fidelity.
Posttranscriptional modifications in transfer RNA (tRNA) are often critical for normal development because they adapt protein synthesis rates to a dynamically changing microenvironment. However, the ...precise cellular mechanisms linking the extrinsic stimulus to the intrinsic RNA modification pathways remain largely unclear. Here, we identified the cytosine-5 RNA methyltransferase NSUN2 as a sensor for external stress stimuli. Exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Using metabolic profiling, we showed that loss of tRNA methylation captured cells in a distinct catabolic state. Mechanistically, loss of NSUN2 altered the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlated with the dynamic repression of global protein synthesis. Finally, NSUN2-driven RNA methylation was functionally required to adapt cell cycle progression to the early stress response. In summary, we revealed that changes in tRNA methylation profiles were sufficient to specify cellular metabolic states and efficiently adapt protein synthesis rates to cell stress.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Although the biological importance of post-transcriptional RNA modifications in gene expression is widely appreciated, methods to directly detect their introduction during RNA biosynthesis are rare ...and do not easily provide information on the temporal nature of events. Here, we introduce the application of NMR spectroscopy to observe the maturation of tRNAs in cell extracts. By following the maturation of yeast tRNA
with time-resolved NMR measurements, we show that modifications are introduced in a defined sequential order, and that the chronology is controlled by cross-talk between modification events. In particular, we show that a strong hierarchy controls the introduction of the T54, Ψ55 and m
A58 modifications in the T-arm, and we demonstrate that the modification circuits identified in yeast extract with NMR also impact the tRNA modification process in living cells. The NMR-based methodology presented here could be adapted to investigate different aspects of tRNA maturation and RNA modifications in general.
Recently, studies about RNA modification dynamics in human RNAs are among the most controversially discussed. As a main reason, we identified the unavailability of a technique which allows the ...investigation of the temporal processing of RNA transcripts. Here, we present nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) for efficient, monoisotopic stable isotope labeling in both RNA and DNA in standard cell culture. We design pulse chase experiments and study the temporal placement of modified nucleosides in tRNA
and 18S rRNA. In existing RNAs, we observe a time-dependent constant loss of modified nucleosides which is masked by post-transcriptional methylation mechanisms and thus undetectable without NAIL-MS. During alkylation stress, NAIL-MS reveals an adaptation of tRNA modifications in new transcripts but not existing ones. Overall, we present a fast and reliable stable isotope labeling strategy which allows in-depth study of RNA modification dynamics in human cell culture.
Nucleic acids, especially RNA, naturally contain a diversity of chemically modified nucleosides. To understand the biological role of these modified nucleosides, nucleic acid scientists need tools to ...specifically label, detect and enrich modified nucleic acids. These tools comprise a diverse set of chemical reagents which have been established in the early years of nucleic acid research. Recent developments in high-throughput sequencing and mass spectrometry utilize these chemical labeling strategies to efficiently detect and localize modifications in nucleic acids. As a consequence the transcriptome-wide distribution of modified nucleosides, especially 5-methylcytosine and pseudouridine, in all domains of life could be analyzed. With the help of these techniques and the gained knowledge, it becomes possible to understand the functions of modifications and even study their connections to human health and disease. Here, the differential chemical reactivity of modified nucleosides and their canonical counterpart is reviewed and discussed.
RNA in yeast, especially rRNA and tRNA are heavily modified to fulfill their function in protein translation. Using biosynthetic stable isotope labeled internal standards we quantified 12 modified ...nucleosides in tRNA from S. cerevisiae over 24 hours. We observed different quantities of modified nucleosides in dependence of the growth phase. To elucidate the underlying mechanism of the observed tRNA modification profile adaptation, it is necessary to distinguish the pre-existing tRNA pool and its modifications from newly-synthesized tRNAs. By combination of 2 differentially isotope labeled media we developed NAIL-MS, nucleic acid isotope labeling coupled mass spectrometry. During the yeast growth cycle we observe dilution of pre-existing tRNAs by newly-synthesized tRNAs by the growing number of cells. tRNA was found to be highly stable with only little degradation over the observed period. The method was further used to quantify the levels of modified nucleosides in the original and new tRNA pools. By addition of deuterium-labeled methionine, we could observe the incorporation of new methyl marks on pre-existing tRNAs. For 2′-O-methylcytidine (Cm) we observed a global increase in log phase. We identified extensive 2′-OH-cytidine methylation of the pre-existing tRNAs and the new tRNAs which masks an actual decrease of pre-existing Cm. In contrast, global 5-methylcytidine (m
5
C) levels decreased during growth due to a drop in m
5
C quantities in the original tRNA pool.
The NAIL-MS data suggests different mechanisms for tRNA modification adaptation depending on the individual modification observed. With this new tool it is possible to follow the fate of methylated RNAs during growth and potentially compare the impact of different stress conditions on the epitranscriptome.
Queuosine (Q) is a hypermodified 7-deaza-guanosine nucleoside that is found at position 34, also known as the wobble position, of tRNAs with a GUN anticodon, and Q ensures faithful translation of the ...respective C- and U-ending codons. While Q is present in tRNAs in most eukaryotes, only bacteria can synthesize it denovo. In contrast, eukaryotes rely on external sources like their food and the gut microbiome in order to Q-modify their tRNAs, and Q therefore can be regarded as a micronutrient. The eukaryotic tRNA guanine transglycosylase (eTGT) uses the base queuine (q) as a substrate to replace G34 by Q in the tRNAs. Eukaryotic cells can uptake both q and Q, raising the question how the Q nucleoside is converted to q for incorporation into the tRNAs. Here, we identified Qng1 (also termed Duf2419) as a queuosine nucleoside glycosylase in Schizosaccharomyces pombe. S. pombe cells with a deletion of qng1+ contained Q-modified tRNAs only when cultured in the presence of the nucleobase q, but not with the nucleoside Q, indicating that the cells are proficient at q incorporation, but not in Q hydrolysis. Furthermore, purified recombinant Qng1 hydrolyzed Q to q in vitro. Qng1 displays homology to DNA glycosylases and has orthologs across eukaryotes, including flies, mice and humans. Qng1 therefore plays an essential role in allowing eukaryotic cells to salvage Q from bacterial sources and to recycle Q from endogenous tRNAs.
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•Eukaryotic tRNAs with a GUN anticodon are modified at the wobble position with queuosine (Q).•Eukaryotes cannot synthesize Q, but salvage queuosine nucleoside from food and the microbiome.•The enzyme Qng1 from Schizosaccharomyces pombe hydrolyzes queuosine to queuine.•Queuine is the substrate for the TGT enzyme for incorporation into tRNAs.
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•NAIL-MS is the first tool which allows the observation of RNA modification dynamics in vivo.•Types of NAIL-MS experiments.•Nucleic acid isotope labeling strategies.•Observing RNA ...methylation damage repair in vivo.
Ribonucleic acids (RNA) are extensively modified. These modifications are quantified by mass spectrometry (LC-MS/MS) to determine the abundance of a modification under certain conditions or in various genetic backgrounds. With LC-MS/MS the steady state of modifications is determined, and thus we only have a static view of the dynamics of RNA modifications. With nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) we overcome this limitation and get access to the dynamics of RNA modifications. We describe labeling techniques for E. coli, S. cerevisiae and human cell culture and the current instrumental limitations. We present the power of NAIL-MS but we also outline validation experiments, which are necessary for correct data interpretation.
As an example, we apply NAIL-MS to study the demethylation of adenine and cytidine, which are methylated by the damaging agent methyl-methanesulfonate in E. coli. With NAIL-MS we exclude the concurrent processes for removal of RNA methylation, namely RNA degradation, turnover and dilution. We use our tool to study the speed and efficiency of 1-methyladenosine and 3-methylcytidine demethylation.
We further outline current limitations of NAIL-MS but also potential future uses for e.g. relative quantification of tRNA isoacceptor abundances.
Queuosine is one of the most complex hypermodified RNA nucleosides found in the Wobble position of tRNAs. In addition to Queuosine itself, several further modified derivatives are known, where the ...cyclopentene ring structure is additionally modified by a galactosyl-, a mannosyl-, or a glutamyl-residue. While sugar-modified Queuosine derivatives are found in the tRNAs of vertebrates, glutamylated Queuosine (gluQ) is only known in bacteria. The exact structure of gluQ, particularly with respect to how and where the glutamyl side chain is connected to the Queuosine cyclopentene side chain, is unknown. Here we report the first synthesis of gluQ and, using UHPLC-MS-
injection and NMR studies, we show that the isolated natural gluQ is the α-allyl-connected gluQ compound.
Toll-like receptor 7 (TLR7) is essential for recognition of RNA viruses and initiation of antiviral immunity. TLR7 contains two ligand-binding pockets that recognize different RNA degradation ...products: pocket 1 recognizes guanosine, while pocket 2 coordinates pyrimidine-rich RNA fragments. We found that the endonuclease RNase T2, along with 5′ exonucleases PLD3 and PLD4, collaboratively generate the ligands for TLR7. Specifically, RNase T2 generated guanosine 2′,3′-cyclic monophosphate-terminated RNA fragments. PLD exonuclease activity further released the terminal 2′,3′-cyclic guanosine monophosphate (2',3'-cGMP) to engage pocket 1 and was also needed to generate RNA fragments for pocket 2. Loss-of-function studies in cell lines and primary cells confirmed the critical requirement for PLD activity. Biochemical and structural studies showed that PLD enzymes form homodimers with two ligand-binding sites important for activity. Previously identified disease-associated PLD mutants failed to form stable dimers. Together, our data provide a mechanistic basis for the detection of RNA fragments by TLR7.
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•RNase T2 and PLD exonucleases process RNA upstream of TLR7•PLD exonucleases release terminal 2′,3′-cyclic GMPs to engage TLR7 pocket 1•PLD enzymes are also critical to generate RNA fragments for TLR7 pocket 2•PLDs dimer formation is needed for RNA substrate processing
TLR7 is critical for recognizing RNA virus infection and initiating antiviral responses. Bérouti et al. demonstrate how RNase T2 and PLD exonucleases generate RNA fragments for TLR7 activation, thus providing insights into immune recognition of exogenous RNAs, with potential therapeutic implications.
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