Analyzing RNA posttranscriptional modifications to decipher the epitranscriptomic code.

The discovery of RNA silencing has revealed that non-protein-coding sequences (ncRNAs) can cover essential roles in regulatory networks and their malfunction may result in severe consequences on human health. These findings have prompted a general reassessment of the significance of RNA as a key player in cellular processes. This reassessment, however, will not be complete without a greater understanding of the distribution and function of the over 170 variants of the canonical ribonucleotides, which contribute to the breathtaking structural diversity of natural RNA. This review surveys the analytical approaches employed for the identification, characterization, and detection of RNA posttranscriptional modifications (rPTMs). The merits of analyzing individual units after exhaustive hydrolysis of the initial biopolymer are outlined together with those of identifying their position in the sequence of parent strands. Approaches based on next generation sequencing and mass spectrometry technologies are covered in depth to provide a comprehensive view of their respective merits. Deciphering the epitranscriptomic code will require not only mapping the location of rPTMs in the various classes of RNAs, but also assessing the variations of expression levels under different experimental conditions. The fact that no individual platform is currently capable of meeting all such demands implies that it will be essential to capitalize on complementary approaches to obtain the desired information. For this reason, the review strived to cover the broadest possible range of techniques to provide readers with the fundamental elements necessary to make informed choices and design the most effective possible strategy to accomplish the task at hand.

m6A Methylation of Transcription Leader Sequence of SARS-CoV-2 Impacts Discontinuous Transcription of Subgenomic mRNAs.

The SARS-CoV-2 genome has been shown to be m6A methylated at several positions in vivo. Strikingly, a DRACH motif, the recognition motif for adenosine methylation, resides in the core of the transcriptional regulatory leader sequence (TRS-L) at position A74, which is highly conserved and essential for viral discontinuous transcription. Methylation at position A74 correlates with viral pathogenicity. Discontinuous transcription produces a set of subgenomic mRNAs that function as templates for translation of all structural and accessory proteins. A74 is base-paired in the short stem-loop structure 5'SL3 that opens during discontinuous transcription to form long-range RNA-RNA interactions with nascent (-)-strand transcripts at complementary TRS-body sequences. A74 can be methylated by the human METTL3/METTL14 complex in vitro. Here, we investigate its impact on the structural stability of 5'SL3 and the long-range TRS-leader:TRS-body duplex formation necessary for synthesis of subgenomic mRNAs of all four viral structural proteins. Methylation uniformly destabilizes 5'SL3 and long-range duplexes and alters their relative equilibrium populations, suggesting that the m6A74 modification acts as a regulator for the abundance of viral structural proteins due to this destabilization.

Influence of the Number, Nature and Position of Methyl Posttranscriptional Modifications on Nucleobase Stacking in RNA.

DFT calculations are employed to quantify the influence of the presence, number, nature, and position of posttranscriptional methylation on stacking strength of RNA bases. We carry out detailed potential energy scans of the variation in stacking energies with characteristic geometrical parameters in three categories of forty stacked dimers - canonical base homodimers (N||N), methylated base homodimers (mN||mN) and heterodimers of canonical bases and methylated counterparts (N||mN). Our analysis reveals that neutral methylation invariably enhances the stacking of bases. Further, N||mN stacking is stronger than mN||mN stacking and charged N||mN exhibit strongest stacking among all dimers. This indicates that methylations greatly enhance stacking when dispersed in RNA sequences containing identical bases. Comparison of stacks involving singly- and doubly-methylated purines reveal that incremental methylation enhances the stacking in neutral dimers. Although methylation at the carbon position of neutral pyrimidine dimers greatly enhances the stacking, methylation on the 5-membered ring imparts better stacking compared to methylation on the 6-membered ring in adenine dimers. However, methylation at the ring nitrogen (N1 ) provides better stacking than the amino group (N2 ) in guanine dimers. Our results thus highlight subtle structural effects of methylation on RNA base stacking and will enhance our understanding of the physicochemical principles of RNA structure and dynamics.

Radical Transfer Dissociation for De†Novo Characterization of Modified Ribonucleic Acids by Mass Spectrometry.

Mass spectrometry (MS) can reliably detect and localize all mass-altering modifications of ribonucleic acids (RNA), but current MS approaches that allow for simultaneous de novo sequencing and modification analysis generally require specialized instrumentation. Here we report a novel RNA dissociation technique, radical transfer dissociation (RTD), that can be used for the comprehensive de novo characterization of ribonucleic acids and their posttranscriptional or synthetic modifications. We demonstrate full sequence coverage for RNA consisting of up to 39 nucleotides and show that RTD is especially useful for RNA with highly labile modifications such as 5-hydroxymethylcytidine and 5-formylcytidine.

Intratumor heterogeneity in epigenetic patterns.

Analogous to life on earth, tumor cells evolve through space and time and adapt to different micro-environmental conditions. As a result, tumors are composed of millions of genetically diversified cells at the time of diagnosis. Profiling these variants contributes to understanding tumors' clonal origins and might help to better understand response to therapy. However, even genetically homogenous cell populations show remarkable diversity in their response to different environmental stimuli, suggesting that genetic heterogeneity does not explain the full spectrum of tumor plasticity. Understanding epigenetic diversity across cancer cells provides important additional information about the functional state of subclones and therefore allows better understanding of tumor evolution and resistance to current therapies.

Crystal structure of tRNAHis guanylyltransferase from Saccharomyces cerevisiae.

tRNA maturation involves several steps, including processing, splicing, CCA addition, and posttranscriptional modifications. tRNAHis guanylyltransferase (Thg1) is the only enzyme known to catalyze templated nucleotide addition in the 3'-5' direction, unlike other DNA and RNA polymerases. For a better understanding of its unique catalytic mechanism at the molecular level, we determined the crystal structure of GTP-bound Thg1 from Saccharomyces cerevisiae at the maximum resolution of 3.0 Å. The structure revealed the enzyme to have a tetrameric conformation that is well conserved among different species, and the GTP molecule was clearly bound at the active site, coordinating with two magnesium ions. In addition, two flexible protomers at the potential binding site (PBS) for tRNAHis were observed. We suggest that the PBS of the tetramer could also be one of the sites for interaction with partner proteins.

The NLRP3 inflammasome is a potential mechanism and therapeutic target for perioperative neurocognitive disorders.

Perioperative neurocognitive disorders (PNDs) are frequent complications associated with cognitive impairment during the perioperative period, including acute postoperative delirium and long-lasting postoperative cognitive dysfunction. There are some risk factors for PNDs, such as age, surgical trauma, anesthetics, and the health of the patient, but the underlying mechanism has not been fully elucidated. Pyroptosis is a form of programmed cell death that is mediated by the gasdermin protein and is involved in cognitive dysfunction disorders. The canonical pathway induced by nucleotide oligomerization domain (NOD)-, leucine-rich repeat (LRR)- and pyrin domain-containing protein 3 (NLRP3) inflammasomes contributes to PNDs, which suggests that targeting NLRP3 inflammasomes may be an effective strategy for the treatment of PNDs. Therefore, inhibiting upstream activators and blocking the assembly of the NLRP3 inflammasome may attenuate PNDs. The present review summarizes recent studies and systematically describes the pathogenesis of NLRP3 activation and regulation and potential therapeutics targeting NLRP3 inflammasomes in PNDs patients.

Quantitative Analysis of Adenosine-to-Inosine RNA Editing.

The conversion of adenosine to inosine (A to I) by RNA editing represents a common posttranscriptional mechanism for diversification of both the transcriptome and proteome, and is a part of the cellular response for innate immune tolerance. Due to its preferential base-pairing with cytosine (C), inosine (I) is recognized as guanosine (G) by reverse transcriptase, as well as the cellular splicing and translation machinery. A-to-I editing events appear as A-G discrepancies between genomic DNA and cDNA sequences. Molecular analyses of RNA editing have leveraged these nucleoside differences to quantify RNA editing in ensemble populations of RNA transcripts and within individual cDNAs using high-throughput sequencing or Sanger sequencing-derived analysis of electropherogram peak heights. Here, we briefly review and compare these methods of RNA editing quantification, as well as provide experimental protocols by which such analyses may be achieved.

A Method to Monitor the Introduction of Posttranscriptional Modifications in tRNAs with NMR Spectroscopy.

During their biosynthesis, transfer RNAs (tRNAs) are decorated with a large number of posttranscriptional chemical modifications. Methods to directly detect the introduction of posttranscriptional modifications during tRNA maturation are rare and do not provide information on the temporality of modification events. Here, we report a methodology, using NMR as a tool to monitor tRNA maturation in a nondisruptive and continuous fashion in cellular extracts. This method requires the production of substrate tRNA transcripts devoid of modifications and active cell extracts containing the necessary cellular enzymatic activities to modify RNA. The present protocol describes these different aspects of our method and reports the time-resolved NMR monitoring of the yeast tRNAPhe maturation as an example. The NMR-based methodology presented here could be adapted to investigate diverse features in tRNA maturation.

Dot Blot Analysis for Measuring Global N6-Methyladenosine Modification of RNA.

Posttranscriptional modification of mRNAs plays an important role in establishing the functional diversity of the proteome. The m6A modification is found in many species of RNA, including tRNA, mRNA, rRNA, and long noncoding RNAs. The physiological role of m6A modification of RNA is not fully explored and is a topic of current research. It is predicted that the major effect of m6A modification of mRNAs is on its stability and/or translation. The global changes in m6A levels in total RNA or particular species of RNAs can be measured by dot blot analysis using m6A specific antibodies or using mass spectrometry following chromatographic separation. The dot blot method for detection of global m6A changes is a relatively straightforward method to quantitate m6A modification but suffers from low sensitivity when the fraction of m6A-modified RNA is small in analyzed samples. Here, we describe a modified dot blot method that is sensitive and quantitative for detecting m6A-modified RNA by adding an immunoprecipitation step to enrich for m6A-modified RNA.

An archaeal aminoacyl-tRNA synthetase complex for improved substrate quality control.

Aminoacyl-tRNA synthetases (aaRSs) decode genetic information by coupling tRNAs with cognate amino acids. In the archaeon Methanothermobacter thermautotrophicus arginyl- and seryl-tRNA synthetase (ArgRS and SerRS, respectively) form a complex which enhances serylation and facilitates tRNASer recycling through its association with the ribosome. Yet, the way by which complex formation participates in Arg-tRNAArg synthesis is still unresolved. Here we utilized pull down and surface plasmon resonance experiments with truncated ArgRS variants to demonstrate that ArgRS uses its N-terminal domain to establish analogous interactions with both SerRS and cognate tRNAArg, providing a rationale for the lack of detectable SerRS•[ArgRS•tRNAArg] complex. In contrast, stable ternary ArgRS•[SerRS•tRNASer] complex was easily detected supporting the model wherein ArgRS operates in serylation by modulating SerRS affinity toward tRNASer. We also found that the interaction with SerRS suppresses arginylation of unmodified tRNAArg by ArgRS, which, by itself, does not discriminate against tRNAArg substrates lacking posttranscriptional modifications. Hence, there is a fundamentally different participation of the protein partners in Arg-tRNA and Ser-tRNA synthesis. Propensity of the ArgRS•SerRS complex to exclude unmodified tRNAs from translation leads to an attractive hypothesis that SerRS•ArgRS complex might act in vivo as a safeguarding switch that improves translation accuracy.

Effects of Heterologous tRNA Modifications on the Production of Proteins Containing Noncanonical Amino Acids.

Synthesis of proteins with noncanonical amino acids (ncAAs) enables the creation of protein-based biomaterials with diverse new chemical properties that may be attractive for material science. Current methods for large-scale production of ncAA-containing proteins, frequently carried out in Escherichia coli, involve the use of orthogonal aminoacyl-tRNA synthetases (o-aaRSs) and tRNAs (o-tRNAs). Although o-tRNAs are designed to be orthogonal to endogenous aaRSs, their orthogonality to the components of the E. coli metabolism remains largely unexplored. We systematically investigated how the E. coli tRNA modification machinery affects the efficiency and orthogonality of o-tRNASep used for production of proteins with the ncAA O-phosphoserine (Sep). The incorporation of Sep into a green fluorescent protein (GFP) in 42 E. coli strains carrying deletions of single tRNA modification genes identified several genes that affect the o-tRNA activity. Deletion of cysteine desulfurase (iscS) increased the yield of Sep-containing GFP more than eightfold, while overexpression of dimethylallyltransferase MiaA and pseudouridine synthase TruB improved the specificity of Sep incorporation. These results highlight the importance of tRNA modifications for the biosynthesis of proteins containing ncAAs, and provide a novel framework for optimization of o-tRNAs.