An ethically guided preclinical device for phenotyping H2 production in laboratory rodents.

BACKGROUND: Dihydrogen (H2) is produced endogenously by the intestinal microbiota through the fermentation of diet carbohydrates. Over the past few years, numerous studies have demonstrated the significant therapeutic potential of H2 in various pathophysiological contexts, making the characterization of its production in laboratory species of major preclinical importance. METHODS: This study proposes an innovative solution to accurately monitor H2 production in free-moving rodents while respecting animal welfare standards. The developed device consisted of a wire rodent cage placed inside an airtight chamber in which the air quality was maintained, and the H2 concentration was continuously analyzed. After the airtightness and efficiency of the systems used to control and maintain air quality in the chamber were checked, tests were carried out on rats and mice with different metabolic phenotypes, over 12 min to 1-h experiments and repeatedly. H2 production rates (HPR) were obtained using an easy calculation algorithm based on a first-order moving average. RESULTS: HPR in hyperphagic Zucker rats was found to be twice as high as in control Wistar rats, respectively, 2.64 and 1.27 nmol.s-1 per animal. In addition, the ingestion of inulin, a dietary fiber, stimulated H2 production in mice. HPRs were 0.46 nmol.s-1 for animals under control diet and 1.99 nmol.s-1 for animals under inulin diet. CONCLUSIONS: The proposed device coupled with our algorithm enables fine analysis of the metabolic phenotype of laboratory rats or mice with regard to their endogenous H2 production.

Structural basis for human mitochondrial tRNA maturation.

The human mitochondrial genome is transcribed into two RNAs, containing mRNAs, rRNAs and tRNAs, all dedicated to produce essential proteins of the respiratory chain. The precise excision of tRNAs by the mitochondrial endoribonucleases (mt-RNase), P and Z, releases all RNA species from the two RNA transcripts. The tRNAs then undergo 3'-CCA addition. In metazoan mitochondria, RNase P is a multi-enzyme assembly that comprises the endoribonuclease PRORP and a tRNA methyltransferase subcomplex. The requirement for this tRNA methyltransferase subcomplex for mt-RNase P cleavage activity, as well as the mechanisms of pre-tRNA 3'-cleavage and 3'-CCA addition, are still poorly understood. Here, we report cryo-EM structures that visualise four steps of mitochondrial tRNA maturation: 5' and 3' tRNA-end processing, methylation and 3'-CCA addition, and explain the defined sequential order of the tRNA processing steps. The methyltransferase subcomplex recognises the pre-tRNA in a distinct mode that can support tRNA-end processing and 3'-CCA addition, likely resulting from an evolutionary adaptation of mitochondrial tRNA maturation complexes to the structurally-fragile mitochondrial tRNAs. This subcomplex can also ensure a tRNA-folding quality-control checkpoint before the sequential docking of the maturation enzymes. Altogether, our study provides detailed molecular insight into RNA-transcript processing and tRNA maturation in human mitochondria.

Beyond the Anticodon: tRNA Core Modifications and Their Impact on Structure, Translation and Stress Adaptation.

Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional chemical modifications. Approximately 100 different modifications have been identified in tRNAs, and each tRNA typically contains 5-15 modifications that are incorporated at specific sites along the tRNA sequence. These modifications may be classified into two groups according to their position in the three-dimensional tRNA structure, i.e., modifications in the tRNA core and modifications in the anticodon-loop (ACL) region. Since many modified nucleotides in the tRNA core are involved in the formation of tertiary interactions implicated in tRNA folding, these modifications are key to tRNA stability and resistance to RNA decay pathways. In comparison to the extensively studied ACL modifications, tRNA core modifications have generally received less attention, although they have been shown to play important roles beyond tRNA stability. Here, we review and place in perspective selected data on tRNA core modifications. We present their impact on tRNA structure and stability and report how these changes manifest themselves at the functional level in translation, fitness and stress adaptation.

Relationship between Car-Sickness Susceptibility and Postural Activity: Could the Re-Weighting Strategy between Signals from Different Body Sensors Be an Underlying Factor?

Postural control characteristics have been proposed as a predictor of Motion Sickness (MS). However, postural adaptation to sensory environment changes may also be critical for MS susceptibility. In order to address this issue, a postural paradigm was used where accurate orientation information from body sensors could be lost and restored, allowing us to infer sensory re-weighting dynamics from postural oscillation spectra in relation to car-sickness susceptibility. Seventy-one participants were standing on a platform (eyes closed) alternating from static phases (proprioceptive and vestibular sensors providing reliable orientation cues) to sway referenced to the ankle-angle phases (proprioceptive sensors providing unreliable orientation cues). The power spectrum density (PSD) on a 10 s sliding window was computed from the antero-posterior displacement of the center of pressure. Energy ratios (ERs) between the high (0.7-1.3 Hz) and low (0.1-0.7 Hz) frequency bands of these PSDs were computed on key time windows. Results showed no difference between MS and non-MS participants following loss of relevant ankle proprioception. However, the reintroduction of reliable ankle signals led, for the non-MS participants, to an increase of the ER originating from a previously up-weighted vestibular information during the sway-referenced situation. This suggests inter-individual differences in re-weighting dynamics in relation to car-sickness susceptibility.

Advances in the Structural and Functional Understanding of m1A RNA Modification.

ConspectusRNA modification is a co- or post-transcriptional process by which specific nucleotides are chemically altered by enzymes after their initial incorporation into the RNA chain, expanding the chemical and functional diversity of RNAs. Our understanding of RNA modifications has changed dramatically in recent years. In the past decade, RNA methyltransferases (MTases) have been highlighted in numerous clinical studies and disease models, modifications have been found to be dynamically regulated by demodification enzymes, and significant technological advances have been made in the fields of RNA sequencing, mass spectrometry, and structural biology. Among RNAs, transfer RNAs (tRNAs) exhibit the greatest diversity and density of post-transcriptional modifications, which allow for potential cross-talks and regulation during their incorporation. N1-methyladenosine (m1A) modification is found in tRNAs at positions 9, 14, 16, 22, 57, and 58, depending on the tRNA and organism.Our laboratory has used and developed a large panel of tools to decipher the different mechanisms used by m1A tRNA MTases to recognize and methylate tRNA. We have solved the structures of TrmI from Thermus thermophilus (m1A58), TrmK from Bacillus subtilis (m1A22), and human TRMT10C (m1A9). These MTases do not share the same structure or organization to recognize tRNAs, but they all modify an adenosine, forming a non-Watson-Crick (WC) interaction. For TrmK, nuclear magnetic resonance (NMR) chemical shift mapping of the binding interface between TrmK and tRNASer was invaluable to build a TrmK/tRNA model, where both domains of TrmK participate in the binding of a full-length L-shaped tRNA and where the non-WC purine 13-A22 base pair positions the A22 N1-atom close to the methyl of the S-adenosyl-l-methionine (SAM) TrmK cofactor. For TRMT10C, cryoEM structures showed the MTase poised to N1-methylate A9 or G9 in tRNA and revealed different steps of tRNA maturation, where TRMT10C acts as a tRNA binding platform for sequential docking of each maturation enzyme. This work confers a role for TRMT10C in tRNA quality control and provides a framework to understand the link between mitochondrial tRNA maturation dysfunction and diseases.Methods to directly detect the incorporation of modifications during tRNA biosynthesis are rare and do not provide easy access to the temporality of their introduction. To this end, we have introduced time-resolved NMR to monitor tRNA maturation in the cellular environment. Combined with genetic and biochemical approaches involving the synthesis of specifically modified tRNAs, our methodology revealed that some modifications are incorporated in a defined sequential order, controlled by cross-talks between modification events. In particular, a strong modification circuit, namely Ψ55 → m5U54 → m1A58, controls the modification process in the T-arm of yeast elongator tRNAs. Conversely, we showed that m1A58 is efficiently introduced on unmodified initiator tRNAiMet without the need of any prior modification. Two distinct pathways are therefore followed for m1A58 incorporation in elongator and initiator tRNAs.We are undoubtedly entering an exciting period for the elucidation of the functions of RNA modifications and the intricate mechanisms by which modification enzymes identify and alter their RNA substrates. These are promising directions for the field of epitranscriptomics.

Modulation of Visually Induced Self-motion Illusions by α Transcranial Electric Stimulation over the Superior Parietal Cortex.

The growing popularity of virtual reality systems has led to a renewed interest in understanding the neurophysiological correlates of the illusion of self-motion (vection), a phenomenon that can be both intentionally induced or avoided in such systems, depending on the application. Recent research has highlighted the modulation of α power oscillations over the superior parietal cortex during vection, suggesting the occurrence of inhibitory mechanisms in the sensorimotor and vestibular functional networks to resolve the inherent visuo-vestibular conflict. The present study aims to further explore this relationship and investigate whether neuromodulating these waves could causally affect the quality of vection. In a crossover design, 22 healthy volunteers received high amplitude and focused α-tACS (transcranial alternating current stimulation) over the superior parietal cortex while experiencing visually induced vection triggered by optokinetic stimulation. The tACS was tuned to each participant's individual α peak frequency, with θ-tACS and sham stimulation serving as controls. Overall, participants experienced better quality vection during α-tACS compared with control θ-tACS and sham stimulations, as quantified by the intensity of vection. The observed neuromodulation supports a causal relationship between parietal α oscillations and visually induced self-motion illusions, with their entrainment triggering overinhibition of the conflict within the sensorimotor and vestibular functional networks. These results confirm the potential of noninvasive brain stimulation for modulating visuo-vestibular conflicts, which could help to enhance the sense of presence in virtual reality environments.

Different modification pathways for m1A58 incorporation in yeast elongator and initiator tRNAs.

As essential components of the protein synthesis machinery, tRNAs undergo a tightly controlled biogenesis process, which include the incorporation of numerous posttranscriptional modifications. Defects in these tRNA maturation steps may lead to the degradation of hypomodified tRNAs by the rapid tRNA decay (RTD) and nuclear surveillance pathways. We previously identified m1A58 as a late modification introduced after modifications Ψ55 and T54 in yeast elongator tRNAPhe. However, previous reports suggested that m1A58 is introduced early during the tRNA modification process, in particular on primary transcripts of initiator tRNAiMet, which prevents its degradation by RNA decay pathways. Here, aiming to reconcile this apparent inconsistency on the temporality of m1A58 incorporation, we examined its introduction into yeast elongator and initiator tRNAs. We used specifically modified tRNAs to report on the molecular aspects controlling the Ψ55 → T54 → m1A58 modification circuit in elongator tRNAs. We also show that m1A58 is efficiently introduced on unmodified tRNAiMet, and does not depend on prior modifications. Finally, we show that m1A58 has major effects on the structural properties of initiator tRNAiMet, so that the tRNA elbow structure is only properly assembled when this modification is present. This observation provides a structural explanation for the degradation of hypomodified tRNAiMet lacking m1A58 by the nuclear surveillance and RTD pathways.

Single-cytosine methylation at W-boxes repels binding of WRKY transcription factors through steric hindrance.

DNA methylation is an epigenetic mark that fine-tunes gene expression, notably by negatively or positively regulating transcription factor (TF)-DNA binding. In plants, DNA methylation has primarily been shown to inhibit TF-DNA binding. However, little is known about the underlying mechanisms. Here, we show that DNA methylation decreases the binding of several Arabidopsis (Arabidopsis thaliana) WRKY TFs to their genomic regions and their binding sites in vitro. We also provide evidence that DNA methylation at a single cytosine located in a functional core W-box motif repels DNA binding of AtWRKY40 in vitro. Using structural modelling, we further demonstrate that this cytosine interacts through van der Waals contacts with the conserved tyrosine of WRKY-DNA binding domains. Importantly, our model predicts steric hindrance when a 5-methyl group is present on this specific cytosine, thereby likely preventing tight binding of WRKY-DNA binding domains. Finally, because the WRKY motif and the residues involved in DNA contacts are conserved across Arabidopsis and rice (Oryza sativa) WRKY TFs, we propose that this methylation-dependent WRKY-DNA binding inhibitory mechanism could be widespread across plant species.

Synthesis of RNA-cofactor conjugates and structural exploration of RNA recognition by an m6A RNA methyltransferase.

Chemical synthesis of RNA conjugates has opened new strategies to study enzymatic mechanisms in RNA biology. To gain insights into poorly understood RNA nucleotide methylation processes, we developed a new method to synthesize RNA-conjugates for the study of RNA recognition and methyl-transfer mechanisms of SAM-dependent m6A RNA methyltransferases. These RNA conjugates contain a SAM cofactor analogue connected at the N6-atom of an adenosine within dinucleotides, a trinucleotide or a 13mer RNA. Our chemical route is chemo- and regio-selective and allows flexible modification of the RNA length and sequence. These compounds were used in crystallization assays with RlmJ, a bacterial m6A rRNA methyltransferase. Two crystal structures of RlmJ in complex with RNA-SAM conjugates were solved and revealed the RNA-specific recognition elements used by RlmJ to clamp the RNA substrate in its active site. From these structures, a model of a trinucleotide bound in the RlmJ active site could be built and validated by methyltransferase assays on RlmJ mutants. The methyl transfer by RlmJ could also be deduced. This study therefore shows that RNA-cofactor conjugates are potent molecular tools to explore the active site of RNA modification enzymes.

Idiosyncratic multisensory reweighting as the common cause for motion sickness susceptibility and adaptation to postural perturbation.

Numerous empirical and modeling studies have been done to find a relationship between postural stability and the susceptibility to motion sickness (MS). However, while the demonstration of a causal relationship between postural stability and the susceptibility to MS is still lacking, recent studies suggest that motion sick individuals have genuine deficits in selecting and reweighting multimodal sensory information. Here we investigate how the adaptation to changing postural situations develops and how the dynamics in multisensory integration is modulated on an individual basis along with MS susceptibility. We used a postural task in which participants stood on a posturographic platform with either eyes open (EO) or eyes closed (EC) during three minutes. The platform was static during the first minute (baseline phase), oscillated harmonically during the second minute (perturbation phase) and returned to its steady state for the third minute (return phase). Principal component (PC) analysis was applied to the sequence of short-term power density spectra of the antero-posterior position of the center of pressure. Results showed that the less motion-sick a participant is, the more similar is his balance between high and low frequencies for EO and EC conditions (as calculated from the eigenvector of the first PC). By fitting exponential decay models to the first PC score in the return phase, we estimated, for each participant in each condition, the sluggishness to return to the baseline spectrum. We showed that the de-adaptation following platform oscillation depends on the susceptibility to MS. These results suggest that non motion-sick participants finely adjust their spectrum in the perturbation phase (i.e. reweighting) and therefore take longer to return to their initial postural control particularly with eyes closed. Thus, people have idiosyncratic ways of doing sensory reweighting for postural control, these processes being tied to MS susceptibility.

Postural responses to specific types of long-term memory during visually induced roll self-motion.

A large body of research has shown that visually induced self-motion (vection) and cognitive processing may interfere with each other. The aim of this study was to assess the interactive effects of a visual motion inducing vection (uniform motion in roll) versus a visual motion without vection (non-uniform motion) and long-term memory processing using the characteristics of standing posture (quiet stance). As the level of interference may be related to the nature of the cognitive tasks used, we examined the effect of visual motion on a memory task which requires a spatial process (episodic recollection) versus a memory task which does not require this process (semantic comparisons). Results confirm data of the literature showing that compensatory postural response in the same direction as background motion. Repeatedly watching visual uniform motion or increasing the cognitive load with a memory task did not decrease postural deviations. Finally, participants were differentially controlling their balance according to the memory task but this difference was significant only in the vection condition and in the plane of background motion. Increased sway regularity (decreased entropy) combined with decreased postural stability (increase variance) during vection for the episodic task would indicate an ineffective postural control. The different interference of episodic and semantic memory on posture during visual motion is consistent with the involvement of spatial processes during episodic memory recollection. It can be suggested that spatial disorientation due to visual roll motion preferentially interferes with spatial cognitive tasks, as spatial tasks can draw on resources expended to control posture.

Brain oscillatory correlates of visuomotor adaptive learning.

Sensorimotor adaptation involves the recalibration of the mapping between motor command and sensory feedback in response to movement errors. Although adaptation operates within individual movements on a trial-to-trial basis, it can also undergo learning when adaptive responses improve over the course of many trials. Brain oscillatory activities related to these "adaptation" and "learning" processes remain unclear. The main reason for this is that previous studies principally focused on the beta band, which confined the outcome message to trial-to-trial adaptation. To provide a wider understanding of adaptive learning, we decoded visuomotor tasks with constant, random or no perturbation from EEG recordings in different bandwidths and brain regions using a multiple kernel learning approach. These different experimental tasks were intended to separate trial-to-trial adaptation from the formation of the new visuomotor mapping across trials. We found changes in EEG power in the post-movement period during the course of the visuomotor-constant rotation task, in particular an increased (i) theta power in prefrontal region, (ii) beta power in supplementary motor area, and (iii) gamma power in motor regions. Classifying the visuomotor task with constant rotation versus those with random or no rotation, we were able to relate power changes in beta band mainly to trial-to-trial adaptation to error while changes in theta band would relate rather to the learning of the new mapping. Altogether, this suggested that there is a tight relationship between modulation of the synchronization of low (theta) and higher (essentially beta) frequency oscillations in prefrontal and sensorimotor regions, respectively, and adaptive learning.

Caenorhabditis elegans RMI2 functional homolog-2 (RMIF-2) and RMI1 (RMH-1) have both overlapping and distinct meiotic functions within the BTR complex.

Homologous recombination is a high-fidelity repair pathway for DNA double-strand breaks employed during both mitotic and meiotic cell divisions. Such repair can lead to genetic exchange, originating from crossover (CO) generation. In mitosis, COs are suppressed to prevent sister chromatid exchange. Here, the BTR complex, consisting of the Bloom helicase (HIM-6 in worms), topoisomerase 3 (TOP-3), and the RMI1 (RMH-1 and RMH-2) and RMI2 scaffolding proteins, is essential for dismantling joint DNA molecules to form non-crossovers (NCOs) via decatenation. In contrast, in meiosis COs are essential for accurate chromosome segregation and the BTR complex plays distinct roles in CO and NCO generation at different steps in meiotic recombination. RMI2 stabilizes the RMI1 scaffolding protein, and lack of RMI2 in mitosis leads to elevated sister chromatid exchange, as observed upon RMI1 knockdown. However, much less is known about the involvement of RMI2 in meiotic recombination. So far, RMI2 homologs have been found in vertebrates and plants, but not in lower organisms such as Drosophila, yeast, or worms. We report the identification of the Caenorhabditis elegans functional homolog of RMI2, which we named RMIF-2. The protein shows a dynamic localization pattern to recombination foci during meiotic prophase I and concentration into recombination foci is mutually dependent on other BTR complex proteins. Comparative analysis of the rmif-2 and rmh-1 phenotypes revealed numerous commonalities, including in regulating CO formation and directing COs toward chromosome arms. Surprisingly, the prevalence of heterologous recombination was several fold lower in the rmif-2 mutant, suggesting that RMIF-2 may be dispensable or less strictly required for some BTR complex-mediated activities during meiosis.

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.

DNA topoisomerase 3 is required for efficient germ cell quality control.

An important quality control mechanism eliminates meiocytes that have experienced recombination failure during meiosis. The culling of defective oocytes in Caenorhabditis elegans meiosis resembles late oocyte elimination in female mammals. Here we show that topoisomerase 3 depletion generates DNA lesions in both germline mitotic and meiotic compartments that are less capable of triggering p53 (cep-1)-dependent apoptosis, despite the activation of DNA damage and apoptosis signaling. Elimination of nonhomologous, alternative end joining and single strand annealing repair factors (CKU-70, CKU-80, POLQ-1, and XPF-1) can alleviate the apoptosis block. Remarkably, the ability of single mutants in the other members of the Bloom helicase-topoisomerase-RMI1 complex to elicit apoptosis is not compromised, and depletion of Bloom helicase in topoisomerase 3 mutants restores an effective apoptotic response. Therefore, uncontrolled Bloom helicase activity seems to direct DNA repair toward normally not used repair pathways, and this counteracts efficient apoptosis. This implicates an as-yet undescribed requirement for topoisomerase 3 in mounting an effective apoptotic response to ensure germ cell quality control.

Improving vision for surgeons during laparoscopy: the Enhanced Laparoscopic Vision System (ELViS).

BACKGROUND: For many abdominal surgical interventions, laparotomy has gradually been replaced by laparoscopy, with numerous benefits for the patient in terms of post-operative recovery. However, during laparoscopy, the endoscope only provides a single viewpoint to the surgeon, leaving numerous blind spots and opening the way to peri-operative adverse events. Alternative camera systems have been proposed, but many lack the requisite resolution/robustness for use during surgery or cannot provide real-time images. Here, we present the added value of the Enhanced Laparoscopic Vision System (ELViS) which overcomes these limitations and provides a broad view of the surgical field in addition to the usual high-resolution endoscope. METHODS: Experienced laparoscopy surgeons performed several typical procedure steps on a live pig model. The time-to-completion for surgical exercises performed by conventional endoscopy and ELViS-assisted surgery was measured. A debriefing interview following each operating session was conducted by an ergonomist, and a System Usability Scale (SUS) score was determined. RESULTS: Proof of concept of ELVIS was achieved in an animal model with seven expert surgeons without peroperative adverse events related to the surgical device. No differences were found in time-to-completion. Mean SUS score was 74.7, classifying the usability of the ELViS as "good". During the debriefing interview, surgeons highlighted several situations where the ELViS provided a real advantage (such as during instrument insertion, exploration of the abdominal cavity or for orientation during close work) and also suggested avenues for improvement of the system. CONCLUSIONS: This first test of the ELViS prototype on a live animal model demonstrated its usability and provided promising and useful feedback for further development.

Transportin-1: A Nuclear Import Receptor with Moonlighting Functions.

Transportin-1 (Trn1), also known as karyopherin-ß2 (Kapß2), is probably the best-characterized nuclear import receptor of the karyopherin-ß family after Importin-ß, but certain aspects of its functions in cells are still puzzling or are just recently emerging. Since the initial identification of Trn1 as the nuclear import receptor of hnRNP A1 ∼25 years ago, several molecular and structural studies have unveiled and refined our understanding of Trn1-mediated nuclear import. In particular, the understanding at a molecular level of the NLS recognition by Trn1 made a decisive step forward with the identification of a new class of NLSs called PY-NLSs, which constitute the best-characterized substrates of Trn1. Besides PY-NLSs, many Trn1 cargoes harbour NLSs that do not resemble the archetypical PY-NLS, which complicates the global understanding of cargo recognition by Trn1. Although PY-NLS recognition is well established and supported by several structures, the recognition of non-PY-NLSs by Trn1 is far less understood, but recent reports have started to shed light on the recognition of this type of NLSs. Aside from its principal and long-established activity as a nuclear import receptor, Trn1 was shown more recently to moonlight outside nuclear import. Trn1 has for instance been caught in participating in virus uncoating, ciliary transport and in modulating the phase separation properties of aggregation-prone proteins. Here, we focus on the structural and functional aspects of Trn1-mediated nuclear import, as well as on the moonlighting activities of Trn1.

Structural studies of RNase M5 reveal two-metal-ion supported two-step dsRNA cleavage for 5S rRNA maturation.

All species transcribe ribosomal RNA in an immature form that requires several enzymes for processing into mature rRNA. The number and types of enzymes utilized for these processes vary greatly between different species. In low G + C Gram-positive bacteria including Bacillus subtilis and Geobacillus stearothermophilus, the endoribonuclease (RNase) M5 performs the final step in 5S rRNA maturation, by removing the 3'- and 5'-extensions from precursor (pre) 5S rRNA. This cleavage activity requires initial complex formation between the pre-rRNA and a ribosomal protein, uL18, making the full M5 substrate a ribonucleoprotein particle (RNP). M5 contains a catalytic N-terminal Toprim domain and an RNA-binding C-terminal domain, respectively, shown to assist in processing and binding of the RNP. Here, we present structural data that show how two Mg2+ ions are accommodated in the active site pocket of the catalytic Toprim domain and investigate the importance of these ions for catalysis. We further perform solution studies that support the previously proposed 3'-before-5' order of removal of the pre-5S rRNA extensions and map the corresponding M5 structural rearrangements during catalysis.

Instrumental analysis of RNA modifications.

Organisms from all domains of life invest a substantial amount of energy for the introduction of RNA modifications into nearly all transcripts studied to date. Instrumental analysis of RNA can focus on the modified residues and reveal the function of these epitranscriptomic marks. Here, we will review recent advances and breakthroughs achieved by NMR spectroscopy, sequencing, and mass spectrometry of the epitranscriptome.