Structures of B. subtilis Maturation RNases Captured on 50S Ribosome with Pre-rRNAs.

The pathways for ribosomal RNA (rRNA) maturation diverge greatly among the domains of life. In the Gram-positive model bacterium, Bacillus subtilis, the final maturation steps of the two large ribosomal subunit (50S) rRNAs, 23S and 5S pre-rRNAs, are catalyzed by the double-strand specific ribonucleases (RNases) Mini-RNase III and RNase M5, respectively. Here we present a protocol that allowed us to solve the 3.0 and 3.1 Å resolution cryoelectron microscopy structures of these RNases poised to cleave their pre-rRNA substrates within the B. subtilis 50S particle. These data provide the first structural insights into rRNA maturation in bacteria by revealing how these RNases recognize and process double-stranded pre-rRNA. Our structures further uncover how specific ribosomal proteins act as chaperones to correctly fold the pre-rRNA substrates and, for Mini-III, anchor the RNase to the ribosome. These r-proteins thereby serve a quality-control function in the process from accurate ribosome assembly to rRNA processing.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Structural characterization of B. subtilis m1A22 tRNA methyltransferase TrmK: insights into tRNA recognition.

1-Methyladenosine (m1A) is a modified nucleoside found at positions 9, 14, 22 and 58 of tRNAs, which arises from the transfer of a methyl group onto the N1-atom of adenosine. The yqfN gene of Bacillus subtilis encodes the methyltransferase TrmK (BsTrmK) responsible for the formation of m1A22 in tRNA. Here, we show that BsTrmK displays a broad substrate specificity, and methylates seven out of eight tRNA isoacceptor families of B. subtilis bearing an A22. In addition to a non-Watson-Crick base-pair between the target A22 and a purine at position 13, the formation of m1A22 by BsTrmK requires a full-length tRNA with intact tRNA elbow and anticodon stem. We solved the crystal structure of BsTrmK showing an N-terminal catalytic domain harbouring the typical Rossmann-like fold of Class-I methyltransferases and a C-terminal coiled-coil domain. We used NMR chemical shift mapping to drive the docking of BstRNASer to BsTrmK in complex with its methyl-donor cofactor S-adenosyl-L-methionine (SAM). In this model, validated by methyltransferase activity assays on BsTrmK mutants, both domains of BsTrmK participate in tRNA binding. BsTrmK recognises tRNA with very few structural changes in both partner, the non-Watson-Crick R13-A22 base-pair positioning the A22 N1-atom close to the SAM methyl group.

Molecular basis for transfer RNA recognition by the double-stranded RNA-binding domain of human dihydrouridine synthase 2.

Double stranded RNA-binding domain (dsRBD) is a ubiquitous domain specialized in the recognition of double-stranded RNAs (dsRNAs). Present in many proteins and enzymes involved in various functional roles of RNA metabolism, including RNA splicing, editing, and transport, dsRBD generally binds to RNAs that lack complex structures. However, this belief has recently been challenged by the discovery of a dsRBD serving as a major tRNA binding module for human dihydrouridine synthase 2 (hDus2), a flavoenzyme that catalyzes synthesis of dihydrouridine within the complex elbow structure of tRNA. We here unveil the molecular mechanism by which hDus2 dsRBD recognizes a tRNA ligand. By solving the crystal structure of this dsRBD in complex with a dsRNA together with extensive characterizations of its interaction with tRNA using mutagenesis, NMR and SAXS, we establish that while hDus2 dsRBD retains a conventional dsRNA recognition capability, the presence of an N-terminal extension appended to the canonical domain provides additional residues for binding tRNA in a structure-specific mode of action. Our results support that this extension represents a feature by which the dsRBD specializes in tRNA biology and more broadly highlight the importance of structural appendages to canonical domains in promoting the emergence of functional diversity.

Modulation of alpha waves in sensorimotor cortical networks during self-motion perception evoked by different visual-vestibular conflicts.

Visually induced illusion of self-motion (vection) has been used as a tool to address neural correlates of visual-vestibular interaction. The extent to which vestibular cortical areas are deactivated during vection varies from one study to another. The main question in this study is whether such deactivation depends on the visual-vestibular conflict induced by visual motion. A visual motion about the line of sight (roll motion) induces a visual-canal conflict in upright and supine observers. An additional visual-otolith conflict arises in the upright position only, with the graviceptive inputs indicating that the head is stationary. A 96-channel electroencephalogram (EEG) was recorded in 21 participants exposed to roll motion in seated and supine positions. Meanwhile, perceptual state of self-motion was recorded. Results showed a transient decrease in the cortical sensorimotor networks' alpha activity at the onset of vection whatever the participant's position, and therefore the visual-vestibular conflict. During vection, an increase in alpha activity over parieto-occipital areas was observed in the upright condition, that is, in a condition of visual-otolith conflict. The modulation of alpha activity may be predictive of the illusion of self-motion but also may reflect the level of inhibition in the sensorimotor networks needed to reduce potential interference from vestibular conflicting inputs.NEW & NOTEWORTHY For the first time, we explored the neural correlates of different visuo-vestibular conflicts induced by visual motion using EEG. Our study highlighted a neuronal signature for illusory self-motion (vection) in the sensorimotor networks. Strong alpha activity may predict successful vection but also reflects the level of inhibition of sensorimotor networks needed to reduce potential interfering vestibular inputs. These findings would be of prime importance for simulator and virtual reality systems that induce vection.

Modulation of the HIV nucleocapsid dynamics finely tunes its RNA-binding properties during virion genesis.

During HIV-1 assembly and budding, Gag protein, in particular the C-terminal domain containing the nucleocapsid domain (NCd), p1 and p6, is the site of numerous interactions with viral and cellular factors. Most in vitro studies of Gag have used constructs lacking p1 and p6. Here, using NMR spectroscopy, we show that the p1-p6 region of Gag (NCp15) is largely disordered, but interacts transiently with the NCd. These interactions modify the dynamic properties of the NCd. Indeed, using isothermal titration calorimetry (ITC), we have measured a higher entropic penalty to RNA-binding for the NCd precursor, NCp15, than for the mature form, NCp7, which lacks p1 and p6. We propose that during assembly and budding of virions, concomitant with Gag oligomerization, transient interactions between NCd and p1-p6 become salient and responsible for (i) a higher level of structuration of p6, which favours recruitment of budding partners; and (ii) a higher entropic penalty to RNA-binding at specific sites that favours non-specific binding of NCd at multiple sites on the genomic RNA (gRNA). The contributions of p6 and p1 are sequentially removed via proteolysis during Gag maturation such that the RNA-binding specificity of the mature protein is governed by the properties of NCd.

RNAi keeps Atf1-bound stress response genes in check at nuclear pores.

RNAi pathways are prevalent throughout the eukaryotic kingdom and are well known to regulate gene expression on a post-transcriptional level in the cytoplasm. Less is known about possible functions of RNAi in the nucleus. In the fission yeast Schizosaccharomyces pombe, RNAi is crucial to establish and maintain centromeric heterochromatin and functions to repress genome activity by a chromatin silencing mechanism referred to as cotranscriptional gene silencing (CTGS). Mechanistic details and the physiological relevance of CTGS are unknown. Here we show that RNAi components interact with chromatin at nuclear pores to keep stress response genes in check. We demonstrate that RNAi-mediated CTGS represses stress-inducible genes by degrading mRNAs under noninduced conditions. Under chronic heat stress conditions, a Dicer thermoswitch deports Dicer to the cytoplasm, thereby disrupting CTGS and enabling expression of genes implicated in the acquisition of thermotolerance. Taken together, our work highlights a role for nuclear pores and the stress response transcription factor Atf1 in coordinating the interplay between the RNAi machinery and the S. pombe genome and uncovers a novel mode of RNAi regulation in response to an environmental cue.

Conformational Stability Adaptation of a Double-Stranded RNA-Binding Domain to Transfer RNA Ligand.

The double-stranded RNA-binding domain (dsRBD) is a broadly distributed domain among RNA-maturing enzymes. Although this domain recognizes dsRNA's structures via a conserved canonical structure adopting an α1-ß1ß2ß3-α2 topology, several dsRBDs can accommodate discrete structural extensions expanding further their functional repertoire. How these structural elements engage cooperative communications with the canonical structure and how they contribute to the dsRBD's overall folding are poorly understood. Here, we addressed these issues using the dsRBD of human dihydrouridine synthase-2 (hDus2) (hDus2-dsRBD) as a model. This dsRBD harbors N- and C-terminal extensions, the former being directly involved in the recognition of tRNA substrate of hDus2. These extensions engage residues that form a long-range hydrophobic network (LHN) outside the RNA-binding interface. We show by coarse-grain Brownian dynamics that the Nt-extension and its residues F359 and Y364 rigidify the major folding nucleus of the canonical structure via an indirect effect. hDus2-dsRBD unfolds following a two-state cooperative model, whereas both F359A and Y364A mutants, designed to destabilize this LHN, unfold irreversibly. Structural and computational analyses show that these mutants are unstable due to an increase in the dynamics of the two extensions favoring solvent exposure of α2-helix and weakening the main folding nucleus rigidity. This LHN appears essential for maintaining a thermodynamic stability of the overall system and eventually a functional conformation for tRNA recognition. Altogether, our findings suggest that functional adaptability of extended dsRBDs is promoted by a cooperative hydrophobic coupling between the extensions acting as effectors and the folding nucleus of the canonical structure.

The NMR signature of gluconoylation: a frequent N-terminal modification of isotope-labeled proteins.

N-terminal gluconoylation is a moderately widespread modification in recombinant proteins expressed in Escherichia coli, in particular in proteins bearing an N-terminal histidine-tag. This post-translational modification has been investigated mainly by mass spectrometry. Although its NMR signals must have been observed earlier in spectra of 13C/15N labeled proteins, their chemical shifts were not yet reported. Here we present the complete 1H and 13C chemical shift assignment of the N-terminal gluconoyl post-translational modification, based on a selection of His-tagged protein constructs (CCL2, hnRNP A1 and Lin28) starting with Met-Gly-...-(His)6. In addition, we show that the modification can hydrolyze over time, resulting in a free N-terminus and gluconate. This leads to the disappearance of the gluconoyl signals and the appearance of gluconate signals during the NMR measurements. The chemical shifts presented here can now be used as a reference for the identification of gluconoylation in recombinant proteins, in particular when isotopically labeled.

Benefits of stable isotope labeling in RNA analysis.

RNAs are key players in life as they connect the genetic code (DNA) with all cellular processes dominated by proteins. They contain a variety of chemical modifications and many RNAs fold into complex structures. Here, we review recent progress in the analysis of RNA modification and structure on the basis of stable isotope labeling techniques. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are the key tools and many breakthrough developments were made possible by the analysis of stable isotope labeled RNA. Therefore, we discuss current stable isotope labeling techniques such as metabolic labeling, enzymatic labeling and chemical synthesis. RNA structure analysis by NMR is challenging due to two major problems that become even more salient when the size of the RNA increases, namely chemical shift overlaps and line broadening leading to complete signal loss. Several isotope labeling strategies have been developed to provide solutions to these major issues, such as deuteration, segmental isotope labeling or site-specific labeling. Quantification of modified nucleosides in RNA by MS is only possible through the application of stable isotope labeled internal standards. With nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS), it is now possible to analyze the dynamic processes of post-transcriptional RNA modification and demodification. The trend, in both NMR and MS RNA analytics, is without doubt shifting from the analysis of snapshot moments towards the development and application of tools capable of analyzing the dynamics of RNA structure and modification profiles.