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.

Although very effective in decreasing NMR relaxation of large proteins, homogeneous deuteration can be costly, and anyway unsuitable for recombinant production in metazoan systems. We sought to explore other deuteration schemes, which would be adapted to protein expression in mammalian cells. Here, we evaluate the benefits of the deuteration on alpha- and beta-positions of amino acids for a typical middle size protein domain, namely the model 40 kDa-large kinase p38α. We report the position-specific deuteration of free amino acids by using enzyme-assisted H/D exchange, executed by the cystathionine gamma-synthase and a newly designed high-performance mutant E325A. Then, we used cell-free expression in bacterial extracts to avoid any scrambling and back-protonation of the tested isotopically labelled amino acids (Ala, Leu, Lys, Ser, Asp, Glu, Gly). Our results show signal enhancements up to three in 1H-15N spectra when these α/β-deuterated amino acids are integrated. Because our approach relies on single 2Hα/β-15N-amino acid labeling, an additional three-fold increase in sensitivity is obtained by the possible use of moderate resolution SOFAST-HMQC instead of the classical HSQC or TROSY experiments. This allows recording residue-resolved solution 1H-15N NMR spectra of 100 μg of p38α in one hour with S/N∼10.

Familial partial lipodystrophy type 2 is an autosomal dominant disorder generally due to heterozygous missense variants in the LMNA gene.In women, the phenotype begins to manifest before puberty, while in men the onset is later.It is striking that most of the cases reported in the literature are women.Specifically, in our cohort only 27% of these patients were men.Considering the pattern of inheritance of an autosomal dominant disease where a similar ratio between males and females would be expected, it is obvious that the majority of men with this disorder do not they are diagnosed.The causes of this discrepancy may seem obvious.On the one hand, the pattern -Patient organizations: Patient experiences

Barrier-to-Autointegration Factor (BAF) is a highly conserved DNA binding protein important for genome integrity. Its localization and function are regulated through phosphorylation. Previously reported structures of BAF suggested that it is fully ordered, but our recent NMR analysis revealed that its N-terminal region is flexible in solution and that S4/T3 di-phosphorylation by VRK1 reduces this flexibility. Here, molecular dynamics (MD) simulation was used to unveil the conformational ensembles accessible to the N-terminal region of BAF either unphosphorylated, mono-phosphorylated on S4 or di-phosphorylated on S4/T3 (pBAF) and to reveal the interactions that contribute to define these ensembles. We show that the intrinsic flexibility observed in the N-terminal region of BAF is reduced by S4 phosphorylation and to a larger extent by S4/T3 di-phosphorylation. Thanks to the atomic description offered by MD supported by the NMR study of several BAF mutants, we identified the dynamic network of salt bridge interactions responsible for the conformational restriction involving pS4 and pT3 with residues located in helix α1 and α6. Using MD, we showed that the flexibility in the N-terminal region of BAF depends on the ionic strength and on the pH. We show that the presence of two negative charges of the phosphoryl groups is required for a substantial decrease in flexibility in pBAF. Using MD supported by NMR, we also showed that H7 deprotonation reduces the flexibility in the N-terminal region of BAF. Thus, the conformation of the intrinsically disordered N-terminal region of BAF is highly tunable, likely related to its diverse functions.

Barrier-to-autointegration factor (BAF) is an essential component of the nuclear lamina. Encoded by BANF1, this DNA binding protein contributes to the regulation of gene expression, cell cycle progression, and nuclear integrity. A rare recessive BAF variant, Ala12Thr, causes the premature aging syndrome, Néstor–Guillermo progeria syndrome (NGPS). Here, we report the first dominant pathogenic BAF variant, Gly16Arg, identified in a patient presenting with progressive neuromuscular weakness. Although disease variants carry nearby amino acid substitutions, cellular and biochemical properties are distinct. In contrast to NGPS, Gly16Arg patient fibroblasts show modest changes in nuclear lamina structure and increases in repressive marks associated with heterochromatin. Structural studies reveal that the Gly16Arg substitution introduces a salt bridge between BAF monomers, reducing the conformation ensemble available to BAF. We show that this structural change increases the double-stranded DNA binding affinity of BAF Gly16Arg. Together, our findings suggest that BAF Gly16Arg has an increased chromatin occupancy that leads to epigenetic changes and impacts nuclear functions. These observations provide a new example of how a missense mutation can change a protein conformational equilibrium to cause a dominant disease and extend our understanding of mechanisms by which BAF function impacts human health.

Protein kinases that phosphorylate histones are ideally-placed to influence the behavior of chromosomes during cell division. Indeed, a number of conserved histone phosphorylation events occur prominently during mitosis and meiosis in most eukaryotes, including on histone H3 at threonine-3 (H3T3ph). At least two kinases, Haspin and VRK1 (NHK-1/ballchen in Drosophila), have been proposed to carry out this modification. Phosphorylation of H3 by Haspin has defined roles in mitosis, but the significance of VRK1 activity towards histones in dividing cells has been unclear. Here, using in vitro kinase assays, KiPIK screening, RNA interference, and CRISPR/Cas9 approaches, we were unable to substantiate a direct role for VRK1, or its paralogue VRK2, in the phosphorylation of threonine-3 or serine-10 of Histone H3 in mitosis, although loss of VRK1 did slow cell proliferation. We conclude that the role of VRKs, and their more recently identified association with neuromuscular disease and importance in cancers of the nervous system, are unlikely to involve mitotic histone kinase activity. In contrast, Haspin is required to generate H3T3ph during mitosis.

Nestor-Guillermo progeria syndrome (NGPS) is caused by a homozygous alanine-to-threonine mutation at position 12 (A12T) in barrier-to-autointegration factor (BAF). It is characterized by accelerated aging with severe skeletal abnormalities. BAF is an essential protein binding to DNA and nuclear envelope (NE) proteins, involved in NE rupture repair. Here, we assessed the impact of BAF A12T on NE integrity using NGPS-derived patient fibroblasts. We observed a strong defect in lamin A/C accumulation to NE ruptures in NGPS cells, restored upon homozygous reversion of the pathogenic BAF A12T mutation with CRISPR/Cas9. By combining in vitro and cellular assays, we demonstrated that while the A12T mutation does not affect BAF 3D structure and phosphorylation by VRK1, it specifically decreases the interaction between BAF and lamin A/C. Finally, we revealed that the disrupted interaction does not prevent repair of NE ruptures but instead generates weak points in the NE that lead to a higher frequency of NE re-rupturing in NGPS cells. We propose that this NE fragility could directly contribute to the premature aging phenotype in patients.

Progeroid laminopathies are characterized by the premature appearance of certain signs of physiological aging in a subset of tissues. They are caused by mutations in genes coding for A-type lamins or lamin-binding proteins. Here, we review how different mutations causing progeroid laminopathies alter protein structure or protein-protein interactions and how these impact on mechanisms that protect cell viability and function. One group of progeroid laminopathies, which includes Hutchinson-Gilford progeria syndrome, is characterized by accumulation of unprocessed prelamin A or variants. These are caused by mutations in the A-type lamin gene (LMNA), altering prelamin A itself, or inZMPSTE24, encoding an endoprotease involved in its processing. The abnormally expressed farnesylated proteins impact on various cellular processes that may contribute to progeroid phenotypes. OtherLMNAmutations lead to the production of nonfarnesylated A-type lamin variants with amino acid substitutions in solvent-exposed hot spots located mainly in coil 1B and the immunoglobulin fold domain. Dominant missense mutations might reinforce interactions between lamin domains, thus giving rise to excessively stabilized filament networks. Recessive missense mutations in A-type lamins and barrier-to-autointegration factor (BAF) causing progeroid disorders are found at the interface between these interacting proteins. The amino acid changes decrease the binding affinity of A-type lamins for BAF, which may contribute to lamina disorganization, as well as defective repair of mechanically induced nuclear envelope rupture. Targeting these molecular alterations in A-type lamins and associated proteins identified through structural biology studies could facilitate the design of therapeutic strategies to treat patients with rare but severe progeroid laminopathies.

Barrier-to-autointegration factor (BAF), encoded by the BANF1 gene, is an abundant and ubiquitously expressed metazoan protein that has multiple functions during the cell cycle. Through its ability to cross-bridge two double-stranded DNA (dsDNA), it favours chromosome compaction, participates in post-mitotic nuclear envelope reassembly and is essential for the repair of large nuclear ruptures. BAF forms a ternary complex with the nuclear envelope proteins lamin A/C and emerin, and its interaction with lamin A/C is defective in patients with recessive accelerated aging syndromes. Phosphorylation of BAF by the vaccinia-related kinase 1 (VRK1) is a key regulator of BAF localization and function. Here, we demonstrate that VRK1 successively phosphorylates BAF on Ser4 and Thr3. The crystal structures of BAF before and after phosphorylation are extremely similar. However, in solution, the extensive flexibility of the N-terminal helix α1 and loop α1α2 in BAF is strongly reduced in di-phosphorylated BAF, due to interactions between the phosphorylated residues and the positively charged C-terminal helix α6. These regions are involved in DNA and lamin A/C binding. Consistently, phosphorylation causes a 5000-fold loss of affinity for dsDNA. However, it does not impair binding to lamin A/C Igfold domain and emerin nucleoplasmic region, which leaves open the question of the regulation of these interactions.

Progeroid laminopathies are characterized by the premature appearance of certain signs of physiological aging in a subset of tissues. They are caused by mutations in genes coding for A‐type lamins or lamin‐binding proteins. Here, we review how different mutations causing progeroid laminopathies alter protein structure or protein–protein interactions and how these impact on mechanisms that protect cell viability and function. One group of progeroid laminopathies, which includes Hutchinson‐Gilford progeria syndrome, is characterized by accumulation of unprocessed prelamin A or variants. These are caused by mutations in the A‐type lamin gene (LMNA), altering prelamin A itself, or in ZMPSTE24, encoding an endoprotease involved in its processing. The abnormally expressed farnesylated proteins impact on various cellular processes that may contribute to progeroid phenotypes. Other LMNA mutations lead to the production of nonfarnesylated A‐type lamin variants with amino acid substitutions in solvent‐exposed hot spots located mainly in coil 1B and the immunoglobulin fold domain. Dominant missense mutations might reinforce interactions between lamin domains, thus giving rise to excessively stabilized filament networks. Recessive missense mutations in A‐type lamins and barrier‐to‐autointegration factor (BAF) causing progeroid disorders are found at the interface between these interacting proteins. The amino acid changes decrease the binding affinity of A‐type lamins for BAF, which may contribute to lamina disorganization, as well as defective repair of mechanically induced nuclear envelope rupture. Targeting these molecular alterations in A‐type lamins and associated proteins identified through structural biology studies could facilitate the design of therapeutic strategies to treat patients with rare but severe progeroid laminopathies.

Emerin is a nuclear envelope protein that contributes to genome organization and cell mechanics. Through its N-terminal LAP2-emerin-MAN1 (LEM)-domain, emerin interacts with the DNA-binding protein barrier-to-autointegration (BAF). Emerin also binds to members of the linker of the nucleoskeleton and cytoskeleton (LINC) complex. Mutations in the gene encoding emerin are responsible for the majority of cases of X-linked Emery-Dreifuss muscular dystrophy (X-EDMD). Most of these mutations lead to an absence of emerin. A few missense and short deletion mutations in the disordered region of emerin are also associated with X-EDMD. More recently, missense and short deletion mutations P22L, ∆K37 and T43I were discovered in emerin LEM-domain, associated with isolated atrial cardiac defects (ACD). Here we reveal which defects, at both the molecular and cellular levels, are elicited by these LEM-domain mutations. Whereas ΔK37 mutation impaired the correct folding of the LEM-domain, P22L and T43I had no impact on the 3D structure of emerin. Surprisingly, all three mutants bound to BAF, albeit with a weaker affinity in the case of ΔK37. In human myofibroblasts derived from a patient’s fibroblasts, emerin ∆K37 was correctly localized at the inner nuclear membrane, but was present at a significantly lower level, indicating that this mutant is abnormally degraded. Moreover, SUN2 was reduced, and these cells were defective in producing actin stress fibers when grown on a stiff substrate and after cyclic stretches. Altogether, our data suggest that the main effect of mutation ΔK37 is to perturb emerin function within the LINC complex in response to mechanical stress.

Etude structurale, dynamique et fonctionnelle de la protéine BAF phosphorylée : un dimère muté dans un syndrome de vieillissement prématuré Les syndromes progéroïdes sont des maladies génétiques induisant un vieillissement prématuré chez de jeunes patients. Certains de ces syndromes sont dus à des mutations de la lamina nucléaire. Afin d’augmenter les chances de développer de nouvelles drogues contre ces laminopathies progéroïdes, l’une des approches est de caractériser les mécanismes qui y sont mis en jeu in vitro. Le complexe formé par le domaine C-terminal de la lamine A/C et la protéine dimérique BAF (Barrier-to-Autointegration Factor) est muté dans plusieurs cas de syndromes progéroïdes. La phosphorylation de BAF par Vaccinia-Related Kinase 1 régule la localisation et certaines fonctions de cette protéine au cours de la mitose. J’ai analysé la structure et la dynamique de BAF phosphorylé (pBAF) en utilisant la spectroscopie par Resonnance Magnetique Nucleaire (RNM) résolue dans le temps et la cristallographie aux rayons X, et conclu que la phosphorylation de BAF modifie son équilibre conformationnel. J’ai étudié en détails les interactions intramoléculaires impliquées dans cet équilibre à l’aide de la spectroscopie RMN et de simulations de dynamique moléculaire. Puis, j’ai mesuré les affinités de pBAF pour ses partenaires in vitro en utilisant le Titrage Calorimétrique Isotherme et la fluorescence. Finalement, j’ai recherché les causes moléculaires des phénotypespathologiques causés par des mutations de BAF, et conclu à un défaut d’interaction (pour le premier mutant), et d’équilibre conformationnel (pour le second), à explorer in vivo.