Mitochondrial impairment and synaptic dysfunction are associated with neurological defects in iPSCs-derived cortical neurons of MERRF patients.

BACKGROUND: Myoclonic epilepsy with ragged-red fibers (MERRF) syndrome is a rare inherited mitochondrial disease mainly caused by the m.8344A > G mutation in mitochondrial tRNALys gene, and usually manifested as complex neurological disorders and muscle weakness. Currently, the pathogenic mechanism of this disease has not yet been resolved, and there is no effective therapy for MERRF syndrome. In this study, MERRF patients-derived iPSCs were used to model patient-specific neurons for investigation of the pathogenic mechanism of neurological disorders in mitochondrial disease. METHODS: MERRF patient-derived iPSCs were differentiated into excitatory glutamatergic neurons to unravel the effects of the m.8344A > G mutation on mitochondrial bioenergetic function, neural-lineage differentiation and neuronal function. By the well-established differentiation protocol and electrophysiological activity assay platform, we examined the pathophysiological behaviors in cortical neurons of MERRF patients. RESULTS: We have successfully established the iPSCs-derived neural progenitor cells and cortical-like neurons of patients with MERRF syndrome that retained the heteroplasmy of the m.8344A > G mutation from the patients' skin fibroblasts and exhibited the phenotype of the mitochondrial disease. MERRF neural cells harboring the m.8344A > G mutation exhibited impaired mitochondrial bioenergetic function, elevated ROS levels and imbalanced expression of antioxidant enzymes. Our findings indicate that neural immaturity and synaptic protein loss led to the impairment of neuronal activity and plasticity in MERRF neurons harboring the m.8344A > G mutation. By electrophysiological recordings, we monitored the in vivo neuronal behaviors of MERRF neurons and found that neurons harboring a high level of the m.8344A > G mutation exhibited impairment of the spontaneous and evoked potential-stimulated neuronal activities. CONCLUSIONS: We demonstrated for the first time the link of mitochondrial impairment and synaptic dysfunction to neurological defects through impeding synaptic plasticity in excitatory neurons derived from iPSCs of MERRF patients harboring the m.8344A > G mutation. This study has provided new insight into the pathogenic mechanism of the tRNALys gene mutation of mtDNA, which is useful for the development of a patient-specific iPSCs platform for disease modeling and screening of new drugs to treat patients with MERRF syndrome.

Pressure pushes tRNALys3 into excited conformational states.

Conformational dynamics play essential roles in RNA function. However, detailed structural characterization of excited states of RNA remains challenging. Here, we apply high hydrostatic pressure (HP) to populate excited conformational states of tRNALys3, and structurally characterize them using a combination of HP 2D-NMR, HP-SAXS (HP-small-angle X-ray scattering), and computational modeling. HP-NMR revealed that pressure disrupts the interactions of the imino protons of the uridine and guanosine U-A and G-C base pairs of tRNALys3. HP-SAXS profiles showed a change in shape, but no change in overall extension of the transfer RNA (tRNA) at HP. Configurations extracted from computational ensemble modeling of HP-SAXS profiles were consistent with the NMR results, exhibiting significant disruptions to the acceptor stem, the anticodon stem, and the D-stem regions at HP. We propose that initiation of reverse transcription of HIV RNA could make use of one or more of these excited states.

Structural Significance of Conformational Preferences and Ribose-Ring-Puckering of Hyper Modified Nucleotide 5'-Monophosphate 2-Methylthio Cyclic N6-Threonylcarbamoyladenosine (p-ms2ct6A) Present at 37th Position in Anticodon Loop of tRNALys.

Structural significance of conformational preferences and ribose ring puckering of newly discovered hyper modified nucleotide, 5'-monophosphate 2-methylthio cyclic N6-threonylcarbamoyladenosine (p-ms2ct6A) have been investigated using quantum chemical semi-empirical RM1 and molecular dynamics simulation techniques. Automated geometry optimization of most stable structure of p-ms2ct6A has also been carried out with the help of abinitio (HF SCF, DFT) as well as semi empirical quantum chemical (RM1, AM1, PM3, and PM6) methods. Most stable structure of p-ms2ct6A is stabilized by intramolecular interactions between N(3)…HC(2'), N(1)…HC(16), O(13)…HC(15), and O(13)…HO(14). The torsion angles alpha (α) and beta (ß) show the significant characteristic patterns with the involvement of intramolecular hydrogen bonding to provide stability to the p-ms2ct6A. Further, molecular dynamics simulations of p-ms2ct6A revealed the role of ribose sugar ring puckering i.e. C2'-endo and C3'-endo on the structural dynamics of ms2ct6A side chain. The modified nucleotide p-ms2ct6A periodically prefers both the C2'-endo and C3'-endo sugar with 'anti' and 'syn' conformations. This property of p-ms2ct6A could be useful to recognize the starting ANN codons. All atom explicit MD simulation of anticodon loop (ACL) of tRNALys of Bacillus subtilis containing ms2ct6A at 37th position showed the U-turn feature, base stacking ability with other adjacent bases and hydrogen bonding interactions similar to the isolated base p-ms2ct6A. The ribose sugar puckering contributes to the orientation of the side chain conformation of p-ms2ct6A. Thus, the present study could be helpful to understand the structure-function relationship of the hypermodified nucleoside, ms2ct6A in recognition of the proper codons AAA/AAG during protein biosynthesis.

Rapamycin rescues mitochondrial dysfunction in cells carrying the m.8344A > G mutation in the mitochondrial tRNALys.

BACKGROUND: Myoclonus, Epilepsy and Ragged-Red-Fibers (MERRF) is a mitochondrial encephalomyopathy due to heteroplasmic mutations in mitochondrial DNA (mtDNA) most frequently affecting the tRNALys gene at position m.8344A > G. Defective tRNALys severely impairs mitochondrial protein synthesis and respiratory chain when a high percentage of mutant heteroplasmy crosses the threshold for full-blown clinical phenotype. Therapy is currently limited to symptomatic management of myoclonic epilepsy, and supportive measures to counteract muscle weakness with co-factors/supplements. METHODS: We tested two therapeutic strategies to rescue mitochondrial function in cybrids and fibroblasts carrying different loads of the m.8344A > G mutation. The first strategy was aimed at inducing mitochondrial biogenesis directly, over-expressing the master regulator PGC-1α, or indirectly, through the treatment with nicotinic acid, a NAD+ precursor. The second was aimed at stimulating the removal of damaged mitochondria through prolonged rapamycin treatment. RESULTS: The first approach slightly increased mitochondrial protein expression and respiration in the wild type and intermediate-mutation load cells, but was ineffective in high-mutation load cell lines. This suggests that induction of mitochondrial biogenesis may not be sufficient to rescue mitochondrial dysfunction in MERRF cells with high-mutation load. The second approach, when administered chronically (4 weeks), induced a slight increase of mitochondrial respiration in fibroblasts with high-mutation load, and a significant improvement in fibroblasts with intermediate-mutation load, rescuing completely the bioenergetics defect. This effect was mediated by increased mitochondrial biogenesis, possibly related to the rapamycin-induced inhibition of the Mechanistic Target of Rapamycin Complex 1 (mTORC1) and the consequent activation of the Transcription Factor EB (TFEB). CONCLUSIONS: Overall, our results point to rapamycin-based therapy as a promising therapeutic option for MERRF.

mRNA and tRNA modification states influence ribosome speed and frame maintenance during poly(lysine) peptide synthesis.

Ribosome speed is dictated by multiple factors including substrate availability, cellular conditions, and product (peptide) formation. Translation slows during the synthesis of cationic peptide sequences, potentially influencing the expression of thousands of proteins. Available evidence suggests that ionic interactions between positively charged nascent peptides and the negatively charged ribosome exit tunnel impede translation. However, this hypothesis was difficult to test directly because of inability to decouple the contributions of amino acid charge from mRNA sequence and tRNA identity/abundance in cells. Furthermore, it is unclear if other components of the translation system central to ribosome function (e.g., RNA modification) influence the speed and accuracy of positively charged peptide synthesis. In this study, we used a fully reconstituted Escherichia coli translation system to evaluate the effects of peptide charge, mRNA sequence, and RNA modification status on the translation of lysine-rich peptides. Comparison of translation reactions on poly(lysine)-encoding mRNAs conducted with either Lys-tRNALys or Val-tRNALys reveals that that amino acid charge, while important, only partially accounts for slowed translation on these transcripts. We further find that in addition to peptide charge, mRNA sequence and both tRNA and mRNA modification status influence the rates of amino acid addition and the ribosome's ability to maintain frame (instead of entering the -2, -1, and +1 frames) during poly(lysine) peptide synthesis. Our observations lead us to expand the model for explaining how the ribosome slows during poly(lysine) peptide synthesis and suggest that posttranscriptional RNA modifications can provide cells a mechanism to precisely control ribosome movements along an mRNA.

Spinach-based RNA mimicking GFP in plant cells.

Spinach RNA-mimicking GFP (S-RMG) has been successfully used to monitor cellular RNAs including microRNAs in bacterium, yeast, and human cells. However, S-RMG has not been established in plants. In this study, we found that like bacterial, yeast, and human cellular tRNAs, plant tRNAs such as tRNALys can protect and/or stabilize the Spinach RNA aptamer interaction with the fluorophore DFHBI enabling detectable levels of green fluorescence to be emitted. The tRNALys-Spinach-tRNALys, once delivered into "chloroplast-free" onion epidermal cells can emit strong green fluorescence in the presence of DFHBI. Our results demonstrate for the first time that Spinach-based RNA visualization has the potential for in vivo monitoring of RNAs in plant cells.

Physical Chemistry of a Single tRNA-Modified Nucleoside Regulates Decoding of the Synonymous Lysine Wobble Codon and Affects Type 2 Diabetes.

The 2-methylthio-modification (ms2-) of N6-threonylcarbonyladenosine (t6A37) at position-37 (ms2t6A37) in tRNAUUULys3 provides the needed stability between the tRNA anticodon and the human insulin mRNA codon AAG during translation, as determined by molecular dynamics simulation. Single-nucleoside polymorphisms of the human gene for the enzyme, Cdkal1 that post-transcriptionally modifies t6A37 to ms2t6A37 in tRNAUUULys3, correlate with type 2 diabetes mellitus. Without the ms2-modification, tRNAUUULys3 is incapable of correctly translating the insulin mRNA AAG codon for lysine at the site of protease cleavage between the A-chain and the C-peptide. By enhancing anticodon/codon cross-strand stacking, the ms2-modification adds stability through van der Waals interactions and dehydration of the ASL loop and cavity of the anticodon/codon minihelix but does not add hydrogen bonding of any consequence. Thus, the modifying enzyme Cdkal1, by adding a crucial ms2-group to tRNAUUULys3-t6A37, facilitates the decoding of the AAG codon and enables human pancreatic islets to correctly translate insulin mRNA.

Cooperative methylation of human tRNA3Lys at positions A58 and U54 drives the early and late steps of HIV-1 replication.

Retroviral infection requires reverse transcription, and the reverse transcriptase (RT) uses cellular tRNA as its primer. In humans, the TRMT6-TRMT61A methyltransferase complex incorporates N1-methyladenosine modification at tRNA position 58 (m1A58); however, the role of m1A58 as an RT-stop site during retroviral infection has remained questionable. Here, we constructed TRMT6 mutant cells to determine the roles of m1A in HIV-1 infection. We confirmed that tRNA3Lys m1A58 was required for in vitro plus-strand strong-stop by RT. Accordingly, infectivity of VSV-G pseudotyped HIV-1 decreased when the virus contained m1A58-deficient tRNA3Lys instead of m1A58-modified tRNA3Lys. In TRMT6 mutant cells, the global protein synthesis rate was equivalent to that of wild-type cells. However, unexpectedly, plasmid-derived HIV-1 expression showed that TRMT6 mutant cells decreased accumulation of HIV-1 capsid, integrase, Tat, Gag, and GagPol proteins without reduction of HIV-1 RNAs in cells, and fewer viruses were produced. Moreover, the importance of 5,2'-O-dimethyluridine at U54 of tRNA3Lys as a second RT-stop site was supported by conservation of retroviral genome-tRNALys sequence-complementarity, and TRMT6 was required for efficient 5-methylation of U54. These findings illuminate the fundamental importance of tRNA m1A58 modification in both the early and late steps of HIV-1 replication, as well as in the cellular tRNA modification network.

High-resolution view of HIV-1 reverse transcriptase initiation complexes and inhibition by NNRTI drugs.

Reverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNALys3 primer bound to the vRNA genome and is the target of key antivirals, such as non-nucleoside reverse transcriptase inhibitors (NNRTIs). Initiation proceeds slowly with discrete pausing events along the vRNA template. Despite prior medium-resolution structural characterization of reverse transcriptase initiation complexes (RTICs), higher-resolution structures of the RTIC are needed to understand the molecular mechanisms that underlie initiation. Here we report cryo-EM structures of the core RTIC, RTIC-nevirapine, and RTIC-efavirenz complexes at 2.8, 3.1, and 2.9 Å, respectively. In combination with biochemical studies, these data suggest a basis for rapid dissociation kinetics of RT from the vRNA-tRNALys3 initiation complex and reveal a specific structural mechanism of nucleic acid conformational stabilization during initiation. Finally, our results show that NNRTIs inhibit the RTIC and exacerbate discrete pausing during early reverse transcription.

The three-way junction structure of the HIV-1 PBS-segment binds host enzyme important for viral infectivity.

HIV-1 reverse transcription initiates at the primer binding site (PBS) in the viral genomic RNA (gRNA). Although the structure of the PBS-segment undergoes substantial rearrangement upon tRNALys3 annealing, the proper folding of the PBS-segment during gRNA packaging is important as it ensures loading of beneficial host factors. DHX9/RNA helicase A (RHA) is recruited to gRNA to enhance the processivity of reverse transcriptase. Because the molecular details of the interactions have yet to be defined, we solved the solution structure of the PBS-segment preferentially bound by RHA. Evidence is provided that PBS-segment adopts a previously undefined adenosine-rich three-way junction structure encompassing the primer activation stem (PAS), tRNA-like element (TLE) and tRNA annealing arm. Disruption of the PBS-segment three-way junction structure diminished reverse transcription products and led to reduced viral infectivity. Because of the existence of the tRNA annealing arm, the TLE and PAS form a bent helical structure that undergoes shape-dependent recognition by RHA double-stranded RNA binding domain 1 (dsRBD1). Mutagenesis and phylogenetic analyses provide evidence for conservation of the PBS-segment three-way junction structure that is preferentially bound by RHA in support of efficient reverse transcription, the hallmark step of HIV-1 replication.

Synthetic post-translational modifications of elongation factor P using the ligase EpmA.

Canonically, tRNA synthetases charge tRNA. However, the lysyl-tRNA synthetase paralog EpmA catalyzes the attachment of (R)-ß-lysine to the ε-amino group of lysine 34 of the translation elongation factor P (EF-P) in Escherichia coli. This modification is essential for EF-P-mediated translational rescue of ribosomes stalled at consecutive prolines. In this study, we determined the kinetics of EpmA and its variant EpmA_A298G to catalyze the post-translational modification of K34 in EF-P with eight noncanonical substrates. In addition, acetylated EF-P was generated using an amber suppression system. The impact of these synthetically modified EF-P variants on in vitro translation of a polyproline-containing NanoLuc luciferase reporter was analyzed. Our results show that natural (R)-ß-lysylation was more effective in rescuing stalled ribosomes than any other synthetic modification tested. Thus, our work not only provides new biochemical insights into the function of EF-P, but also opens a new route to post-translationally modify proteins using EpmA.

Posttranscriptional modifications at the 37th position in the anticodon stem-loop of tRNA: structural insights from MD simulations.

Transfer RNA (tRNA) is the most diversely modified RNA. Although the strictly conserved purine position 37 in the anticodon stem-loop undergoes modifications that are phylogenetically distributed, we do not yet fully understand the roles of these modifications. Therefore, molecular dynamics simulations are used to provide molecular-level details for how such modifications impact the structure and function of tRNA. A focus is placed on three hypermodified base families that include the parent i6A, t6A, and yW modifications, as well as derivatives. Our data reveal that the hypermodifications exhibit significant conformational flexibility in tRNA, which can be modulated by additional chemical functionalization. Although the overall structure of the tRNA anticodon stem remains intact regardless of the modification considered, the anticodon loop must rearrange to accommodate the bulky, dynamic hypermodifications, which includes changes in the nucleotide glycosidic and backbone conformations, and enhanced or completely new nucleobase-nucleobase interactions compared to unmodified tRNA or tRNA containing smaller (m1G) modifications at the 37th position. Importantly, the extent of the changes in the anticodon loop is influenced by the addition of small functional groups to parent modifications, implying each substituent can further fine-tune tRNA structure. Although the dominant conformation of the ASL is achieved in different ways for each modification, the molecular features of all modified tRNA drive the ASL domain to adopt the functional open-loop conformation. Importantly, the impact of the hypermodifications is preserved in different sequence contexts. These findings highlight the likely role of regulating mRNA structure and translation.

Structural and functional characterization of peptidyl-tRNA hydrolase from Klebsiella pneumoniae.

Klebsiella pneumoniae is a member of the ESKAPE panel of pathogens that are top priority to tackle AMR. Bacterial peptidyl tRNA hydrolase (Pth), an essential, ubiquitous enzyme, hydrolyzes the peptidyl-tRNAs that accumulate in the cytoplasm because of premature termination of translation. Pth cleaves the ester bond between 2' or 3' hydroxyl of the ribose in the tRNA and C-terminal carboxylate of the peptide, thereby making free tRNA available for repeated cycles of protein synthesis and preventing cell death by alleviating tRNA starvation. Pth structures have been determined in peptide-bound or peptide-free states. In peptide-bound state, highly conserved residues F67, N69 and N115 adopt a conformation that is conducive to their interaction with peptide moiety of the substrate. While, in peptide-free state, these residues move away from the catalytic center, perhaps, in order to facilitate release of hydrolysed peptide. Here, we present a novel X-ray crystal structure of Pth from Klebsiella pneumoniae (KpPth), at 1.89 Å resolution, in which out of the two molecules in the asymmetric unit, one reflects the peptide-bound while the other reflects peptide-free conformation of the conserved catalytic site residues. Each molecule of the protein has canonical structure with seven stranded ß-sheet structure surrounded by six α-helices. MD simulations indicate that both the forms converge over 500 ns simulation to structures with wider opening of the crevice at peptide-binding end. In solution, KpPth is monomeric and its 2D-HSQC spectrum displays a single set of well dispersed peaks. Further, KpPth was demonstrated to be enzymatically active on BODIPY-Lys-tRNALys3.

Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity.

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3'-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

A substrate binding model for the KEOPS tRNA modifying complex.

The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway-Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3' CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential.

"Myo-neuropathy" is commonly associated with mitochondrial tRNALysine mutation.

The mitochondrial tRNALys (mt-tRNALys) mutation is initially associated with myoclonic epilepsy and ragged-red fibers (MERRF). The clinical, laboratory, morphologic and molecular findings from 22 mt-tRNALys mutation carriers from local database in East China were analyzed retrospectively. We identified 13 symptomatic and 9 asymptomatic individuals with a known pathogenic mitochondrial tRNALys mutation. The most common mutations were m.8344 A>G (81.8%), m.8363G>A (9.1%), m.8356 T>C (4.5%) and m.8356 T>G (4.5%). The degree of mutation heteroplasmy in blood was high both in symptomatic (mean 64.5%, range 41-82%) and asymptomatic individuals (mean 53.1%, range 21-78%). Age at onset ranged from 6 year-old to the age of 66 years (mean 35.8 ± 16.4 years old). The most frequent symptoms were muscle weakness (76.9%), exercise intolerance (76.9%), elevated creatine kinase levels (61.5%), peripheral neuropathy (69.2%) and cerebellar ataxia (61.5%), while myoclonus was only present in 23.1% of symptomatic patients. A diagnosis of mitochondrial myopathy (MM) and neuropathy ataxia and retinitis pigmentosa (NARP/NARP-like) syndrome was made in 77% of symptomatic patients, whereas the classic syndrome of myoclonic epilepsy with ragged-red fibers (MERRF) was rare (23%). In this cohort of patients with mt-tRNALys mutation, more than one third of our patients did not develop signs and symptoms of central nervous system involvement even in later stages of the disease, indicating the necessity to investigate the mt-tRNALys gene in 'pure' mitochondrial 'myo-neuropathy'.

Hearing impairment-associated KARS mutations lead to defects in aminoacylation of both cytoplasmic and mitochondrial tRNALys.

Aminoacyl-tRNA synthetases (aaRSs) are ubiquitously expressed, essential enzymes, synthesizing aminoacyl-tRNAs for protein synthesis. Functional defects of aaRSs frequently cause various human disorders. Human KARS encodes both cytosolic and mitochondrial lysyl-tRNA synthetases (LysRSs). Previously, two mutations (c.1129G>A and c.517T>C) were identified that led to hearing impairment; however, the underlying biochemical mechanism is unclear. In the present study, we found that the two mutations have no impact on the incorporation of LysRS into the multiple-synthetase complex in the cytosol, but affect the cytosolic LysRS level, its tertiary structure, and cytosolic tRNA aminoacylation in vitro. As for mitochondrial translation, the two mutations have little effect on the steady-state level, mitochondrial targeting, and tRNA binding affinity of mitochondrial LysRS. However, they exhibit striking differences in charging mitochondrial tRNALys, with the c.517T>C mutant being completely deficient in vitro and in vivo. We constructed two yeast genetic models, which are powerful tools to test the in vivo aminoacylation activity of KARS mutations at both the cytosolic and mitochondrial levels. Overall, our data provided biochemical insights into the potentially molecular pathological mechanism of KARS c.1129G>A and c.517T>C mutations and provided yeast genetic bases to investigate other KARS mutations in the future.

Irp2 regulates insulin production through iron-mediated Cdkal1-catalyzed tRNA modification.

Regulation of cellular iron homeostasis is crucial as both iron excess and deficiency cause hematological and neurodegenerative diseases. Here we show that mice lacking iron-regulatory protein 2 (Irp2), a regulator of cellular iron homeostasis, develop diabetes. Irp2 post-transcriptionally regulates the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-storage protein ferritin, and dysregulation of these proteins due to Irp2 loss causes functional iron deficiency in ß cells. This impairs Fe-S cluster biosynthesis, reducing the function of Cdkal1, an Fe-S cluster enzyme that catalyzes methylthiolation of t6A37 in tRNALysUUU to ms2t6A37. As a consequence, lysine codons in proinsulin are misread and proinsulin processing is impaired, reducing insulin content and secretion. Iron normalizes ms2t6A37 and proinsulin lysine incorporation, restoring insulin content and secretion in Irp2-/- ß cells. These studies reveal a previously unidentified link between insulin processing and cellular iron deficiency that may have relevance to type 2 diabetes in humans.

Conformational Changes in HIV-1 Reverse Transcriptase that Facilitate Its Maturation.

HIV-1 reverse transcriptase (RT) is translated as part of the Gag-Pol polyprotein that is proteolytically processed by HIV-1 protease (PR) to finally become a mature heterodimer, composed of a p66 and a p66-derived 51-kDa subunit, p51. Our previous work suggested that tRNALys3 binding to p66/p66 introduces conformational changes in the ribonuclease (RNH) domain of RT that facilitate efficient cleavage of p66 to p51 by PR. In this study, we characterized the conformational changes in the RNH domain of p66/p66 imparted by tRNALys3 using NMR. Moreover, the importance of tRNALys3 in RT maturation was confirmed in cellulo by modulating the levels of Lys-tRNA synthetase, which affects recruitment of tRNALys3 to the virus. We also employed nonnucleoside RT inhibitors, to modulate the p66 dimer-monomer equilibrium and monitor the resulting structural changes. Taken together, our data provide unique insights into the conformational changes in p66/p66 that drive PR cleavage.

Structure of HIV-1 RT/dsRNA initiation complex prior to nucleotide incorporation.

The initiation phase of HIV reverse transcription has features that are distinct from its elongation phase. The first structure of a reverse transcription initiation complex (RTIC) that trapped the complex after incorporation of one ddCMP nucleotide was published recently [Larsen KP, et al. (2018) Nature 557:118-122]. Here we report a crystal structure of a catalytically active HIV-1 RT/dsRNA complex that mimics the state of the RTIC before the first nucleotide incorporation. The structure reveals that the dsRNA-bound conformation of RT is closer to that of RT bound to a nonnucleoside RT inhibitor (NNRTI) and dsDNA; a hyperextended thumb conformation helps to accommodate the relatively wide dsRNA duplex. The RNA primer 3' end is positioned 5 Å away from the polymerase site; however, unlike in an NNRTI-bound state in which structural elements of RT restrict the movement of the primer, the primer terminus of dsRNA is not blocked from reaching the active site of RT. The observed structural changes and energetic cost of bringing the primer 3' end to the priming site are hypothesized to explain the slower nucleotide incorporation rate of the RTIC. An unusual crystal lattice interaction of dsRNA with its symmetry mate is reminiscent of the RNA architecture within the extended vRNA-tRNALys3 in the RTIC. This RT/dsRNA complex captures the key structural characteristics and components of the RTIC, including the RT conformational changes and interactions with the dsRNA primer-binding site region, and these features have implications for better understanding of RT initiation.