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.

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.

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.

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.

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.

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.

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.

RNA modification landscape of the human mitochondrial tRNALys regulates protein synthesis.

Post-transcriptional RNA modifications play a critical role in the pathogenesis of human mitochondrial disorders, but the mechanisms by which specific modifications affect mitochondrial protein synthesis remain poorly understood. Here we used a quantitative RNA sequencing approach to investigate, at nucleotide resolution, the stoichiometry and methyl modifications of the entire mitochondrial tRNA pool, and establish the relevance to human disease. We discovered that a N1-methyladenosine (m1A) modification is missing at position 58 in the mitochondrial tRNALys of patients with the mitochondrial DNA mutation m.8344 A > G associated with MERRF (myoclonus epilepsy, ragged-red fibers). By restoring the modification on the mitochondrial tRNALys, we demonstrated the importance of the m1A58 to translation elongation and the stability of selected nascent chains. Our data indicates regulation of post-transcriptional modifications on mitochondrial tRNAs is finely tuned for the control of mitochondrial gene expression. Collectively, our findings provide novel insight into the regulation of mitochondrial tRNAs and reveal greater complexity to the molecular pathogenesis of MERRF.

Architecture of an HIV-1 reverse transcriptase initiation complex.

Reverse transcription of the HIV-1 RNA genome into double-stranded DNA is a central step in viral infection 1 and a common target of antiretroviral drugs 2 . The reaction is catalysed by viral reverse transcriptase (RT)3,4 that is packaged in an infectious virion with two copies of viral genomic RNA 5 each bound to host lysine 3 transfer RNA (tRNALys3), which acts as a primer for initiation of reverse transcription6,7. Upon viral entry into cells, initiation is slow and non-processive compared to elongation8,9. Despite extensive efforts, the structural basis of RT function during initiation has remained a mystery. Here we use cryo-electron microscopy to determine a three-dimensional structure of an HIV-1 RT initiation complex. In our structure, RT is in an inactive polymerase conformation with open fingers and thumb and with the nucleic acid primer-template complex shifted away from the active site. The primer binding site (PBS) helix formed between tRNALys3 and HIV-1 RNA lies in the cleft of RT and is extended by additional pairing interactions. The 5' end of the tRNA refolds and stacks on the PBS to create a long helical structure, while the remaining viral RNA forms two helical stems positioned above the RT active site, with a linker that connects these helices to the RNase H region of the PBS. Our results illustrate how RNA structure in the initiation complex alters RT conformation to decrease activity, highlighting a potential target for drug action.

Characterization of association of human mitochondrial lysyl-tRNA synthetase with HIV-1 Pol and tRNA3Lys.

BACKGROUND: An important step in human immunodeficiency virus type 1 (HIV-1) replication is the packaging of tRNA3Lys from the host cell, which plays the role of primer RNA in the process of initiation of reverse transcription. The viral GagPol polyprotein precursor, and the human mitochondrial lysyl-tRNA synthetase (mLysRS) from the host cell, have been proposed to be involved in the packaging process. More specifically, the catalytic domain of mLysRS is supposed to interact with the transframe (TF or p6*) and integrase (IN) domains of the Pol region of the GagPol polyprotein. RESULTS: In this work, we report a quantitative characterization of the protein:protein interactions between mLysRS and its viral partners, the Pol polyprotein, and the isolated integrase and transframe domains of Pol. A dissociation constant of 1.3 ± 0.2 nM was determined for the Pol:mLysRS interaction, which exemplifies the robustness of this association. The protease and reverse transcriptase domains of GagPol are dispensable in this association, but the TF and IN domains have to be connected by a linker polypeptide to recapitulate a high affinity partner for mLysRS. The binding of the viral proteins to mLysRS does not dramatically enhance the binding affinity of mLysRS for tRNA3Lys. CONCLUSIONS: These data support the conclusion that the complex formed between GagPol, mLysRS and tRNA3Lys, which involves direct interactions between the IN and TF domains of Pol with mLysRS, is more robust than suggested by the previous models supposed to be involved in the packaging of tRNA3Lys into HIV-1 particles.

The t6A modification acts as a positive determinant for the anticodon nuclease PrrC, and is distinctively nonessential in Streptococcus mutans.

Endoribonuclease toxins (ribotoxins) are produced by bacteria and fungi to respond to stress, eliminate non-self competitor species, or interdict virus infection. PrrC is a bacterial ribotoxin that targets and cleaves tRNALysUUU in the anticodon loop. In vitro studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (t6A) is required for PrrC activity but this prediction had never been validated in vivo. Here, by using t6A-deficient yeast derivatives, it is shown that t6A is a positive determinant for PrrC proteins from various bacterial species. Streptococcus mutans is one of the few bacteria where the t6A synthesis gene tsaE (brpB) is dispensable and its genome encodes a PrrC toxin. We had previously shown using an HPLC-based assay that the S. mutans tsaE mutant was devoid of t6A. However, we describe here a novel and a more sensitive hybridization-based t6A detection method (compared to HPLC) that showed t6A was still present in the S. mutans ΔtsaE, albeit at greatly reduced levels (93% reduced compared with WT). Moreover, mutants in 2 other S. mutans t6A synthesis genes (tsaB and tsaC) were shown to be totally devoid of the modification thus confirming its dispensability in this organism. Furthermore, analysis of t6A modification ratios and of t6A synthesis genes mRNA levels in S. mutans suggest they may be regulated by growth phase.

Factors beyond Enolase 2 and Mitochondrial Lysyl-tRNA Synthetase Precursor Are Required for tRNA Import into Yeast Mitochondria.

In yeast, the import of tRNALys with CUU anticodon (tRK1) relies on a complex mechanism where interaction with enolase 2 (Eno2p) dictates a deep conformational change of the tRNA. This event is believed to mask the tRNA from the cytosolic translational machinery to re-direct it towards the mitochondria. Once near the mitochondrial outer membrane, the precursor of the mitochondrial lysyl-tRNA synthetase (preMsk1p) takes over enolase to carry the tRNA within the mitochondrial matrix, where it is supposed to participate in translation following correct refolding. Biochemical data presented in this report focus on the role of enolase. They show that despite the inability of Eno2p alone to form a complex with tRK1, mitochondrial import can be recapitulated in vitro using fractions of yeast extracts sharing either recombinant or endogenous yeast Eno2p as one of the main components. Taken together, our data suggest the existence of a protein complex containing Eno2p that is involved in RNA mitochondrial import.

Hydroxylation of a conserved tRNA modification establishes non-universal genetic code in echinoderm mitochondria.

The genetic code is not frozen but still evolving, which can result in the acquisition of 'dialectal' codons that deviate from the universal genetic code. RNA modifications in the anticodon region of tRNAs play a critical role in establishing such non-universal genetic codes. In echinoderm mitochondria, the AAA codon specifies asparagine instead of lysine. By analyzing mitochondrial (mt-) tRNALys isolated from the sea urchin (Mesocentrotus nudus), we discovered a novel modified nucleoside, hydroxy-N6-threonylcarbamoyladenosine (ht6A), 3' adjacent to the anticodon (position 37). Biochemical analysis revealed that ht6A37 has the ability to prevent mt-tRNALys from misreading AAA as lysine, thereby indicating that hydroxylation of N6-threonylcarbamoyladenosine (t6A) contributes to the establishment of the non-universal genetic code in echinoderm mitochondria.

HIV-1 Exploits a Dynamic Multi-aminoacyl-tRNA Synthetase Complex To Enhance Viral Replication.

A hallmark of retroviruses such as human immunodeficiency virus type 1 (HIV-1) is reverse transcription of genomic RNA to DNA, a process that is primed by cellular tRNAs. HIV-1 recruits human tRNALys3 to serve as the reverse transcription primer via an interaction between lysyl-tRNA synthetase (LysRS) and the HIV-1 Gag polyprotein. LysRS is normally sequestered in a multi-aminoacyl-tRNA synthetase complex (MSC). Previous studies demonstrated that components of the MSC can be mobilized in response to certain cellular stimuli, but how LysRS is redirected from the MSC to viral particles for packaging is unknown. Here, we show that upon HIV-1 infection, a free pool of non-MSC-associated LysRS is observed and partially relocalized to the nucleus. Heat inactivation of HIV-1 blocks nuclear localization of LysRS, but treatment with a reverse transcriptase inhibitor does not, suggesting that the trigger for relocalization occurs prior to reverse transcription. A reduction in HIV-1 infection is observed upon treatment with an inhibitor to mitogen-activated protein kinase that prevents phosphorylation of LysRS on Ser207, release of LysRS from the MSC, and nuclear localization. A phosphomimetic mutant of LysRS (S207D) that lacked the capability to aminoacylate tRNALys3 localized to the nucleus, rescued HIV-1 infectivity, and was packaged into virions. In contrast, a phosphoablative mutant (S207A) remained cytosolic and maintained full aminoacylation activity but failed to rescue infectivity and was not packaged. These findings suggest that HIV-1 takes advantage of the dynamic nature of the MSC to redirect and coopt cellular translation factors to enhance viral replication.IMPORTANCE Human tRNALys3, the primer for reverse transcription, and LysRS are essential host factors packaged into HIV-1 virions. Previous studies found that tRNALys3 packaging depends on interactions between LysRS and HIV-1 Gag; however, many details regarding the mechanism of tRNALys3 and LysRS packaging remain unknown. LysRS is normally sequestered in a high-molecular-weight multi-aminoacyl-tRNA synthetase complex (MSC), restricting the pool of free LysRS-tRNALys Mounting evidence suggests that LysRS is released under a variety of stimuli to perform alternative functions within the cell. Here, we show that HIV-1 infection results in a free pool of LysRS that is relocalized to the nucleus of target cells. Blocking this pathway in HIV-1-producing cells resulted in less infectious progeny virions. Understanding the mechanism by which LysRS is recruited into the viral assembly pathway can be exploited for the development of specific and effective therapeutics targeting this nontranslational function.

The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs.

Cryptic polyadenylation within coding sequences (CDS) triggers ribosome-associated quality control (RQC), followed by degradation of the aberrant mRNA and polypeptide, ribosome disassembly and recycling. Although ribosomal subunit dissociation and nascent peptide degradation are well-understood, the molecular sensors of aberrant mRNAs and their mechanism of action remain unknown. We studied the Zinc Finger Protein 598 (ZNF598) using PAR-CLIP and revealed that it cross-links to tRNAs, mRNAs and rRNAs, thereby placing the protein on translating ribosomes. Cross-linked reads originating from AAA-decoding tRNALys(UUU) were 10-fold enriched over its cellular abundance, and poly-lysine encoded by poly(AAA) induced RQC in a ZNF598-dependent manner. Encounter with translated polyA segments by ZNF598 triggered ubiquitination of several ribosomal proteins, requiring the E2 ubiquitin ligase UBE2D3 to initiate RQC. Considering that human CDS are devoid of >4 consecutive AAA codons, sensing of prematurely placed polyA tails by a specialized RNA-binding protein is a novel nucleic-acid-based surveillance mechanism of RQC.

Structure of the MazF-mt9 toxin, a tRNA-specific endonuclease from Mycobacterium tuberculosis.

Tuberculosis (TB) is a severe disease caused by Mycobacterium tuberculosis (M. tb) and the well-characterized M. tb MazE/F proteins play important roles in stress adaptation. Recently, the MazF-mt9 toxin has been found to display endonuclease activities towards tRNAs but the mechanism is unknown. We hereby present the crystal structure of apo-MazF-mt9. The enzyme recognizes tRNALys with a central UUU motif within the anticodon loop, but is insensitive to the sequence context outside of the loop. Based on our crystallographic and biochemical studies, we identified key residues for catalysis and proposed the potential tRNA-binding site.