Lysosomal cystine governs ferroptosis sensitivity in cancer via cysteine stress response.

The amino acid cysteine and its oxidized dimeric form cystine are commonly believed to be synonymous in metabolic functions. Cyst(e)ine depletion not only induces amino acid response but also triggers ferroptosis, a non-apoptotic cell death. Here, we report that unlike general amino acid starvation, cyst(e)ine deprivation triggers ATF4 induction at the transcriptional level. Unexpectedly, it is the shortage of lysosomal cystine, but not the cytosolic cysteine, that elicits the adaptative ATF4 response. The lysosome-nucleus signaling pathway involves the aryl hydrocarbon receptor (AhR) that senses lysosomal cystine via the kynurenine pathway. A blockade of lysosomal cystine efflux attenuates ATF4 induction and sensitizes ferroptosis. To potentiate ferroptosis in cancer, we develop a synthetic mRNA reagent, CysRx, that converts cytosolic cysteine to lysosomal cystine. CysRx maximizes cancer cell ferroptosis and effectively suppresses tumor growth in vivo. Thus, intracellular nutrient reprogramming has the potential to induce selective ferroptosis in cancer without systematic starvation.

Human SAMD9 is a poxvirus-activatable anticodon nuclease inhibiting codon-specific protein synthesis.

As a defense strategy against viruses or competitors, some microbes use anticodon nucleases (ACNases) to deplete essential tRNAs, effectively halting global protein synthesis. However, this mechanism has not been observed in multicellular eukaryotes. Here, we report that human SAMD9 is an ACNase that specifically cleaves phenylalanine tRNA (tRNAPhe), resulting in codon-specific ribosomal pausing and stress signaling. While SAMD9 ACNase activity is normally latent in cells, it can be activated by poxvirus infection or rendered constitutively active by SAMD9 mutations associated with various human disorders, revealing tRNAPhe depletion as an antiviral mechanism and a pathogenic condition in SAMD9 disorders. We identified the N-terminal effector domain of SAMD9 as the ACNase, with substrate specificity primarily determined by a eukaryotic tRNAPhe-specific 2'-O-methylation at the wobble position, making virtually all eukaryotic tRNAPhe susceptible to SAMD9 cleavage. Notably, the structure and substrate specificity of SAMD9 ACNase differ from known microbial ACNases, suggesting convergent evolution of a common immune defense strategy targeting tRNAs.

NAD+ supplement potentiates tumor-killing function by rescuing defective TUB-mediated NAMPT transcription in tumor-infiltrated T cells.

Although tumor-infiltrating lymphocytes (TILs) maintain their ability to proliferate, persist, and eradicate tumors, they are frequently dysfunctional in situ. By performing both whole-genome CRISPR and metabolic inhibitor screens, we identify that nicotinamide phosphoribosyltransferase (NAMPT) is required for T cell activation. NAMPT is low in TILs, and its expression is controlled by the transcriptional factor Tubby (TUB), whose activity depends on the T cell receptor-phospholipase C gamma (TCR-PLCγ) signaling axis. The intracellular level of NAD+, whose synthesis is dependent on the NAMPT-mediated salvage pathway, is also decreased in TILs. Liquid chromatography-mass spectrometry (LC-MS) and isotopic labeling studies confirm that NAD+ depletion led to suppressed glycolysis, disrupted mitochondrial function, and dampened ATP synthesis. Excitingly, both adoptive CAR-T and anti-PD1 immune checkpoint blockade mouse models demonstrate that NAD+ supplementation enhanced the tumor-killing efficacy of T cells. Collectively, this study reveals that an impaired TCR-TUB-NAMPT-NAD+ axis leads to T cell dysfunction in the tumor microenvironment, and an over-the-counter nutrient supplement of NAD+ could boost T-cell-based immunotherapy.

A heat shock-responsive lncRNA Heat acts as a HSF1-directed transcriptional brake via m6A modification.

Long noncoding RNAs (lncRNAs) are key regulators of gene expression in diverse cellular contexts and biological processes. Given the surprising range of shapes and sizes, how distinct lncRNAs achieve functional specificity remains incompletely understood. Here, we identified a heat shock-inducible lncRNA, Heat, in mouse cells that acts as a transcriptional brake to restrain stress gene expression. Functional characterization reveals that Heat directly binds to heat shock transcription factor 1 (HSF1), thereby targeting stress genes in a trans-acting manner. Intriguingly, Heat is heavily methylated in the form of m6A. Although dispensable for HSF1 binding, Heat methylation is required for silencing stress genes to attenuate heat shock response. Consistently, m6A depletion results in prolonged activation of stress genes. Furthermore, Heat mediates these effects via the nuclear m6A reader YTHDC1, forming a transcriptional silencing complex for stress genes. Our study reveals a crucial role of nuclear epitranscriptome in the transcriptional regulation of heat shock response.

Decoding mRNA translatability and stability from the 5' UTR.

Precise control of protein synthesis by engineering sequence elements in 5' untranslated regions (5' UTRs) remains a fundamental challenge. To accelerate our understanding of the cis-regulatory code embedded in 5' UTRs, we devised massively parallel reporter assays from a synthetic messenger RNA library composed of over one million 5' UTR variants. A completely randomized 10-nucleotide sequence preceding an upstream open reading frame (uORF) and downstream GFP drives a broad range of translational outputs and mRNA stability in mammalian cells. While efficient translation protects mRNA from degradation, uORF translation triggers mRNA decay in a UPF1-dependent manner. We also identified translational inhibitory elements with G-quadruplexes as marks for mRNA decay in P-bodies. Unexpectedly, an unstructured A-rich element in 5' UTRs destabilizes mRNAs in the absence of translation, although it enables cap-independent translation. Our results not only identify diverse sequence features of 5' UTRs that control mRNA translatability, but they also reveal ribosome-dependent and ribosome-independent mRNA-surveillance pathways.

Programmable RNA N6-methyladenosine editing by CRISPR-Cas9 conjugates.

RNA modification in the form of N6-methyladenosine (m6A) regulates nearly all the post-transcriptional processes. The asymmetric m6A deposition suggests that regional methylation may have distinct functional consequences. However, current RNA biology tools do not distinguish the contribution of individual m6A modifications. Here we report the development of 'm6A editing', a powerful approach that enables m6A installation and erasure from cellular RNAs without changing the primary sequence. We engineered fusions of CRISPR-Cas9 and a single-chain m6A methyltransferase that can be programmed with a guide RNA. The resultant m6A 'writers' allow functional comparison of single site methylation in different messenger RNA regions. We further engineered m6A 'erasers' by fusing CRISPR-Cas9 with ALKBH5 or FTO to achieve site-specific demethylation of RNAs. The development of programmable m6A editing not only expands the scope of RNA engineering, but also facilitates mechanistic understanding of epitranscriptome.

LeuRS can leucylate type I and type II tRNALeus in Streptomyces coelicolor.

Transfer RNAs (tRNAs) are divided into two types, type I with a short variable loop and type II with a long variable loop. Aminoacylation of type I or type II tRNALeu is catalyzed by their cognate leucyl-tRNA synthetases (LeuRSs). However, in Streptomyces coelicolor, there are two types of tRNALeu and only one LeuRS (ScoLeuRS). We found that the enzyme could leucylate both types of ScotRNALeu, and had a higher catalytic efficiency for type II ScotRNALeu(UAA) than for type I ScotRNALeu(CAA). The results from tRNA and enzyme mutagenesis showed that ScoLeuRS did not interact with the canonical discriminator A73. The number of nucleotides, rather than the type of base of the variable loop in the two types of ScotRNALeus, was determined as important for aminoacylation. In vitro and in vivo assays showed that the tertiary structure formed by the D-loop and TψC-loop is more important for ScotRNALeu(UAA). We showed that the leucine-specific domain (LSD) of ScoLeuRS could help LeuRS, which originally only leucylates type II tRNALeu, to aminoacylate type I ScotRNALeu(CAA) and identified the crucial amino acid residues at the C-terminus of the LSD to recognize type I ScotRNALeu(CAA). Overall, our findings identified a rare recognition mechanism of LeuRS to tRNALeu.

A natural non-Watson-Crick base pair in human mitochondrial tRNAThr causes structural and functional susceptibility to local mutations.

Six pathogenic mutations have been reported in human mitochondrial tRNAThr (hmtRNAThr); however, the pathogenic molecular mechanism remains unclear. Previously, we established an activity assay system for human mitochondrial threonyl-tRNA synthetase (hmThrRS). In the present study, we surveyed the structural and enzymatic effects of pathogenic mutations in hmtRNAThr and then focused on m.15915 G > A (G30A) and m.15923A > G (A38G). The harmful evolutionary gain of non-Watson-Crick base pair A29/C41 caused hmtRNAThr to be highly susceptible to mutations disrupting the G30-C40 base pair in various ways; for example, structural integrity maintenance, modification and aminoacylation of tRNAThr, and editing mischarged tRNAThr. A similar phenomenon was observed for hmtRNATrp with an A29/C41 non-Watson-Crick base pair, but not in bovine mtRNAThr with a natural G29-C41 base pair. The A38G mutation caused a severe reduction in Thr-acceptance and editing of hmThrRS. Importantly, A38 is a nucleotide determinant for the t6A modification at A37, which is essential for the coding properties of hmtRNAThr. In summary, our results revealed the crucial role of the G30-C40 base pair in maintaining the proper structure and function of hmtRNAThr because of A29/C41 non-Watson-Crick base pair and explained the molecular outcome of pathogenic G30A and A38G mutations.

Self-protective responses to norvaline-induced stress in a leucyl-tRNA synthetase editing-deficient yeast strain.

The editing function of aminoacyl-tRNA synthetases (aaRSs) is indispensible for formation of the correct aminoacyl-tRNAs. Editing deficiency may lead to growth inhibition and the pathogenesis of various diseases. Herein, we confirmed that norvaline (Nva) but not isoleucine or valine is the major threat to the editing function of Saccharomyces cerevisiae leucyl-tRNA synthetase (ScLeuRS), both in vitro and in vivo. Nva could be misincorporated into the proteome of the LeuRS editing-deficient yeast strain (D419A/ScΔleuS), potentially resulting in dysfunctional protein folding and growth delay. Furthermore, the exploration of the Nva-induced intracellular stress response mechanism in D419A/ScΔleuS revealed that Hsp70 chaperones were markedly upregulated in response to the potential protein misfolding. Additionally, proline (Pro), glutamate (Glu) and glutamine (Gln), which may accumulate due to the conversion of Nva, collectively contributed to the reduction of reactive oxygen species (ROS) levels in Nva-treated D419A/ScΔleuS cells. In conclusion, our study highlights the significance of the editing function of LeuRS and provides clues for understanding the intracellular stress protective mechanisms that are triggered in aaRS editing-deficient organisms.

Acetylation of lysine ϵ-amino groups regulates aminoacyl-tRNA synthetase activity in Escherichia coli.

Previous proteomic analyses have shown that aminoacyl-tRNA synthetases in many organisms can be modified by acetylation of Lys. In this present study, leucyl-tRNA synthetase and arginyl-tRNA synthetase from Escherichia coli (EcLeuRS and EcArgRS) were overexpressed and purified and found to be acetylated on Lys residues by MS. Gln scanning mutagenesis revealed that Lys619, Lys624, and Lys809 in EcLeuRS and Lys126 and Lys408 in EcArgRS might play important roles in enzyme activity. Furthermore, we utilized a novel protein expression system to obtain enzymes harboring acetylated Lys at specific sites and investigated their catalytic activity. Acetylation of these Lys residues could affect their aminoacylation activity by influencing amino acid activation and/or the affinity for tRNA. In vitro assays showed that acetyl-phosphate nonenzymatically acetylates EcLeuRS and EcArgRS and suggested that the sirtuin class deacetylase CobB might regulate acetylation of these two enzymes. These findings imply a potential regulatory role for Lys acetylation in controlling the activity of aminoacyl-tRNA synthetases and thus protein synthesis.

C-terminal Domain of Leucyl-tRNA Synthetase from Pathogenic Candida albicans Recognizes both tRNASer and tRNALeu.

Leucyl-tRNA synthetase (LeuRS) is a multidomain enzyme that catalyzes Leu-tRNA(Leu) formation and is classified into bacterial and archaeal/eukaryotic types with significant diversity in the C-terminal domain (CTD). CTDs of both bacterial and archaeal LeuRSs have been reported to recognize tRNA(Leu) through different modes of interaction. In the human pathogen Candida albicans, the cytoplasmic LeuRS (CaLeuRS) is distinguished by its capacity to recognize a uniquely evolved chimeric tRNA(Ser) (CatRNA(Ser)(CAG)) in addition to its cognate CatRNA(Leu), leading to CUG codon reassignment. Our previous study showed that eukaryotic but not archaeal LeuRSs recognize this peculiar tRNA(Ser), suggesting the significance of their highly divergent CTDs in tRNA(Ser) recognition. The results of this study provided the first evidence of the indispensable function of the CTD of eukaryotic LeuRS in recognizing non-cognate CatRNA(Ser) and cognate CatRNA(Leu). Three lysine residues were identified as involved in mediating enzyme-tRNA interaction in the leucylation process: mutation of all three sites totally ablated the leucylation activity. The importance of the three lysine residues was further verified by gel mobility shift assays and complementation of a yeast leuS gene knock-out strain.

Degenerate connective polypeptide 1 (CP1) domain from human mitochondrial leucyl-tRNA synthetase.

The connective polypeptide 1 (CP1) editing domain of leucyl-tRNA synthetase (LeuRS) from various species either harbors a conserved active site to exclude tRNA mis-charging with noncognate amino acids or is evolutionarily truncated or lost because there is no requirement for high translational fidelity. However, human mitochondrial LeuRS (hmtLeuRS) contains a full-length but degenerate CP1 domain that has mutations in some residues important for post-transfer editing. The significance of such an inactive CP1 domain and a translational accuracy mechanism with different noncognate amino acids are not completely understood. Here, we identified the essential role of the evolutionarily divergent CP1 domain in facilitating hmtLeuRS's catalytic efficiency and endowing enzyme with resistance to AN2690, a broad-spectrum drug acting on LeuRSs. In addition, the canonical core of hmtLeuRS is not stringent for noncognate norvaline (Nva) and valine (Val). hmtLeuRS has a very weak tRNA-independent pre-transfer editing activity for Nva, which is insufficient to remove mis-activated Nva. Moreover, hmtLeuRS chimeras fused with a functional CP1 domain from LeuRSs of other species, regardless of origin, showed restored post-transfer editing activity and acquired fidelity during aminoacylation. This work offers a novel perspective on the role of the CP1 domain in optimizing aminoacylation efficiency.

The mRNA of human cytoplasmic arginyl-tRNA synthetase recruits prokaryotic ribosomes independently.

There are two isoforms of cytoplasmic arginyl-tRNA synthetase (hcArgRS) in human cells. The long form is a component of the multiple aminoacyl-tRNA synthetase complex, and the other is an N-terminal truncated form (NhcArgRS), free in the cytoplasm. It has been shown that the two forms of ArgRS arise from alternative translational initiation in a single mRNA. The short form is produced from the initiation at a downstream, in-frame AUG start codon. Interestingly, our data suggest that the alternative translational initiation of hcArgRS mRNA also takes place in Escherichia coli transformants. When the gene encoding full-length hcArgRS was overexpressed in E. coli, two forms of hcArgRS were observed. The N-terminal sequencing experiment identified that the short form was identical to the NhcArgRS in human cytoplasm. By constructing a bicistronic system, our data support that the mRNA encoding the N-terminal extension of hcArgRS has the capacity of independently recruiting E. coli ribosomes. Furthermore, two critical elements for recruiting prokaryotic ribosomes were identified, the "AGGA" core of the Shine-Dalgarno sequence and the "A-rich" sequence located just proximal to the alternative in-frame initiation site. Although the mechanisms of prokaryotic and eukaryotic translational initiation are distinct, they share some common features. The ability of the hcArgRS mRNA to recruit the prokaryotic ribosome may provide clues for shedding light on the mechanism of alternative translational initiation of hcArgRS mRNA in eukaryotic cells.