Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease.

Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we show that Nsp5 binds and cleaves human tRNA methyltransferase 1 (TRMT1), a host enzyme required for a prevalent post-transcriptional modification in tRNAs. Human cells infected with SARS-CoV-2 exhibit a decrease in TRMT1 protein levels and TRMT1-catalyzed tRNA modifications, consistent with TRMT1 cleavage and inactivation by Nsp5. Nsp5 cleaves TRMT1 at a specific position that matches the consensus sequence of SARS-CoV-2 polyprotein cleavage sites, and a single mutation within the sequence inhibits Nsp5-dependent proteolysis of TRMT1. The TRMT1 cleavage fragments exhibit altered RNA binding activity and are unable to rescue tRNA modification in TRMT1-deficient human cells. Compared to wild-type human cells, TRMT1-deficient human cells infected with SARS-CoV-2 exhibit reduced levels of intracellular viral RNA. These findings provide evidence that Nsp5-dependent cleavage of TRMT1 and perturbation of tRNA modification patterns contribute to the cellular pathogenesis of SARS-CoV-2 infection.

The virus responsible for COVID-19 infections is known as SARS-CoV-2. Like all viruses, SARS-CoV-2 carries instructions to make proteins and other molecules that play essential roles in enabling the virus to multiply and spread. Viruses are unable to make these molecules themselves, so they infect cells and trick them into making the molecules and assembling new virus particles on their behalf instead. When SARS-CoV2 infects cells, the host cells are reprogrammed to make chains containing several virus proteins that need to be severed from each other by a virus enzyme, known as Nsp5, to enable the proteins to work properly. Previous studies suggested that Nsp5 may also interact with a human protein known as TRMT1, which helps with the production of new proteins in cells. However, it was not clear how Nsp5 may bind to TRMT1 or how this interaction may affect the host cell. Zhang et al. used biochemical and molecular techniques in human cells to study how Nsp5 interacts with TRMT1. The experiments found that the virus enzyme cuts TRMT1 into fragments that are inactive and are subsequently destroyed by the cells. Moreover, Nsp5 cuts TRMT1 at exactly the same position corresponding to the cleavage sites of the viral proteins. Mutation of the sequence in TRMT1 renders Nsp5 ineffective at cutting the protein. SARS-CoV-2 infection caused TRMT1 levels to decrease inside the cells, in turn, leading to a drop in TRMT1 activity. The virus multiplied less in cells that were unable to produce TRMT1 compared to normal human cells, suggesting that the virus benefits from TRMT1 early during infection, before inactivating it at a later point. These findings suggest that one way SARS-CoV-2 causes disease is by decreasing the levels of a human protein that regulates protein production. In the future, the work of Zhang et al. may provide new markers for detecting infections of SARS-CoV-2 and other similar viruses and guide efforts to make more effective therapies against them.

Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease.

Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we show that Nsp5 binds and cleaves human tRNA methyltransferase 1 (TRMT1), a host enzyme required for a prevalent post-transcriptional modification in tRNAs. Human cells infected with SARS-CoV-2 exhibit a decrease in TRMT1 protein levels and TRMT1-catalyzed tRNA modifications, consistent with TRMT1 cleavage and inactivation by Nsp5. Nsp5 cleaves TRMT1 at a specific position that matches the consensus sequence of SARS-CoV-2 polyprotein cleavage sites, and a single mutation within the sequence inhibits Nsp5-dependent proteolysis of TRMT1. The TRMT1 cleavage fragments exhibit altered RNA binding activity and are unable to rescue tRNA modification in TRMT1-deficient human cells. Compared to wildtype human cells, TRMT1-deficient human cells infected with SARS-CoV-2 exhibit reduced levels of intracellular viral RNA. These findings provide evidence that Nsp5-dependent cleavage of TRMT1 and perturbation of tRNA modification patterns contribute to the cellular pathogenesis of SARS-CoV-2 infection.

ADATscan - A flexible tool for scanning exomes for wobble inosine-dependent codons reveals a neurological bias for genes enriched in such codons in humans and mice.

The conversion of adenosine to inosine at the wobble position of select tRNAs is essential for decoding specific codons in bacteria and eukarya. In eukarya, wobble inosine modification is catalyzed by the heterodimeric ADAT complex containing ADAT2 and ADAT3. Human individuals homozygous for loss of function variants in ADAT3 exhibit intellectual disability disorders. We created a flexible computational tool to scan the human, mouse, nematode, fruit fly, and yeast exomes for genes either enriched or depleted in ADAT-dependent codons as compared to background models of codon bias derived from the exomes themselves. We find that many genes are enriched or depleted for ADAT-dependent codons as compared to the genomic background in all five species. Among those genes enriched for ADAT-dependent codons in humans, we find there is significant Gene Ontology (GO) enrichment for genes involved in diverse neurological processes. This pattern persists in the mouse exome but not the fruit fly or nematode exome. In the nematode exome, genes enriched in ADAT-dependent codons are GO enriched for translation associated genes, and in yeast there is GO enrichment for genes involved in metabolic functions. There is also GO-term overlap between yeast and fruit flies. Importantly, in its generalized form, ADATscan can also be used to scan any exome for genes enriched in any subset of codons specified by the user.

RNA modifications: an overview of select web-based tools.

The field of epitranscriptomics has expanded dramatically in recent years, both in the number of identified RNA modifications and the number of researchers studying them. As knowledge of post-transcriptional modifications continues to expand, numerous new methods have been developed to detect these modifications. Additionally, modifications are being extended to therapeutic settings, such as with recent mRNA vaccines. With this increase in knowledge and use, the community is recognizing the necessity for user-friendly databases to (i) store information from both high- and low-throughput studies and (ii) provide prediction software on how RNA modifications contribute to RNA function and disease. This mini-review highlights select RNA modification databases and their key attributes with the aim of providing a resource to researchers in the field of epitranscriptomics.

Detection of tRNA-specific adenosine deaminase activity and wobble inosine modification in human cell lysates.

The wobble inosine modification plays a central role in translation by enabling a single tRNA to decode multiple synonymous codons. In eukaryotes, the formation of wobble inosine is catalyzed by a heterodimeric adenosine deaminase complex comprised of the ADAT2 and ADAT3 subunits. Notably, pathogenic variants in the ADAT3 subunit have been identified as the cause of autosomal recessive intellectual disability in the human population by impacting wobble inosine levels. Here, we describe approaches for monitoring adenosine deaminase activity and inosine modification status at the wobble position of cellular tRNAs. To detect adenosine deaminase activity, we provide protocols for preparing extracts from human cells followed by enzymatic assays with in vitro transcribed tRNA substrates. Furthermore, we describe a method to monitor wobble inosine status of individual tRNAs using cDNA sequencing. These assays can be used to decipher the molecular basis for neurodevelopmental disorders linked to wobble inosine deficiency and disease-associated ADAT2/3 variants.

Identification and rescue of a tRNA wobble inosine deficiency causing intellectual disability disorder.

The deamination of adenosine to inosine at the wobble position of tRNA is an essential post-transcriptional RNA modification required for wobble decoding in bacteria and eukaryotes. In humans, the wobble inosine modification is catalyzed by the heterodimeric ADAT2/3 complex. Here, we describe novel pathogenic ADAT3 variants impairing adenosine deaminase activity through a distinct mechanism that can be corrected through expression of the heterodimeric ADAT2 subunit. The variants were identified in a family in which all three siblings exhibit intellectual disability linked to biallelic variants in the ADAT3 locus. The biallelic ADAT3 variants result in a missense variant converting alanine to valine at a conserved residue or the introduction of a premature stop codon in the deaminase domain. Fibroblast cells derived from two ID-affected individuals exhibit a reduction in tRNA wobble inosine levels and severely diminished adenosine tRNA deaminase activity. Notably, the ADAT3 variants exhibit impaired interaction with the ADAT2 subunit and alterations in ADAT2-dependent nuclear localization. Based upon these findings, we find that tRNA adenosine deaminase activity and wobble inosine modification can be rescued in patient cells by overexpression of the ADAT2 catalytic subunit. These results uncover a key role for the inactive ADAT3 deaminase domain in proper assembly with ADAT2 and demonstrate that ADAT2/3 nuclear import is required for maintaining proper levels of the wobble inosine modification in tRNA.

Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro.

RNAs are post-transcriptionally modified by dedicated writer or eraser enzymes that add or remove specific modifications, respectively. Mass spectrometry (MS) of RNA is a useful tool to study the modification state of an oligonucleotide (ON) in a sensitive manner. Here, we developed an ion-pairing reagent free chromatography for positive ion detection of ONs by low- and high-resolution MS, which does not interfere with other types of small compound analyses done on the same instrument. We apply ON-MS to determine the ONs from an RNase T1 digest of in vitro transcribed tRNA, which are purified after ribozyme-fusion transcription by automated size exclusion chromatography. The thus produced tRNAValAAC is substrate of the human tRNA ADAT2/3 enzyme and we confirm the deamination of adenosine to inosine and the formation of tRNAValIACin vitro by ON-MS. Furthermore, low resolution ON-MS is used to monitor the demethylation of ONs containing 1-methyladenosine by bacterial AlkB in vitro. The power of high-resolution ON-MS is demonstrated by the detection and mapping of modified ONs from native total tRNA digested with RNase T1. Overall, we present an oligonucleotide MS method which is broadly applicable to monitor in vitro RNA (de-)modification processes and native RNA.

Formation of tRNA Wobble Inosine in Humans Is Disrupted by a Millennia-Old Mutation Causing Intellectual Disability.

The formation of inosine at the wobble position of eukaryotic tRNAs is an essential modification catalyzed by the ADAT2/ADAT3 complex. In humans, a valine-to-methionine mutation (V144M) in ADAT3 that originated ∼1,600 years ago is the most common cause of autosomal recessive intellectual disability (ID) in Arabia. While the mutation is predicted to affect protein structure, the molecular and cellular effects of the V144M mutation are unknown. Here, we show that cell lines derived from ID-affected individuals expressing only ADAT3-V144M exhibit decreased wobble inosine in certain tRNAs. Moreover, extracts from the same cell lines of ID-affected individuals display a severe reduction in tRNA deaminase activity. While ADAT3-V144M maintains interactions with ADAT2, the purified ADAT2/3-V144M complexes exhibit defects in activity. Notably, ADAT3-V144M exhibits an increased propensity to form aggregates associated with cytoplasmic chaperonins that can be suppressed by ADAT2 overexpression. These results identify a key role for ADAT2-dependent folding of ADAT3 in wobble inosine modification and indicate that proper formation of an active ADAT2/3 complex is crucial for proper neurodevelopment.

The emerging impact of tRNA modifications in the brain and nervous system.

A remarkable number of neurodevelopmental disorders have been linked to defects in tRNA modifications. These discoveries place tRNA modifications in the spotlight as critical modulators of gene expression pathways that are required for proper organismal growth and development. Here, we discuss the emerging molecular and cellular functions of the diverse tRNA modifications linked to cognitive and neurological disorders. In particular, we describe how the structure and location of a tRNA modification influences tRNA folding, stability, and function. We then highlight how modifications in tRNA can impact multiple aspects of protein translation that are instrumental for maintaining proper cellular proteostasis. Importantly, we describe how perturbations in tRNA modification lead to a spectrum of deleterious biological outcomes that can disturb neurodevelopment and neurological function. Finally, we summarize the biological themes shared by the different tRNA modifications linked to cognitive disorders and offer insight into the future questions that remain to decipher the role of tRNA modifications. This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert.

Phosphorylation of human TRM9L integrates multiple stress-signaling pathways for tumor growth suppression.

The human transfer RNA methyltransferase 9-like gene (TRM9L, also known as KIAA1456) encodes a negative regulator of tumor growth that is frequently silenced in many forms of cancer. While TRM9L can inhibit tumor cell growth in vivo, the molecular mechanisms underlying the tumor inhibition activity of TRM9L are unknown. We show that oxidative stress induces the rapid and dose-dependent phosphorylation of TRM9L within an intrinsically disordered domain that is necessary for tumor growth suppression. Multiple serine residues are hyperphosphorylated in response to oxidative stress. Using a chemical genetic approach, we identified a key serine residue in TRM9L that undergoes hyperphosphorylation downstream of the oxidative stress-activated MEK (mitogen-activated protein kinase kinase)-ERK (extracellular signal-regulated kinase)-RSK (ribosomal protein S6 kinase) signaling cascade. Moreover, we found that phosphorylated TRM9L interacts with the 14-3-3 family of proteins, providing a link between oxidative stress and downstream cellular events involved in cell cycle control and proliferation. Mutation of the serine residues required for TRM9L hyperphosphorylation and 14-3-3 binding abolished the tumor inhibition activity of TRM9L. Our results uncover TRM9L as a key downstream effector of the ERK signaling pathway and elucidate a phospho-signaling regulatory mechanism underlying the tumor inhibition activity of TRM9L.

Monitoring the 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification in eukaryotic tRNAs via the γ-toxin endonuclease.

The post-transcriptional modification of tRNA at the wobble position is a universal process occurring in all domains of life. In eukaryotes, the wobble uridine of particular tRNAs is transformed to the 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification which is critical for proper mRNA decoding and protein translation. However, current methods to detect mcm5s2U are technically challenging and/or require specialized instrumental expertise. Here, we show that γ-toxin endonuclease from the yeast Kluyveromyces lactis can be used as a probe for assaying mcm5s2U status in the tRNA of diverse eukaryotic organisms ranging from protozoans to mammalian cells. The assay couples the mcm5s2U-dependent cleavage of tRNA by γ-toxin with standard molecular biology techniques such as northern blot analysis or quantitative PCR to monitor mcm5s2U levels in multiple tRNA isoacceptors. The results gained from the γ-toxin assay reveals the evolutionary conservation of the mcm5s2U modification across eukaryotic species. Moreover, we have used the γ-toxin assay to verify uncharacterized eukaryotic Trm9 and Trm112 homologs that catalyze the formation of mcm5s2U. These findings demonstrate the use of γ-toxin as a detection method to monitor mcm5s2U status in diverse eukaryotic cell types for cellular, genetic, and biochemical studies.

Loss of miR-203 regulates cell shape and matrix adhesion through ROBO1/Rac/FAK in response to stiffness.

Breast tumor progression is accompanied by changes in the surrounding extracellular matrix (ECM) that increase stiffness of the microenvironment. Mammary epithelial cells engage regulatory pathways that permit dynamic responses to mechanical cues from the ECM. Here, we identify a SLIT2/ROBO1 signaling circuit as a key regulatory mechanism by which cells sense and respond to ECM stiffness to preserve tensional homeostasis. We observed that Robo1 ablation in the developing mammary gland compromised actin stress fiber assembly and inhibited cell contractility to perturb tissue morphogenesis, whereas SLIT2 treatment stimulated Rac and increased focal adhesion kinase activity to enhance cell tension by maintaining cell shape and matrix adhesion. Further investigation revealed that a stiff ECM increased Robo1 levels by down-regulating miR-203. Consistently, patients whose tumor expressed a low miR-203/high Robo1 expression pattern exhibited a better overall survival prognosis. These studies show that cells subjected to stiffened environments up-regulate Robo1 as a protective mechanism that maintains cell shape and facilitates ECM adherence.