Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression.

Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expression at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells. Using chemically induced dimerization domains, we developed split o-aaRSs that mediate gene expression by conditionally suppressing stop codons in the presence of the small molecules rapamycin and abscisic acid. By activating o-aaRSs, these molecular switches induce stop codon suppression, and in their absence stop codon suppression is turned off. We demonstrate, in Escherichia coli and in human cells, that split o-aaRSs function as genetically encoded AND gates where stop codon suppression is controlled by two distinct molecular inputs. In addition, we show that split o-aaRSs can be used as versatile biosensors to detect therapeutically relevant protein-protein interactions, including those involved in cancer, and those that mediate severe acute respiratory syndrome-coronavirus-2 infection.

Selective degradation of tRNASer(AGY) is the primary driver for mitochondrial seryl-tRNA synthetase-related disease.

Mitochondrial translation is of high significance for cellular energy homeostasis. Aminoacyl-tRNA synthetases (aaRSs) are crucial translational components. Mitochondrial aaRS variants cause various human diseases. However, the pathogenesis of the vast majority of these diseases remains unknown. Here, we identified two novel SARS2 (encoding mitochondrial seryl-tRNA synthetase) variants that cause a multisystem disorder. c.654-14T > A mutation induced mRNA mis-splicing, generating a peptide insertion in the active site; c.1519dupC swapped a critical tRNA-binding motif in the C-terminus due to stop codon readthrough. Both mutants exhibited severely diminished tRNA binding and aminoacylation capacities. A marked reduction in mitochondrial tRNASer(AGY) was observed due to RNA degradation in patient-derived induced pluripotent stem cells (iPSCs), causing impaired translation and comprehensive mitochondrial function deficiencies. These impairments were efficiently rescued by wild-type SARS2 overexpression. Either mutation caused early embryonic fatality in mice. Heterozygous mice displayed reduced muscle tissue-specific levels of tRNASers. Our findings elucidated the biochemical and cellular consequences of impaired translation mediated by SARS2, suggesting that reduced abundance of tRNASer(AGY) is a key determinant for development of SARS2-related diseases.

Aminoacyl-tRNA Synthetase: A Non-Negligible Molecule in RNA Viral Infection.

Infectious diseases such as the ongoing coronavirus disease 2019 (COVID-19) continue to have a huge impact on global health, and the host-virus interaction remains incompletely understood. To address the global threat, in-depth investigations in pathogenesis are essential for interventions in infectious diseases and vaccine development. Interestingly, aminoacyl-transfer RNA (tRNA) synthetases (aaRSs), an ancient enzyme family that was once considered to play housekeeping roles in protein synthesis, are involved in multiple viral infectious diseases. Many aaRSs in eukaryotes present as the components of a cytoplasmic depot system named the multi-synthetase complex (MSC). Upon viral infections, several components of the MSC are released and exert nonenzymatic activities. Host aaRSs can also be utilized to facilitate viral entry and replication. In addition to their intracellular roles, some aaRSs and aaRS-interacting multi-functional proteins (AIMPs) are secreted as active cytokines or function as "molecule communicators" on the cell surface. The interactions between aaRSs and viruses ultimately affect host innate immune responses or facilitate virus invasion. In this review, we summarized the latest advances of the interactions between aaRSs and RNA viruses, with a particular emphasis on the therapeutic potentials of aaRSs in viral infectious diseases.

The endless frontier of tRNA synthetases.

This chapter calls out the following contributed articles, and gives a sense of why the tRNA synthetases are an endless frontier for scientific research and the unveiling of a vast world of new biology.