Mechanisms and Delivery of tRNA Therapeutics.

Transfer ribonucleic acid (tRNA) therapeutics will provide personalized and mutation specific medicines to treat human genetic diseases for which no cures currently exist. The tRNAs are a family of adaptor molecules that interpret the nucleic acid sequences in our genes into the amino acid sequences of proteins that dictate cell function. Humans encode more than 600 tRNA genes. Interestingly, even healthy individuals contain some mutant tRNAs that make mistakes. Missense suppressor tRNAs insert the wrong amino acid in proteins, and nonsense suppressor tRNAs read through premature stop signals to generate full length proteins. Mutations that underlie many human diseases, including neurodegenerative diseases, cancers, and diverse rare genetic disorders, result from missense or nonsense mutations. Thus, specific tRNA variants can be strategically deployed as therapeutic agents to correct genetic defects. We review the mechanisms of tRNA therapeutic activity, the nature of the therapeutic window for nonsense and missense suppression as well as wild-type tRNA supplementation. We discuss the challenges and promises of delivering tRNAs as synthetic RNAs or as gene therapies. Together, tRNA medicines will provide novel treatments for common and rare genetic diseases in humans.

Mistranslating the genetic code with leucine in yeast and mammalian cells.

Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNALeu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNALeu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs.

Delivery of Active AKT1 to Human Cells.

Protein kinase B (AKT1) is a serine/threonine kinase and central transducer of cell survival pathways. Typical approaches to study AKT1 biology in cells rely on growth factor or insulin stimulation that activates AKT1 via phosphorylation at two key regulatory sites (Thr308, Ser473), yet cell stimulation also activates many other kinases. To produce cells with specific AKT1 activity, we developed a novel system to deliver active AKT1 to human cells. We recently established a method to produce AKT1 phospho-variants from Escherichia coli with programmed phosphorylation. Here, we fused AKT1 with an N-terminal cell penetrating peptide tag derived from the human immunodeficiency virus trans-activator of transcription (TAT) protein. The TAT-tag did not alter AKT1 kinase activity and was necessary and sufficient to rapidly deliver AKT1 protein variants that persisted in human cells for 24 h without the need to use transfection reagents. TAT-pAKT1T308 induced selective phosphorylation of the known AKT1 substrate GSK-3α, but not GSK-3ß, and downstream stimulation of the AKT1 pathway as evidenced by phosphorylation of ribosomal protein S6 at Ser240/244. The data demonstrate efficient delivery of AKT1 with programmed phosphorylation to human cells, thus establishing a cell-based model system to investigate signaling that is dependent on AKT1 activity.

A novel fluorescent reporter sensitive to serine mis-incorporation.

High-fidelity translation was considered a requirement for living cells. The frozen accident theory suggested that any deviation from the standard genetic code should result in the production of so much mis-made and non-functional proteins that cells cannot remain viable. Studies in bacterial, yeast, and mammalian cells show that significant levels of mistranslation (1-10% per codon) can be tolerated or even beneficial under conditions of oxidative stress. Single tRNA mutants, which occur naturally in the human population, can lead to amino acid mis-incorporation at a codon or set of codons. The rate or level of mistranslation can be difficult or impossible to measure in live cells. We developed a novel red fluorescent protein reporter that is sensitive to serine (Ser) mis-incorporation at proline (Pro) codons. The mCherry Ser151Pro mutant is efficiently produced in Escherichia coli but non-fluorescent. We demonstrated in cells and with purified mCherry protein that the fluorescence of mCherry Ser151Pro is rescued by two different tRNASer gene variants that were mutated to contain the Pro (UGG) anticodon. Ser mis-incorporation was confirmed by mass spectrometry. Remarkably, E. coli tolerated mistranslation rates of ~10% per codon with negligible reduction in growth rate. Conformational sampling simulations revealed that the Ser151Pro mutant leads to significant changes in the conformational freedom of the chromophore precursor, which is indicative of a defect in chromophore maturation. Together our data suggest that the mCherry Ser151 mutants may be used to report Ser mis-incorporation at multiple other codons, further expanding the ability to measure mistranslation in living cells.