Making Sense of “Nonsense” and More: Challenges and Opportunities in the Genetic Code Expansion, in the World of tRNA Modifications

The evolutional development of the RNA translation process that leads to protein synthesis based on naturally occurring amino acids has its continuation via synthetic biology, the so-called rational bioengineering. Genetic code expansion (GCE) explores beyond the natural translational processes to further enhance the structural properties and augment the functionality of a wide range of proteins. Prokaryotic and eukaryotic ribosomal machinery have been proven to accept engineered tRNAs from orthogonal organisms to efficiently incorporate noncanonical amino acids (ncAAs) with rationally designed side chains. These side chains can be reactive or functional groups, which can be extensively utilized in biochemical, biophysical, and cellular studies. Genetic code extension offers the contingency of introducing more than one ncAA into protein through frameshift suppression, multi-site-specific incorporation of ncAAs, thereby increasing the vast number of possible applications. However, different mediating factors reduce the yield and efficiency of ncAA incorporation into synthetic proteins. In this review, we comment on the recent advancements in genetic code expansion to signify the relevance of systems biology in improving ncAA incorporation efficiency. We discuss the emerging impact of tRNA modifications and metabolism in protein design. We also provide examples of the latest successful accomplishments in synthetic protein therapeutics and show how codon expansion has been employed in various scientific and biotechnological applications.

A versatile tRNA modification-sensitive northern blot method with enhanced performance

The ~22 mitochondrial and ~45 cytosolic tRNAs contain several dozen different posttranscriptional modified nucleotides such that each carries a unique constellation that complements its function. Many tRNA modifications are linked to altered gene expression and their deficiencies due to mutations in tRNA modification enzymes (TMEs) are responsible for numerous diseases. Easily accessible methods to detect tRNA hypomodifications can facilitate progress in advancing such molecular studies. Our lab developed a northern blot method that can quantify relative levels of base modifications on multiple specific tRNAs ~10 years ago which has been used to characterize four different TME deficiencies and is likely further extendable. The assay method depends on differential annealing efficiency of an DNA-oligo probe to the modified versus unmodified tRNA. The signal of this probe is then normalized by a second probe elsewhere on the same tRNA. This positive hybridization in the absence of modification (PHAM) assay has proven useful for i6A37, t6A37, m3C32 and m2,2G26 in multiple laboratories. Yet, over the years we have observed idiosyncratic inconsistency and variability in the assay. Here we document these for some tRNAs and probes and illustrate principles and practices for improved reliability and uniformity in performance. We provide an overview of the method and illustrate benefits of the improved conditions. This is followed by data that demonstrate quantitative validation of PHAM using a TME deletion control, and that nearby modifications can falsely alter the calculated apparent modification efficiency. Finally, we include a calculator tool for matching probe and hybridization conditions.

MLC-Seq: de novo Sequencing of Full-Length tRNAs and Quantitative Mapping of Multiple RNA Modifications

Despite the extensive use of next-generation sequencing of RNA, simultaneous sequencing and quantitative mapping of multiple RNA modifications remain challenging. Herein, we develop MLC-Seq, a mass spectrometry-based direct sequencing method allowing for simultaneously unravelling the RNA sequences and quantitatively mapping different tRNA nucleotide modifications site-specifically. Importantly, MLC-Seq reveals the stoichiometric changes of tRNA modifications upon treatment with the dealkylating enzyme AlkB, and led to the discovery of new nucleotide modifications.

Dynamic changes in tRNA modifications and abundance during T cell activation

The tRNA pool determines the efficiency, throughput, and accuracy of translation. Previous studies have identified dynamic changes in the tRNA (transfer RNA) supply and mRNA (messenger RNA) demand during cancerous proliferation. Yet dynamic changes may also occur during physiologically normal proliferation, and these are less well characterized. We examined the tRNA and mRNA pools of T cells during their vigorous proliferation and differentiation upon triggering their antigen receptor. We observed a global signature of switch in demand for codons at the early proliferation phase of the response, accompanied by corresponding changes in tRNA expression levels. In the later phase, upon differentiation, the response of the tRNA pool relaxed back to the basal level, potentially restraining excessive proliferation. Sequencing of tRNAs allowed us to evaluate their diverse base-modifications. We found that two types of tRNA modifications, wybutosine and ms2t6A, are reduced dramatically during T cell activation. These modifications occur in the anticodon loops of two tRNAs that decode “slippery codons,” which are prone to ribosomal frameshifting. Attenuation of these frameshift-protective modifications is expected to increase the potential for proteome-wide frameshifting during T cell proliferation. Indeed, human cell lines deleted of a wybutosine writer showed increased ribosomal frameshifting, as detected with an HIV gag-pol frameshifting site reporter. These results may explain HIV’s specific tropism toward proliferating T cells since it requires ribosomal frameshift exactly on the corresponding codon for infection. The changes in tRNA expression and modifications uncover a layer of translation regulation during T cell proliferation and expose a potential tradeoff between cellular growth and translation fidelity.

Adaptor Molecules Epitranscriptome Reprograms Bacterial Pathogenicity

The strong decoration of tRNAs with post-transcriptional modifications provides an unprecedented adaptability of this class of non-coding RNAs leading to the regulation of bacterial growth and pathogenicity. Accumulating data indicate that tRNA post-transcriptional modifications possess a central role in both the formation of bacterial cell wall and the modulation of transcription and translation fidelity, but also in the expression of virulence factors. Evolutionary conserved modifications in tRNA nucleosides ensure the proper folding and stability redounding to a totally functional molecule. However, environmental factors including stress conditions can cause various alterations in tRNA modifications, disturbing the pathogen homeostasis. Post-transcriptional modifications adjacent to the anticodon stem-loop, for instance, have been tightly linked to bacterial infectivity. Currently, advances in high throughput methodologies have facilitated the identification and functional investigation of such tRNA modifications offering a broader pool of putative alternative molecular targets and therapeutic avenues against bacterial infections. Herein, we focus on tRNA epitranscriptome shaping regarding modifications with a key role in bacterial infectivity including opportunistic pathogens of the human microbiome.

Abundances of transfer RNA modifications and transcriptional levels for tRNA-modifying enzymes are sex-specific in mosquitoes

As carriers of multiple human diseases, understanding the mechanisms behind mosquito reproduction may have implications for remediation strategies. Transfer RNA (tRNA) acts as the adapter molecule of amino acids and are key components in protein synthesis and a critical factor in the function of tRNAs is chemical modifications. Here, we provide an assessment of tRNA modifications between sexes for three mosquito species and examine correlated transcript levels underlying key proteins involved in tRNA modification. Thirty-three tRNA modifications were detected among mosquito species and most of these modifications are higher in females compared to males. Analysis of previous male and female RNAseq datasets indicated a similar increase in tRNA modifying enzymes in females, supporting our observed female enrichment of tRNA modifications. Tissues-specific expressional studies revealed high transcript levels for tRNA modifying enzymes in the ovaries for Aedes aegypti, but not male reproductive tissues. These studies suggest that tRNA modifications may be critical to reproduction in mosquitoes, representing a potential novel target for control.

Regulatory roles of RNA modifications in breast cancer

Collectively referred to as the epitranscriptome, RNA modifications play important roles in gene expression control regulating relevant cellular processes. In the last few decades, growing numbers of RNA modifications have been identified not only in abundant ribosomal (rRNA) and transfer RNA (tRNA) but also in messenger RNA (mRNA). In addition, many writers, erasers and readers that dynamically regulate the chemical marks have also been characterized. Correct deposition of RNA modifications is prerequisite for cellular homeostasis, and its alteration results in aberrant transcriptional programs that dictate human disease, including breast cancer, the most frequent female malignancy, and the leading cause of cancer-related death in women. In this review, we emphasize the major RNA modifications that are present in tRNA, rRNA and mRNA. We have categorized breast cancer-associated chemical marks and summarize their contribution to breast tumorigenesis. In addition, we describe less abundant tRNA modifications with related pathways implicated in breast cancer. Finally, we discuss current limitations and perspectives on epitranscriptomics for use in therapeutic strategies against breast and other cancers.

Identification of a motif in Trm732 required for 2′-O-methylation of the tRNA anticodon loop by Trm7

Posttranscriptional tRNA modifications are essential for proper gene expression, and defects in the enzymes that perform tRNA modifications are associated with numerous human disorders. Throughout eukaryotes, 2′-O-methylation of residues 32 and 34 of the anticodon loop of tRNA is important for proper translation, and in humans, lack of these modifications results in non-syndromic X-linked intellectual disability. In yeast, the methyltransferase Trm7 forms a complex with Trm732 to 2′-O-methylate tRNA residue 32 and with Trm734 to 2′-O-methylate tRNA residue 34. Trm732 and Trm734 are required for the methylation activity of Trm7, but the role of these auxiliary proteins is not clear. Additionally, Trm732 and Trm734 homologs are implicated in biological processes not directly related to translation, suggesting that these proteins may have additional cellular functions. To identify critical amino acids in Trm732, we generated variants and tested their ability to function in yeast cells. We identified a conserved RRSAGLP motif in the conserved DUF2428 domain of Trm732 that is required for tRNA modification activity by both yeast Trm732 and its human homolog THADA. The identification of Trm732 variants that lack tRNA modification activity will help to determine if other biological functions ascribed to Trm732 and THADA are directly due to tRNA modification, or to secondary effects due to other functions of these proteins.