A simple post-transcriptional modification of tRNA, deamination of adenosine to inosine at the first, or wobble, position of the anticodon, inspired Francis Crick's Wobble Hypothesis 50 years ago. Many more naturally-occurring modifications have been elucidated and continue to be discovered. The post-transcriptional modifications of tRNA's anticodon domain are the most diverse and chemically complex of any RNA modifications. Their contribution with regards to chemistry, structure and dynamics reveal individual and combined effects on tRNA function in recognition of cognate and wobble codons. As forecast by the Modified Wobble Hypothesis 25 years ago, some individual modifications at tRNA's wobble position have evolved to restrict codon recognition whereas others expand the tRNA's ability to read as many as four synonymous codons. Here, we review tRNA wobble codon recognition using specific examples of simple and complex modification chemistries that alter tRNA function. Understanding natural modifications has inspired evolutionary insights and possible innovation in protein synthesis.

RNAs are central to all gene expression through the control of protein synthesis. Four major nucleosides, adenosine, guanosine, cytidine and uridine, compose RNAs and provide sequence variation, but are limited in contributions to structural variation as well as distinct chemical properties. The ability of RNAs to play multiple roles in cellular metabolism is made possible by extensive variation in length, conformational dynamics, and the over 100 post-transcriptional modifications. There are several reviews of the biochemical pathways leading to RNA modification, but the physicochemical nature of modified nucleosides and how they facilitate RNA function is of keen interest, particularly with regard to the contributions of modified nucleosides. Transfer RNAs (tRNAs) are the most extensively modified RNAs. The diversity of modifications provide versatility to the chemical and structural environments. The added chemistry, conformation and dynamics of modified nucleosides occurring at the termini of stems in tRNA’s cloverleaf secondary structure affect the global three-dimensional conformation, produce unique recognition determinants for macromolecules to recognize tRNAs, and affect the accurate and efficient decoding ability of tRNAs. This review will discuss the impact of specific chemical moieties on the structure, stability, electrochemical properties, and function of tRNAs.

The posttranscriptional modifications of tRNA's anticodon stem and loop (ASL) domain represent a third level, a third code, to the accuracy and efficiency of translating mRNA codons into the correct amino acid sequence of proteins. Modifications of tRNA's ASL domain are enzymatically synthesized and site specifically located at the anticodon wobble position-34 and 3'-adjacent to the anticodon at position-37. Degeneracy of the 64 Universal Genetic Codes and the limitation in the number of tRNA species require some tRNAs to decode more than one codon. The specific modification chemistries and their impact on the tRNA's ASL structure and dynamics enable one tRNA to decode cognate and "wobble codons" or to expand recognition to synonymous codons, all the while maintaining the translational reading frame. Some modified nucleosides' chemistries prestructure tRNA to read the two codons of a specific amino acid that shares a twofold degenerate codon box, and other chemistries allow a different tRNA to respond to all four codons of a fourfold degenerate codon box. Thus, tRNA ASL modifications are critical and mutations in genes for the modification enzymes and tRNA, the consequences of which is a lack of modification, lead to mistranslation and human disease. By optimizing tRNA anticodon chemistries, structure, and dynamics in all organisms, modifications ensure translational fidelity of mRNA transcripts.

Human Genome Wide Association Studies found a significant risk of Type 2 Diabetes Mellitus (T2DM) in single nucleotide polymorphisms in the cdkal1 gene. The cdkal1 gene is remote from the insulin gene and with the surprising function of a specific tRNA modification. Population studies and case control studies acquired evidences of the connection between Cdkal1 protein and insulin production over the years. To obtain biochemical proofs directly linking potential SNPs to their roles in insulin production and availability is challenging, but the development of Cdkal1 knock out mice and knock out cell lines made it possible to extend our knowledge towards therapeutic field of diabetic research. Supporting the evidences, here we show that knock down of the cdkal1 gene using small interfering and short hairpin RNA in the NIT-1 cell line, a β-cell line inducible for insulin resulted in reduced levels of cdkal1 and mature insulin mRNAs, increased the level of precursor insulin mRNA, decreased Cdkal1 and insulin proteins, and diminished modification of tRNALys3 from t6A37 to ms2t6A37, the specified function of Cdkal1. tRNALys3 lacking ms2- is incapable of establishing sufficient hydrogen bonding energy and hydrophobic stabilization to decode the wobble codon AAG.

BackgroundPlasminogen activator inhibitor‐1 (PAI‐1), a secreted glycoprotein and member of the serine protease inhibitor family, has been implicated to have a role in Alzheimer’s disease (AD) pathogenesis by regulating amyloid‐beta (Aβ) accumulation (Neurobiol Aging 32:1079,2011; J Alzheimers Dis. 64:447,2018). A recent study found that plasma PAI‐1 was differentially expressed in subjects with Aβ pathology and neurodegeneration (A+TN+) compared to A‐TN‐ subjects, with decreased plasma PAI‐1 levels in clinical AD compared to cognitively normal controls (Alzheimers Dement 17:1452,2021). However, whether circulating PAI‐1 levels are altered during the preclinical stage of AD, where there is Aβ and tau pathology but no significant cognitive impairment, remains unclear.MethodCognitively normal (Clinical Dementia Rating of 0) volunteers from the Healthy Aging & Senile Dementia and Adult Children Study (Missouri, USA) with body mass index < 30, fasting plasma and cerebrospinal fluid (CSF) samples were included in this cross‐sectional study. Preclinical AD was defined by previously established CSF criteria (preclinical AD: 29 men, 26 women; control: 35 men, 65 women). Plasma PAI‐1 levels were measured by a commercially available immunoassay (MilliporeSigma) and log transformed.ResultIn men, plasma PAI‐1 levels were significantly lower in preclinical AD compared to control subjects (p=0.029) and were associated with CSF Aβ42 and p‐tau181 levels (CSF Aβ42: age‐adjusted beta coefficient 0.38, p=0.007; CSF p‐tau181: age‐adjusted beta coefficient ‐0.32, p=0.026) but not CSF tau levels (p>0.05). In women, there was no significant difference in plasma PAI‐1 levels between preclinical AD and control subjects (p>0.05).ConclusionThese studies raise the possibility that, in men, alterations in peripheral PAI‐1 occur during the earliest stages of AD. Studies in larger cohorts will be needed to confirm these observations and to determine their clinical utility and the underlying mechanisms of any sex differences.