The quality of life is dependent on the quality of protein synthesis, which is determined by how individual codons are translated into amino acid building blocks by tRNAs as the adaptors. Synonymous codons are not translated equally, due to differences in the quality of codon pairing with the anticodon in cognate tRNAs. These differences contribute to “codon-specific translation”, in which the speed and quality of translation of each codon is regulated by the tRNA anticodon and, more importantly, the nature of post-transcriptional epigenetic modifications associated with the anticodon. The biology of post-transcriptional modifications is complex and subject to stress response. Codon-specific translation is therefore emerging as an important regulator of protein expression and the composition of the proteome in each cell.
Methylation of tRNA is the most common form of post-transcriptional modification. With the addition of just one methyl group to a nucleobase or a backbone group, tRNA can gain structural stability or decoding specificity upon pairing with an mRNA codon. However, how each methylation confers a unique strength to tRNA is poorly understood. tRNA research is in a new era, in which the primary challenge is no longer to simply know what methylation is expressed and where, but rather to determine how each methylation impacts translation of codons and expression of genomes. We have focused on the m 1 G37-tRNA methylation, which is necessary to maintain protein synthesis reading frame. Loss of m 1 G37-tRNA leads to accumulation of +1 frameshift errors, resulting in premature termination of translation and cell death. We are exploring the biology of this methylation, which has the potential to switch cells from the healthy to the disease state.
tRNA Wobble Modifications in Stress Response
The tRNA wobble nucleotide (at position 34 of the anticodon) is often extensively modified post-transcriptionally as a mechanism to control the quality of pairing with cognate codons. Many wobble modifications are produced in response to stress, linking the quality of translation to stress response. Additionally, many wobble modifications are co-occurring with modifications at position 37 on the 3'-side of the anticodon. For example, the UGG isoacceptor of E. coli tRNAPro contains the mcmo5 modification at the wobble base and the m1G37 methylation at position 37. How does the wobble modification respond to stress? How does this stress response coordinate with the m1G37 methylation and vice versa? Will this coordination prepare cells for better response to stress?
tRNA Post-Transcriptional Modifications in Mitochondria Disorders
Mitochondria are the powerhouse of our cells. The most common form of mitochondrial genetic disorders is due to mutations in mitochondria-encoded tRNAs (mt-tRNAs), resulting in complex and systemic symptoms. There are no cures for these disorders at present. Mt-tRNAs are A-U rich and require post-transcriptional modifications to stabilize their structure. Such modifications are important determinants in the life cycle of individual mt-tRNAs. How do post-transcriptional modifications determine the quality of mt-tRNAs? How do disease-associated mutations change post-transcriptional modifications? What is the pathway from mutations in mt-tRNAs to mitochondrial disorders?
tRNA in Neurodegenerate Diseases
While tRNA is ubiquitously expressed in all cells and tissues, its dysfunction is often specifically linked to neurodegenerate disorders, affecting the brain and the central and peripheral nervous systems. The molecular basis for this specific manifestation is unclear, but it demonstrates the broad impact of tRNA biology in human health. In the case of Charcot-Marie-Tooth (CMT) disease, the most common form of human peripheral neuropathy, pathogenic mutations are found in ARS enzymes. We show that the majority of these mutations impair tRNA charging, providing a framework to address how decreased charging leads to dysfunction of neuronal cells.
Also, the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is extension of the G4C2 (sense) C4G2 (antisense) motif in repeated sequences in the first intron of the C9orf72 gene. This discovery genetically links these two diseases (C9-ALS/FTD) to a larger group of repeat-expansion diseases, such as fragile X tremor ataxia syndrome (FXTAS), myotonic dystrophy type 1 (DM1), spinocerebellar ataxia 8 (SCA8)3, and Huntington’s disease (HD). Three mechanisms are proposed for C9-ALS/FTD: (1) haplo-insufficiency of the C9 gene; (2) RNA toxicity caused by nucleo-protein sequestration; and (3) repeat-associated non-AUG (RAN) translation, generating dipeptide repeats that produce toxicity to neurons. We are focusing on RAN translation and investigating how tRNA plays a role in such unusual translation that impacts on the development of C9-ALS/FTD.
We are using kinetic tools to address these questions. These are unique tools that give us special insights into the problems.