The Biological Roles of tRNA Modifications
RNA(Phe) with its 13 modified residues highlighted
tRNA is the most highly modified class of RNA species, and modifications are found in tRNAs from all organisms that have been examined. Yeast cytoplasmic tRNAs have 25 biochemically distinct modifications, and the average tRNA has 13 modified residues. These modifications are highly conserved in eukaryotes, including humans, and many of the corresponding genes have been identified in this and other labs. It is known from previous work in the field that several modifications in and around the anticodon loop have crucial roles in various aspects of translation, while several other modifications remote from the anticodon loop have specific roles in tRNA folding and/or stability. However, the precise roles of many other individual modifications are only poorly understood, and are under investigation in our lab.
Investigation of a rapid tRNA decay (RTD) quality control pathway that acts on several hypomodified mature tRNA species
Two quality control pathways are known to act on specific tRNA species lacking modifications. The nuclear surveillance pathway acts on pre-tRNAiMet lacking m1A, and the rapid tRNA decay pathway acts on several specific mature tRNA species lacking one or more different modifications.
We initially found that trm8-Δ trm4-Δ mutants (which lack m7G46 and m5C) are temperature sensitive at 37 °C because mature tRNAVal(AAC) is rapidly inactivated due to degradation and loss of charging. Subsequent genetic analysis showed that this rapid tRNA decay (RTD) pathway is mediated by the 5'-3' exonucleases Rat1 and Xrn1, and by Met22, which likely acts indirectly through its substrate pAp to inhibit Rat1 and Xrn1. Since we also showed that the RTD pathway acts on tRNASer(CGA) and tRNASer(UGA) lacking ac4C12 and Um44, as well as other hypomodified species, we suggested that this pathway might be a general quality control pathway monitoring the integrity of tRNA.
Our recent research has focused on understanding how specific undermodified tRNAs are recognized and targeted for degradation, whereas other tRNA species lacking the same modifications are not targeted. Genetic and biochemical results suggest that specific tRNAs are targeted for RTD because their acceptor and T-stems are less stable, resulting in increased exposure of the 5' end to probes and to Xrn1, and suggest that modifications exert their effect on RTD indirectly through their effect on tertiary structure.
Current research uses a variety of genetic, genomic and biochemical approaches to examine the role of translation in the RTD pathway, to identify other components of the pathway and analyze their roles, and to examine the role of the pathway in wild type cells and on different types of damaged tRNA.
Investigation of the role of modifications of C32
We are interested in modification of residue C32, because this residue is in the anticodon loop and is implicated in translation, because C32 can be modified to m3C or Cm in different tRNAs, and because little is known about the precise role of this residue and either of its modifications.
We recently found that the actin binding protein Abp140 (now called Trm140) is required for formation of m3C32 of the three tRNAThr species and three tRNASer species that are known to have this modification, and is sufficient for modification activity in vitro. Our discovery that a trm140 mutant also has a mild but distinct defect in translation provides a first step in uncovering the precise role of this modification. In addition, we are examining the role of Cm32 in the cell, since mutations in the gene responsible for its formation result in cells with a severe defect in growth and translation.
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