Anticodon Wobble Uridine Modification by Elongator at the Crossroad of Cell Signaling, Differentiation, and Diseases

Abstract

First identified 20 years ago as an RNA polymerase II-associated putative histone acetyltransferase, the conserved Elongator complex has since been recognized as the central player of a complex, regulated, and biologically relevant epitranscriptomic pathway targeting the wobble uridine of some tRNAs. Numerous studies have contributed to three emerging concepts resulting from anticodon modification by Elongator: the codon-specific control of translation, the ability of reprogramming translation in various physiological or pathological contexts, and the maintenance of proteome integrity by counteracting protein aggregation. These three aspects of tRNA modification by Elongator constitute a new layer of regulation that fundamentally contributes to gene expression and are now recognized as being critically involved in various human diseases.

Keywords: anticodon; modification; tRNA; wobble; yeast.

Figure 1

Elongator-dependent modifications of tRNAs. (A) tRNA modifications found in cytoplasmic tRNAs of S. cerevisiae. Bold circles indicate modified positions in some or all tRNAs. Black circles represent the anticodon corresponding to positions 34, 35, and 36. The highly modified position 37 is also indicated. The Elongator-dependent modifications are underlined (modified from Phizicky and Hopper, 2010 [15]). (B) Tri- (left) or bidimensional (right) models of uridine (insert) and its doubly modified mcm⁵s² version. (C)The genetic code with codons, anticodons, and the elongator-dependent modifications, ncm⁵, mcm⁵, and mcm⁵s² found on 11 tRNAs. Note that the mcm⁵s²-modified tRNAs recognize codons in the split boxes. For glutamine (Gln), lysine (Lys), and glutamic acid (Glu), the codon usage of S. pombe is indicated. The A-ended codon is favored due to the low G-C content of the fission yeast genome. “I” stands for inosine and the asterisk for pseudouridine. (D) Steps in the synthesis of the mcm⁵s² modification and the number of required proteins. The six subunits of Elongator are included in the 13 proteins required to synthetize cm⁵U. Trm12 is a physical partner of the Trm9 methylase.

Figure 2

Identification of the antisuppressor sin3-193 mutant as an allele of elp3 in fission yeast. (A) The ade7-413 allele of the fission yeast ade7 gene contains an ochre STOP codon (UAA). The mutation results in adenine auxotrophy and red color (not shown). (B) The suppressor tRNA sup3-18 contains an anticodon mutation able to read the UAA STOP codon and incorporate a serine, which suppresses adenine auxotrophy and red color (not shown). The sin3 (Elp3) gene product is required for both the anticodon modification (mcm⁵s²) and efficient suppression. Note the consistent 5′-3′polarity of the tRNA and mRNA molecules, resulting in the reverse orientation of the codon and the anticodon. (C) The sin3-193 allele of sin3 (Elp3) is a loss-of-function allele and results in anti-suppression. Therefore, despite the presence of the suppressor tRNA, the strain is red and an auxotroph for adenine (not shown), which are easily followed phenotypes.

Figure 3

Identification of the genes necessary for the synthesis of the mcm⁵s² modification in budding yeast using zymocin. (A) Zymocin is a tRNA endonuclease that cleaves the anticodon of tRNAs bearing the mcm⁵s² modification. (B) Screening set-up. The zymocin-encoding gene is expressed from the GAL promoter that is repressed by raffinose and induced by galactose. The growth of a wild-type strain on galactose (middle) results in death, due to the cleavage of the target tRNAs. An Elongator mutant (elp3::kanR) is resistant to zymocin due to the absence of this modification. Expression of zymocin in the yeast deletion library (a collection of yeast strains where all viable haploid strains deleted for a given gene are represented) allows the identification of all the non-essential genes required for the mcm⁵s² modification, which was confirmed by HPLC analysis of tRNAs.

Figure 4

High protein expression in fission yeast is correlated with a skewed codon content excluding the AAA and GAA codons. Codon bias between G- and A-ending mcm⁵s²-modified codons for lysine, glutamic acid, and glutamine in genes as a function of mRNA level (upper panel) or ribosome occupancy (lower panel). Modified from Bauer et al., 2012 [37].

Figure 5

Schematic view of the integration of the Trm9 and Elongator-dependent mcm⁵ modification with cellular signaling pathways. (A) Elongator is a positive regulator of TORC2 in fission yeast by promoting the translation of Rictor and Tor1 components of the complex. In turn, TORC2 downregulates the activity of Gsk3, which is a negative regulator of Elongator through the phosphorylation of Elp4 on S114. This creates the positive feedback loop required to activate TORC2 and cell differentiation in nitrogen starvation conditions. (B) Phosphorylations of human TRM9L by various signalling cascades constitute a critical regulator of oxidative stress survival. Oxidative stress induces the rapid and dose-dependent phosphorylation of TRM9L on serine 380 downstream of the oxidative stress-activated MEK (mitogen-activated protein kinase kinase)-ERK (extracellular signal-regulated kinase)-RSK (ribosomal protein S6 kinase) signaling cascade. In addition, serine 255 phosphorylation is similarly responding to oxidative stress through an unknown pathway.

Figure 6

The remarkable asymmetrical structure of the Elongator complex. Elp1 (orange), Elp2 (yellow), Elp3 (pink), Elp4 (green), Elp5 (blue), and Elp6 (sand) are shown in a topological model of the two-lobed Elp1-2-3 subcomplex and asymmetrical binding of the hexameric RecA-like Elp4-5-6 ring. Individual domains of Elp1 (WD40, NTD and CTD) and the catalytic Elp3 subunit (KAT, SAM) are also indicated. Modified from Dauden et al., 2017 [52,53].