Naturally occurring dual recognition of tRNA^Hissubstrates with and without a universal identity element

Abstract

The extra 5ʹ guanine nucleotide (G-1) on tRNA His is a nearly universal feature that specifies tRNA His identity. The G-1 residue is either genome encoded or post-transcriptionally added by tRNA His guanylyltransferase (Thg1). Despite Caenorhabditis elegans being a Thg1-independent organism, its cytoplasmic tRNA His (CetRNA n His) retains a genome-encoded G-1. Our study showed that this eukaryote possesses a histidyl-tRNA synthetase (CeHisRS) gene encoding two distinct HisRS isoforms that differ only at their N-termini. Most interestingly, its mitochondrial tRNA His (CetRNA m His ) lacks G-1, a scenario never observed in any organelle. This tRNA, while lacking the canonical identity element, can still be efficiently aminoacylated in vivo. Even so, addition of G-1 to CetRNA m His strongly enhanced its aminoacylation efficiency in vitro. Overexpression of CeHisRS successfully bypassed the requirement for yeast THG1 in the presence of CetRNA n His without G-1. Mutagenesis assays showed that the anticodon takes a primary role in CetRNA His identity recognition, being comparable to the universal identity element. Consequently, simultaneous introduction of both G-1 and the anticodon of tRNA His effectively converted a non-cognate tRNA to a tRNA His -like substrate. Our study suggests that a new balance between identity elements of tRNA His relieves HisRS from the absolute requirement for G-1.

Transfer RNA Recognition and Aminoacylation by Synthetases

“…This suggests a new balance of minor His determinants overcoming the absence of G-1 for HisRS recognition, with e.g. yeast HisRS recognizing both A73 and C73 minor determinants while mt-HisRS prefers A73 (Lee et al, 2019). Taken together, these data prove a functional plasticity of tRNA identity rules in modern systems that, in extreme cases, is correlated with a huge structural plasticity of tRNA molecules.…”

“…Thus, in humans, efficient histidylation of mt-tRNA His primarily relies on a C73 discriminator base and on the N-terminal WHEP sequence [present in 3 aaRSs, i.e. TprRS, HisRS and EPRS (fused GluRS-ProRS)] of mt-HisRS (Lee et al, 2019). 4.…”

Gain of C-Ala enables AlaRS to target the L-shaped tRNAAla

“…This result suggests that CeAlaRS m can charge the yeast mitochondrial tRNA Ala and mitochondria require only a minimal AlaRS activity to maintain their normal function. In addition, it implies that the mature CeAlaRS m carries a cryptic MTS, which allows a portion of the enzyme to be imported into mitochondria to function there, a scenario often seen in aaRS rescue assays ( 7 , 30 , 34 ). Unfortunately, no transformants carrying the CeAlaRS m -(C-Ala) fusion were available for the rescue assays.…”

“…For example, Vanderwaltozyma polyspora , Homo sapiens and Caenorhabditis elegans each possess two distinct nuclear alanyl-tRNA synthetase (AlaRS) genes, one encoding the cytoplasmic form and the other encoding its mitochondrial counterpart ( 4–6 ). Nevertheless, in some cases, both isoforms of a given aaRS are produced from a single gene through alternative transcription and translation, examples of which include the histidyl-tRNA synthetase genes of C. elegans and Saccharomyces cerevisiae ( 7 , 8 ).…”

Analysis of GTP addition in the reverse (3′–5′) direction by human tRNAHis guanylyltransferase

“…The enzymes tRNA His guanyltransferase (Thg1) and Thg1-like protein (TLP) catalyze the addition of nucleotides to the 5′-end of tRNA in the reverse direction (3′–5′) ( Heinemann et al 2012 ; Jackman et al 2012 ; Chen et al 2019 ). In the cytoplasm of eukaryotes Thg1 catalyzes the addition of a guanosine residue to the 5′-end of tRNA His ( Gu et al 2003 ) at the −1 position (G −1 ), which serves as a major recognition element of histidyl-tRNA synthetase (HisRS) ( Cooley et al 1982 ; Himeno et al 1989 ; Rudinger et al 1994 ; Rosen and Musier-Forsyth 2004 ), with rare exceptions ( Rao and Jackman 2015 ; Lee et al 2019 ). Thg1 recognizes a His anticodon (GUG) in a 5′-monophosphorylated tRNA His (p-tRNA His ), which is cleaved by RNase P from pre-tRNA His ( Jackman and Phizicky 2006 ).…”