Identification of distinct biological functions for four 3′-5′ RNA polymerases

Abstract

The superfamily of 3′-5′ polymerases synthesize RNA in the opposite direction to all other DNA/RNA polymerases, and its members include eukaryotic tRNAHis guanylyltransferase (Thg1), as well as Thg1-like proteins (TLPs) of unknown function that are broadly distributed, with family members in all three domains of life. Dictyostelium discoideum encodes one Thg1 and three TLPs (DdiTLP2, DdiTLP3 and DdiTLP4). Here, we demonstrate that depletion of each of the genes results in a significant growth defect, and that each protein catalyzes a unique biological reaction, taking advantage of specialized biochemical properties. DdiTLP2 catalyzes a mitochondria-specific tRNAHis maturation reaction, which is distinct from the tRNAHis maturation reaction typically catalyzed by Thg1 enzymes on cytosolic tRNA. DdiTLP3 catalyzes tRNA repair during mitochondrial tRNA 5′-editing in vivo and in vitro, establishing template-dependent 3′-5′ polymerase activity of TLPs as a bona fide biological activity for the first time since its unexpected discovery more than a decade ago. DdiTLP4 is cytosolic and, surprisingly, catalyzes robust 3′-5′ polymerase activity on non-tRNA substrates, strongly implying further roles for TLP 3′-5′ polymerases in eukaryotes.

Minimal requirements for reverse polymerization and tRNA repair by tRNAHis guanylyltransferase

“…Detailed biochemical experiments have established that archaeal-type Thg1 adds nucleotides to the 5' end of immature or incorrectly processed tRNA. 1,3,7,[12][13][14][15][16] Taken together these studies show that Thg1 can uniquely perform 3'-5' nucleotide addition and elongation, and that nucleotide polymerization in nature can proceed both in forward and reverse directions.…”

“…1,3,8 Bacterial and archaeal (archaeal-type) Thg1 are biochemically and phylogenetically distinct from their eukaryotic (eukaryotic-type) counterparts. 1,3,[12][13][14][15][16][17] Major differences between the two are found in RNA-template preference, ATP dependence and the capability to promote extended reverse nucleotide polymerization. 1,3,7,12,[14][15][16] While the eukaryote-type Thg1 exclusively adds G ¡1 to the 5'-end of tRNA His in a non-templated fashion 10,18,19 , archaeal-type Thg1 exhibits extended reverse polymerase activity with template-dependent nucleotide addition.…”

Repairing tRNA termini: News from the 3' end

“…Here an enzyme more distantly related to the canonical Thg1, a Thg1-like protein (TLP), was shown to catalyze the G ¡1 addition in mitochondria. 54 Interestingly, there are also examples of organisms with tRNA His lacking G ¡1 . A group of a-proteobacteria has been shown to neither encode the G ¡1 residue nor any Thg1 enzyme.…”

“…56,67 In Dictyostelium discoideum, one of the 3 TLPs present in the genome was identified as the mitochondrial enzyme responsible for tRNA 5 0 editing, whereas another one functions as a mitochondrial tRNA His guanylyltransferase. 54 The third TLP, however, acts as a 3 0 -5 0 polymerase on nontRNA substrates and TLPs have also been identified in organisms not containing tRNAs with 5 0 mismatches, suggesting that they may either function in general (t)RNA repair, or similarly act on unrelated RNA substrates. 54 Mismatches in the acceptor stem of tRNAs can also be repaired by exchanges/editing of nucleotides in the 3 0 end.…”

“…54 The third TLP, however, acts as a 3 0 -5 0 polymerase on nontRNA substrates and TLPs have also been identified in organisms not containing tRNAs with 5 0 mismatches, suggesting that they may either function in general (t)RNA repair, or similarly act on unrelated RNA substrates. 54 Mismatches in the acceptor stem of tRNAs can also be repaired by exchanges/editing of nucleotides in the 3 0 end. This reaction could in principle be performed by a more conventional, template-dependent 5 0 -3 0 RNA polymerase.…”