Identification and Functional Characterization of Divergent 3'-Phosphate tRNA Ligase From Entamoeba histolytica.

Ruofan Peng,Tomoyoshi Nozaki,Shigeo Yoshinari,Tetsuro Kawano-Sugaya,Ghulam Jeelani

doi:10.3389/fcimb.2021.746261

Ruofan Peng, Tomoyoshi Nozaki + Show 3 more

Open Access

https://doi.org/10.3389/fcimb.2021.746261

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Abstract

HSPC117/RtcB, 3’-phosphate tRNA ligase, is a critical enzyme involved in tRNA splicing and maturation. HSPC117/RtcB is also involved in mRNA splicing of some protein-coding genes including XBP-1. Entamoeba histolytica, a protozoan parasite responsible for human amebiasis, possesses two RtcB proteins (EhRtcB1 and 2), but their biological functions remain unknown. Both RtcBs show kinship with mammalian/archaeal type, and all amino acid residues present in the active sites are highly conserved, as suggested by protein alignment and phylogenetic analyses. EhRtcB1 was demonstrated to be localized to the nucleus, while EhRtcB2 was in the cytosol. EhRtcB1, but not EhRtcB2, was required for optimal growth of E. histolytica trophozoites. Both EhRtcB1 (in cooperation with EhArchease) and EhRtcB2 showed RNA ligation activity in vitro. The predominant role of EhRtcB1 in tRNAIle(UAU) processing in vivo was demonstrated in EhRtcB1- and 2-gene silenced strains. Taken together, we have demonstrated the conservation of tRNA splicing and functional diversification of RtcBs in this amoebozoan lineage.

Highlights

Transfer RNAs are crucial adapter molecules involved in protein translation by delivering specific amino acids to translational machinery, according to a 3-nucleotide anticodon that complements the corresponding codon in messenger RNA (Grosjean, 2009)
Note that an ortholog of transfer RNA (tRNA) endonuclease and an ortholog of archease (EHI_028560) that has been known to be required for full enzyme activity of HSPC117 were found in the E. histolytica genome
We have shown in this study that the E. histolytica genome encodes 2,670 tRNA genes, which include 42 tRNATyr(GUA) and 52 tRNAIle(UAU) intron-containing genes, similar to the human genome (GTCh37/hg19) containing 5 tRNAIle(UAU) and 13 tRNATyr(GUA) intron-containing genes (Chan and Lowe, 2009; Chan and Lowe, 2016)

Summary

Introduction

Transfer RNAs (tRNAs) are crucial adapter molecules involved in protein translation by delivering specific amino acids to translational machinery, according to a 3-nucleotide anticodon that complements the corresponding codon in messenger RNA (Grosjean, 2009). In all three domains of life, nascent tRNAs require numerous modifications to exert their functions in protein translation (Koh and Sarin, 2018). RtcB RNA Ligases in Entamoeba some tRNA gene transcripts containing an intron are processed and matured to be fully functional (Hartmann et al, 2009; Popow et al, 2012). TRNA endonucleases, responsible for the first step of tRNAs maturation, are well characterized. Their origin and the oligomeric architecture are well conserved in eukaryotes and their archaea (Trotta et al, 1997; Li and Abelson, 2000; Paushkin et al, 2004; TocchiniValentini et al, 2005). Molecular mechanisms of the second step, tRNA ligation, are significantly diversified between three domains of life (Silber et al, 1972; Konarska et al, 1981; Filipowicz and Shatkin, 1983; Greer et al, 1983a; Laski et al, 1983; Kjems and Garrett, 1988)

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