The N-terminal half-domain of the long form of tRNase Z is required for the RNase 65 activity

Abstract

Transfer RNA (tRNA) 3' processing endoribonuclease (tRNase Z) is an enzyme responsible for the removal of a 3' trailer from pre-tRNA. There exists two types of tRNase Z: one is a short form (tRNase ZS) that consists of 300-400 amino acids, and the other is a long form (tRNase ZL) that contains 800-900 amino acids. Here we investigated whether the short and long forms have different preferences for various RNA substrates. We examined three recombinant tRNase ZSs from human, Escherichia coli and Thermotoga maritima, two recombinant tRNase ZLs from human and Saccharomyces cerevisiae, one tRNase ZL from pig liver, and the N- and C-terminal half regions of human tRNase ZL for cleavage of human micro-pre-tRNA(Arg) and the RNase 65 activity. All tRNase ZLs cleaved the micro-pre-tRNA and showed the RNase 65 activity, while all tRNase ZSs and both half regions of human tRNase ZL failed to do so with the exception of the C-terminal half, which barely cleaved the micro-pre-tRNA. We also show that only the long forms of tRNase Z can specifically cleave a target RNA under the direction of a new type of small guide RNA, hook RNA. These results indicate that indeed tRNase ZL and tRNase ZS have different substrate specificities and that the differences are attributed to the N-terminal half-domain of tRNase ZL. Furthermore, the optimal concentrations of NaCl, MgCl2 and MnCl2 differed between tRNase ZSs and tRNase ZLs, and the K(m) values implied that tRNase ZLs interact with pre-tRNA substrates more strongly than tRNase ZSs.

Partitioning of the Nuclear and Mitochondrial tRNA 3′-End Processing Activities between Two different Proteins in Schizosaccharomyces pombe

“…In addition, a role for ELAC2 in mitochondrial pre-tRNA 3Ј-end processing has recently been demonstrated in vivo (30,32). Although tRNase Z L s from some species, including S. cerevisiae (37), S. pombe (38), and A. thaliana (34), have been shown to possess tRNA 3Ј-end processing activity in vitro, their nuclear and mitochondrial tRNA 3Ј-end processing activity in vivo remains to be experimentally demonstrated.…”

Gene silencing by the tRNA maturase tRNase ZL under the direction of small-guide RNA

“…This observation is consistent with the in vitro observation that tRNase Z S cannot cleave pre-tRNA-like complexes. 6,13 In the same manner, the downregulation level of the luciferase activity by the linear-type sgRNA sgLucL1 was enhanced B3-fold in the tRNase Z L -overproducing cells, and was relieved B2.5-fold in the tRNase Z L -knockdown cells ( Figure 3c). The downregulation level was enhanced only slightly (B1.3-fold) in the cell lines producing altered levels of tRNase Z S .…”

“…This, together with the fact that tRNase Z L has the extra N-terminal region that is dispensable for pre-tRNA processing, 4,6 implies an additional role of this enzyme. There exist a huge number of microRNAs (miRNAs) that are believed to work as translational regulators, 21 and, curiously, their length is B21-23 nt, which is similar to the length of hook-type sgRNA.…”

“…5 tRNase Zs can be divided into two groups: a short form (tRNase Z S ) that consists of 300-400 amino acids and a long form (tRNase Z L ) that contains 800-900 amino acids. 6 Bacteria and archaea genomes contain a tRNase Z S gene only, whereas eucaryotic genomes encode either only tRNase Z L or both forms. Interestingly, the human tRNase Z L is encoded by a prostate cancer-associated gene on chromosome 17, 7 and the human tRNase Z S gene is on chromosome 18.…”

“…In contrast, tRNase Z S can process pre-tRNAs but not these pre-tRNA-like or micro-pre-tRNA-like complexes in vitro. 4,6 We have shown the efficacy of this RNA-targeting method in the living cells to some degree by introducing sgRNAs either as their expression plasmids or as 2 0 -Omethyl RNAs. 15,16 The expression of exogenous reporter genes for Escherichia coli chloramphenicol acetyltransferase (CAT) and firefly luciferase were downregulated by appropriately designed sgRNAs in mammalian culture cells, 15 and the HIV-1 expression in Jurkat cells was almost completely suppressed by a 5 0 -half-tRNA-type sgRNA up to 18 days.…”