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.

Figure 1

Structures of tRNase ZS and tRNase ZL. (A) Structural difference between tRNase ZS and tRNase ZL and distribution of their genes among various species. The locations of the pseudo-histidine motif, the Walker A motif and the histidine motif are indicated by a gray box, a hatched box and a black box, respectively. The presence of the ELAC1/2 genes in Homo sapiens (Hsa), A.thaliana (Ath), D.melanogaster (Dme), C.elegans (Cel), S.cerevisiae (Sce), Escherichia coli (Eco), T.maritima (Tma), Thermoplasma acidophilum (Tac) and Pyrobaculum aerophilum (Pae) is indicated by their GenBank/EMBL accession number. The sizes of these tRNase ZSs and tRNase ZLs are 280–363 and 789–942 amino acids, respectively. Asterisks denote that the tRNase Z activities are not yet confirmed experimentally. (B) Multiple protein sequence alignment of histidine motif regions in tRNase ZSs, histidine motif regions in the C-terminal domains of tRNase ZLs, and pseudo-histidine motif regions in the N-terminal domains of tRNase ZLs. The computer program ClustalW was used for the alignment. Identical amino acids are shaded in black and similar amino acids are in gray.

Figure 2

Tests of the long and short forms of tRNase Z from various organisms for in vitro tRNA 3′ processing activity. (A) A secondary structure of the human pre-tRNA^Arg R-6TUUU. Discontinuous and continuous arrows denote the primary cleavage sites by T.maritima tRNase ZS and by the other active enzymes, respectively. (B) The in vitro tRNA 3′ processing assays. Each recombinant protein (50 ng) or pig liver tRNase ZL (20 ng) was incubated with the fluorescein-labeled pre-tRNA^Arg (0.1 pmol) in the absence or presence of the N-terminal half (50 ng) of human tRNase ZL at 50°C for 15 min under the standard assay conditions. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea sequencing gel. The pre-tRNA^Arg and the primary 5′ cleavage products are indicated by a bar and arrows, respectively, with their nucleotide size. I, input RNA; P, pig; H, human; Y, yeast; Tm, T.maritima; Ec, E.coli; ΔN, the C-terminal half of human tRNase ZL; ΔC, the N-terminal half of human tRNase ZL.

Figure 3

Optimal conditions for in vitro pre-tRNA^Arg R-6TUUU processing by tRNase Zs from various organisms. (A) Relative cleavage activities of pig (circle), human (square) and yeast (triangle) tRNase ZLs at various temperatures. In each case, activities are normalized against the maximum activity as 100%. (B) Relative cleavage activities of pig, human, and yeast tRNase ZLs at various pHs. (C) Relative cleavage activities of pig, human, and yeast tRNase ZLs at various NaCl concentrations. (D) Relative cleavage activities of human (diamond), T.maritima (square), and E.coli (triangle) tRNase ZSs and the C-terminal half region (circle) of human tRNase ZL at various temperatures. (E) Relative cleavage activities of human, T.maritima, and E.coli tRNase ZSs and the C-terminal half region of human tRNase ZL at various pHs. (F) Relative cleavage activities of human, T.maritima, and E.coli tRNase ZSs and the C-terminal half region of human tRNase ZL at various NaCl concentrations.

Figure 4

Enzymatic activities of various tRNase Zs against the human pre-tRNA^Arg R-6TUUU at various concentrations of MgCl₂ or MnCl₂. (A) Relative cleavage activities of pig (circle), human (square) and yeast (triangle) tRNase ZLs at various MgCl₂ concentrations. In each case, activities are normalized against the maximum activity as 100%. (B) Relative cleavage activities of pig, human and yeast tRNase ZLs at various MnCl₂ concentrations. (C) Relative cleavage activities of human (diamond), T.maritima (square) and E.coli (triangle) tRNase ZSs and the C-terminal half region (circle) of human tRNase ZL at various MgCl₂ concentrations. (D) Relative cleavage activities of human, T.maritima, and E.coli tRNase ZSs at various MnCl₂ concentrations.

Figure 5

In vitro assays for micro-pre-tRNA^Arg cleavage by tRNase ZL and tRNase ZS from various organisms. (A) A secondary structure of the human micro-pre-tRNA^Arg R-ATM5. An arrow denotes the primary cleavage site by tRNase Z. (B) The in vitro cleavage assays. Each recombinant protein (50 ng) or pig liver tRNase ZL (20 ng) was incubated with the fluorescein-labeled human micro-pre-tRNA^Arg (0.1 pmol) in the absence or presence of the N-terminal half (50 ng) of human tRNase ZL at 50°C for 15 min under the standard assay conditions. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea gel. The micro-pre-tRNA^Arg and the primary 5′ cleavage product are indicated by a bar and an arrow, respectively, with their nucleotide size. L, the alkaline ladder of the fluorescein-labeled substrate; I, input RNA; P, pig; H, human; Y, yeast; Tm, T.maritima; Ec, E.coli; ΔN, the C-terminal half of human tRNase ZL; ΔC, the N-terminal half of human tRNase ZL.

Figure 6

The RNase 65 assays for various tRNase ZLs and tRNase ZSs. (A) A secondary structure of the complex of the target RNA and the 3′-truncated tRNA. The 5′-terminal sequence 5′-GAAUACGCAUGCUAGC-3′ and the 3′-terminal sequence 5′-AAAGCUUGAUGU-3′ are omitted in the target RNA. An arrow denotes the primary cleavage site by tRNase ZL. (B) The RNase 65 assays. Each recombinant protein (50 ng) or pig liver tRNase ZL (20 ng) was incubated with the fluorescein-labeled target RNA (0.1 pmol) in the presence of the unlabeled 3′-truncated tRNA (5 pmol) in the absence or presence of the N-terminal half (50 ng) of human tRNase ZL at 50°C for 15 min under the standard assay conditions. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea sequencing gel. The target RNA and the primary 5′ cleavage product are indicated by a bar and an arrow, respectively, with their nucleotide size. L, the alkaline ladder of the fluorescein-labeled target RNA; I, input RNA; P, pig; H, human; Y, yeast; Tm, T.maritima; Ec, E.coli; ΔN, the C-terminal half of human tRNase ZL; ΔC, the N-terminal half of human tRNase ZL.

Figure 7

Hook RNA-guided specific RNA cleavage by the long form of tRNase Z from various organisms. (A) A secondary structure of the complex of the target RNA and the hook RNA. The 5′ terminal sequence 5′-GAAUACGCAUGCUAGC-3′ and the 3′ terminal sequence 5′-AAAGCUUGAUGU-3′ are omitted in the target RNA. An arrow denotes the primary cleavage site by tRNase Z. (B) The specific RNA cleavage assays. Each recombinant protein (50 ng) or pig liver tRNase ZL (20 ng) was incubated with the fluorescein-labeled target RNA (0.1 pmol) in the presence of the unlabeled hook RNA (5 pmol) in the absence or presence of the N-terminal half (50 ng) of human tRNase ZL at 50°C for 15 min under the standard assay conditions. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea sequencing gel. The target RNA and the primary 5′ cleavage product are indicated by a bar and an arrow, respectively, with their nucleotide size. L, the alkaline ladder of the fluorescein-labeled target RNA; I, input RNA; P, pig; H, human; Y, yeast; Tm, T.maritima; Ec, E.coli; ΔN, the C-terminal half of human tRNase ZL; ΔC, the N-terminal half of human tRNase ZL.