The T Loop Structure Is Dispensable for Substrate Recognition by tRNase ZL

Abstract

tRNA 3-processing endoribonucleases (tRNase Z, or 3-tRNase; EC 3.1.26.11) are enzymes that remove 3-trailers from pre-tRNAs. An about 12-base-pair stem, a T loop-like structure, and a 3-trailer were considered to be the minimum requirements for recognition by the long form (tRNase ZL) of tRNase Z; tRNase ZL can recognize and cleave a micro-pre-tRNA or a hooker/target RNA complex that resembles a micro-pre-tRNA. We examined four hook RNAs containing systematically weakened T stems for directing target RNA cleavage by tRNase ZL. As expected, the cleavage efficiency decreased with the decrease in T stem stability, and to our surprise, even the hook RNA that forms no T stem-loopdirected slight cleavage of the target RNA, suggesting that the T stem-loop structure is important but dispensable for substrate recognition by tRNase ZL. To analyze the effect of the T loop on substrate recognition, we compared the cleavage reaction for a micro-pre-tRNA with that for a 12-base-pair double-stranded RNA, which is the same as the micro-pre-tRNA except for the lack of the T loop structure. The observed rate constant value for the double-stranded RNA was comparable with that for the micro-pre-tRNA, whereas the K d value for the complex with the double-stranded RNA was much higher than that for the complex with the micro-pretRNA. These results suggest that the T loop structure is not indispensable for the recognition, although the interaction between the T loop and the enzyme exists. Cleavage assays for such double-stranded RNA substrates of various lengths suggested that tRNase ZL can recognize and cleave double-stranded RNA substrates that are longer than 5 base pairs and shorter than 20 base pairs. We also showed that double-stranded RNA is not a substrate for the short form of tRNase Z.The genome of almost every organism encodes the short form (tRNase ZS) 1 and/or the long form (tRNase ZL) of tRNA 3Ј-processing endoribonucleases (tRNase Z, or 3Ј-tRNase; EC 3.1.26.11) (1-6). tRNase ZSs consist of 300 -400 amino acids, whereas tRNase ZLs contain 800 -900 amino acids. These enzymes are responsible for the removal of 3Ј-trailers from pretRNAs (Fig. 1A), which are transcribed as larger forms. In vitro tRNA 3Ј-processing assays have shown that in most cases, the enzymes cleave pre-tRNAs immediately downstream of a discriminator nucleotide, onto which the CCA residues are added to produce mature tRNA in vivo, whereas in some cases, additional cleavages occur 1 nt upstream or 1 or 2 nt downstream (7-14). As an exceptional case, tRNase Z from Thermotoga maritima cleaves pre-tRNAs containing the 74 CCA 76 sequence precisely after the A 76 residue to create the mature CCA 3Ј-termini of tRNA molecules (5).Bacterial and archaeal genomes contain a gene for the short form of tRNase Z only, whereas eukaryote genomes encode either only the long form or both the short and long forms (15). The C-terminal half region of tRNase ZL has high similarity to the whole region of tRNase ZS (1). These regions contain a well conserved histidine motif, whic...

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 .…”

“…Each linear-type sgRNA is a 14-nt sequence complementary to a sequence 5 0 to the desired cleavage site, and can form a 14-bp doublestranded RNA with the target RNA, which roughly corresponds to a combination of the acceptor and T stems. 13 An siRNA, siRLuc, targeting downstream to one of the sites, which has been shown to work very efficiently, 17 was used as a positive control, and an unrelated 14-nt linear RNA, sgGSKL2 (5 0 -CUUAGUCCA AGGAU-3 0 ), was used as a negative control.…”

“…10 A 14-nt linear RNA guided target RNA cleavage by tRNase Z L through forming a 14 base pair (bp) double-stranded RNA. 13 Amazingly, tRNase Z L can also recognize a 12-bp hairpin RNA complex between a separate target RNA and an RNA heptamer, and cleave the target. 11,12 In this case, however, the target sites are restricted to immediate downstream regions of stable hairpin structures resembling the T stem/loop.…”

“…5,8 We have demonstrated in vitro that mammalian tRNase Z L can cleave any target RNA at any desired site by recognizing a pre-tRNA-like or micro-pre-tRNAlike complex formed between the target RNA and artificial small-guide RNA (sgRNA). [9][10][11][12] sgRNA is divided into four categories: 5 0 -half-tRNA, 10 14-nucleotide (nt) linear RNA, 13 RNA heptamer 11,12 and hook RNA. 14 For example, with the aid of appropriate 5 0 -half-tRNAs, the two partial HIV-1 RNA targets ENV and GAG were cleaved site-specifically by tRNase Z L .…”

Human cytosolic tRNase ZL can downregulate gene expression through miRNA

“…1) [8][9][10][11][12][13]. sgRNA is divided into four groups, 5 0 -half-tRNA, $12-16-nt linear RNA, RNA heptamer, and hook RNA ( Fig.…”