The flexible arm of tRNase Z is not essential for pre-tRNA binding but affects cleavage site selection

tRNase Z is an enzyme responsible for removing a 3′ trailer from pre-tRNA. Although most tRNase Zs cleave pre-tRNAs immediately after the discriminator nucleotide with the exception of Thermotoga maritima tRNase Z, which cleaves after the ₇₄CCA₇₆ sequence, our knowledge was limited about how the cleavage site in pre-tRNA is selected. Bacterial tRNase Zs contain a unique domain termed flexible arm, which extends from the core domain. Using various tRNase Z variants, here we examined how the flexible arm affects the cleavage site selection. T. maritima tRNase Z variants with modified flexible arms shifted the cleavage site and a Bacillus subtilis tRNase Z variant with no flexible arm showed an anomalous cleavage activity. Some of the T. maritima/B. subtilis chimeric enzymes had both properties: they recognized ₇₄CCA₇₆-containing pre-tRNA and cleaved it after the discriminator. Taken together, the present data indicate that the flexible arm is not essential for pre-tRNA binding but affects the cleavage site selection probably by pushing the distal region of the T arm in such a way that the discriminator nucleotide becomes closer to the catalytic site.     

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

tRNase Z is an essential endonuclease responsible for tRNA 3′-end maturation. tRNase Z exists in a short form (tRNase Z^S) and a long form (tRNase Z^L). Prokaryotes have only tRNase Z^S, whereas eukaryotes can have both forms of tRNase Z. Most eukaryotes characterized thus far, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and humans, contain only one tRNase Z^L gene encoding both nuclear and mitochondrial forms of tRNase Z^L. In contrast, Schizosaccharomyces pombe contains two essential tRNase Z^L genes (trz1 and trz2) encoding two tRNase Z^L proteins, which are targeted to the nucleus and mitochondria, respectively. Trz1 protein levels are notably higher than Trz2 protein levels. Here, using temperature-sensitive mutants of trz1 and trz2, we provide in vivo evidence that trz1 and trz2 are involved in nuclear and mitochondrial tRNA 3′-end processing, respectively. In addition, trz2 is also involved in generation of the 5′-ends of other mitochondrial RNAs, whose 5′-ends coincide with the 3′-end of tRNA. Thus, our results provide a rare example showing partitioning of the nuclear and mitochondrial tRNase Z^L activities between two different proteins in S. pombe. The evolution of two tRNase Z^L genes and their differential expression in fission yeast may avoid toxic off-target effects.     

Modulation of Gene Expression by Human Cytosolic tRNase ZL through 5′-Half-tRNA

A long form (tRNase Z^L) of tRNA 3′ processing endoribonuclease (tRNase Z, or 3′ tRNase) can cleave any target RNA at any desired site under the direction of artificial small guide RNA (sgRNA) that mimics a 5′-half portion of tRNA. Based on this enzymatic property, a gene silencing technology has been developed, in which a specific mRNA level can be downregulated by introducing into cells a synthetic 5′-half-tRNA that is designed to form a pre-tRNA-like complex with a part of the mRNA. Recently 5′-half-tRNA fragments have been reported to exist stably in various types of cells, although little is know about their physiological roles. We were curious to know if endogenous 5′-half-tRNA works as sgRNA for tRNase Z^L in the cells. Here we show that human cytosolic tRNase Z^L modulates gene expression through 5′-half-tRNA. We found that 5′-half-tRNA^Glu, which co-immunoprecipitates with tRNase Z^L, exists predominantly in the cytoplasm, functions as sgRNA in vitro, and downregulates the level of a luciferase mRNA containing its target sequence in human kidney 293 cells. We also demonstrated that the PPM1F mRNA is one of the genuine targets of tRNase Z^L guided by 5′-half-tRNA^Glu. Furthermore, the DNA microarray data suggested that tRNase Z^L is likely to be involved in the p53 signaling pathway and apoptosis.     

Potential Small Guide RNAs for tRNase ZL from Human Plasma,Peripheral Blood Mononuclear Cells,and Cultured Cell Lines

Several pieces of evidence suggest that small RNA degradation products together with tRNase Z^L appear to form another layer of the whole gene regulatory network. The degraded RNA such as a 5′-half-tRNA and an rRNA fragment function as small guide RNA (sgRNA) to guide the enzyme to target RNA. We were curious whether there exist RNAs in plasma that can function as sgRNAs for tRNase Z^L, whether these RNAs are working as signaling molecules between cells to fulfill physiological roles, and whether there are any differences in plasma sgRNA species and levels between normal and pathological conditions. Here, we analyzed small plasma RNAs from three healthy persons and three multiple myeloma patients for potential sgRNAs by deep sequencing. We also examined small RNAs from peripheral blood mononuclear cells (PBMC) of three healthy persons and three myeloma patients and from various cultured human cell lines for sgRNAs. We found that read-number distribution patterns of plasma and PBMC RNAs differ between persons in the range of 5–40 nt and that there are many RNA species that exist significantly more or less abundantly in the plasma or PBMC of the myeloma patients than those of the healthy persons. Furthermore, we found that there are many potential sgRNAs in the 5–40-nt RNAs and that, among them, a 31-nt RNA fragment derived from 94-nt Y4-RNA, which can function as a 5′-half-tRNA-type sgRNA, is overwhelmingly abundant in the plasma of 2/3 of the examinees. These observations suggest that the gene regulatory network via tRNase Z^L and sgRNA may be extended intercellularly.     

Elimination of specific miRNAs by naked 14-nt sgRNAs

tRNase Z(L)-utilizing efficacious gene silencing (TRUE gene silencing) is a newly developed technology to suppress mammalian gene expression. TRUE gene silencing works on the basis of a unique enzymatic property of mammalian tRNase Z(L), which is that it can recognize a pre-tRNA-like or micro-pre-tRNA-like complex formed between target RNA and artificial small guide RNA (sgRNA) and can cleave any target RNA at any desired site. There are four types of sgRNA, 5'-half-tRNA, RNA heptamer, hook RNA, and ~14-nt linear RNA. Here we show that a 14-nt linear-type sgRNA against human miR-16 can guide tRNase Z(L) cleavage of miR-16 in vitro and can downregulate the miR-16 level in HEK293 cells. We also demonstrate that the 14-nt sgRNA can be efficiently taken up without any transfection reagents by living cells and can exist stably in there for at least 24 hours. The naked 14-nt sgRNA significantly reduced the miR-16 level in HEK293 and HL60 cells. Three other naked 14-nt sgRNAs against miR-142-3p, miR-206, and miR-19a/b are also shown to downregulate the respective miRNA levels in various mammalian cell lines. Our observations suggest that in general we can eliminate a specific cellular miRNA at least by ~50% by using a naked 14-nt sgRNA on the basis of TRUE gene silencing.     

The heptamer sgRNA targeting the human OCT4 mRNA can upregulate the OCT4 expression

TRUE gene silencing is one of the gene suppression technologies. This technology exploits the enzymatic property of the tRNA 3′ processing endoribonuclease tRNase Z^L, which is that it can cleave a target RNA under the direction of a small guide RNA (sgRNA). We have been working on the development of therapeutic sgRNAs for hematological malignancies. In the course of an experiment to examine the ability of the heptamer-type sgRNA H15792 targeting the OCT4 mRNA to differentiate human amnion stem cells, we observed unexpectedly that the amnion cells exhibited a morphology resembling initialized cells. Here we investigated the effect of H15792 on human HL60 leukemia cells, and found that H15792 can upregulate the OCT4 expression and the expression of alkaline phosphatase in the cells.     

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

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.     

Expanding the utility of heptamer-type sgRNA for TRUE gene silencing

tRNase Z(L)-utilizing efficacious gene silencing (TRUE gene silencing) is a novel technology for suppressing gene expression. TRUE gene silencing is based on a unique enzymatic property of mammalian tRNase Z(L), which is that it can cleave any target RNA at any desired site by recognizing a pre-tRNA-like or micro-pre-tRNA-like complex formed between the target RNA and artificial small guide RNA (sgRNA). sgRNA is divided into four groups, 5'-half-tRNA, RNA heptamer, hook RNA, and ～14-nt linear RNA. One of the final destinations of TRUE gene silencing is to generate cancer therapeutic sgRNAs, and from a pharmacological point of view, heptamer-type sgRNA appears to be the most appropriate for this purpose. In this paper, we present two strategies to expand the utility of heptamer-type sgRNA: one is about locked nucleic acid (LNA) modifications of heptamers and the other is about usage of double heptamers. We showed that RNA heptamers with LNA modifications can work as sgRNA in vitro and in vivo. We also demonstrated that two consecutively aligned heptamers can guide target RNA cleavage by human tRNase Z(L) as efficiently as a corresponding 14-nt sgRNA in vitro and that a double heptamer can work much better than a 14-nt sgRNA in vivo.     

tRNA 3′ processing in yeast involves tRNase Z,Rex1, and Rrp6

Mature tRNA 3′ ends in the yeast Saccharomyces cerevisiae are generated by two pathways: endonucleolytic and exonucleolytic. Although two exonucleases, Rex1 and Rrp6, have been shown to be responsible for the exonucleolytic trimming, the identity of the endonuclease has been inferred from other systems but not confirmed in vivo. Here, we show that the yeast tRNA 3′ endonuclease tRNase Z, Trz1, is catalyzing endonucleolytic tRNA 3′ processing. The majority of analyzed tRNAs utilize both pathways, with a preference for the endonucleolytic one. However, 3′-end processing of precursors with long 3′ trailers depends to a greater extent on Trz1. In addition to its function in the nucleus, Trz1 processes the 3′ ends of mitochondrial tRNAs, contributing to the general RNA metabolism in this organelle.     

Inhibition of HIV-1 gene expression by retroviral vector-mediated small-guide RNAs that direct specific RNA cleavage by tRNase ZL

The tRNA 3′-processing endoribonuclease (tRNase Z or 3′ tRNase; EC 3.1.26.11) is an essential enzyme that removes the 3′ trailer from pre-tRNA. The long form (tRNase ZL) can cleave a target RNA in vitro at the site directed by an appropriate small-guide RNA (sgRNA). Here, we investigated whether this sgRNA/tRNase ZL strategy could be applied to gene therapy for AIDS. We tested the ability of four sgRNA-expression plasmids to inhibit HIV-1 gene expression in COS cells, using a transient-expression assay. The three sgRNAs guide inhibition of HIV-1 gene expression in cultured COS cells. Analysis of the HIV-1 mRNA levels suggested that sgRNA directed the tRNase ZL to mediate the degradation of target RNA. The observation that sgRNA was localized primarily in nuclei suggests that tRNase ZL cleaves the HIV-1 mRNA when complexed with sgRNA in this location. We also examined the ability of two retroviral vectors expressing sgRNA to suppress HIV-1 expression in HIV-1-infected Jurkat T cells. sgRNA-SL4 suppressed HIV-1 expression almost completely in infected cells for up to 18 days. These results suggest that the sgRNA/tRNase ZL approach is effective in downregulating HIV-1 gene expression.     

CFTR mediates bicarbonate-dependent activation of miR-125b in preimplantation embryo development

Although HCO₃⁻ is known to be required for early embryo development, its exact role remains elusive. Here we report that HCO₃⁻ acts as an environmental cue in regulating miR-125b expression through CFTR-mediated influx during preimplantation embryo development. The results show that the effect of HCO₃⁻ on preimplantation embryo development can be suppressed by interfering the function of a HCO₃⁻-conducting channel, CFTR, by a specific inhibitor or gene knockout. Removal of extracellular HCO₃⁻ or inhibition of CFTR reduces miR-125b expression in 2 cell-stage mouse embryos. Knockdown of miR-125b mimics the effect of HCO₃⁻ removal and CFTR inhibition, while injection of miR-125b precursor reverses it. Downregulation of miR-125b upregulates p53 cascade in both human and mouse embryos. The activation of miR-125b is shown to be mediated by sAC/PKA-dependent nuclear shuttling of NF-κB. These results have revealed a critical role of CFTR in signal transduction linking the environmental HCO₃⁻ to activation of miR-125b during preimplantation embryo development and indicated the importance of ion channels in regulation of miRNAs.     

ADAR1 regulates ARHGAP26 gene expression through RNA editing by disrupting miR-30b-3p and miR-573 binding

Rho GTPase activating protein 26 (ARHGAP26) is a negative regulator of the Rho family that converts the small G proteins RhoA and Cdc42 to their inactive GDP-bound forms. It is essential for the CLIC/GEEC endocytic pathway, cell spreading, and muscle development. The present study shows that ARHGAP26 mRNA undergoes extensive A-to-I RNA editing in the 3′ UTR that is specifically catalyzed by ADAR1. Furthermore, the mRNA and protein levels of ARHGAP26 were decreased in cells in which ADAR1 was knocked down. Conversely, ADAR1 overexpression increased the abundance of ARHGAP26 mRNA and protein. In addition, we found that both miR-30b-3p and miR-573 target the ARHGAP26 gene and that RNA editing of ARHGAP26 mediated by ADAR1 abolished the repression of its expression by miR-30b-3p or miR-573. When ADAR1 was overexpressed, the reduced abundance of ARHGAP26 protein mediated by miR-30b-3p or miR-573 was rescued. Importantly, we also found that knocking down ADAR1 elevated RhoA activity, which was consistent with the reduced level of ARHGAP26. Conversely, when ADAR1 was overexpressed, the amount of RhoA-GTP decreased. The similar expression patterns of ARHGAP26 and ADAR1 in human tissue samples further confirmed our findings. Taken together, our results suggest that ADAR1 regulates the expression of ARHGAP26 through A-to-I RNA editing by disrupting the binding of miR-30b-3p and miR-573 within the 3′ UTR of ARHGAP26. This study provides a novel insight into the mechanism by which ADAR1 and its RNA editing function regulate microRNA-mediated modulation of target genes.