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.     

Growth Inhibition of Head and Neck Squamous Cell Carcinoma Cells by sgRNA Targeting the Cyclin D1 mRNA Based on TRUE Gene Silencing

Head and neck squamous cell carcinoma (HNSCC) exhibits increased expression of cyclin D1 (CCND1). Previous studies have shown a correlation between poor prognosis of HNSCC and cyclin D1 overexpression. tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is one of the RNA-mediated gene expression control technologies that have therapeutic potential. This technology is based on a unique enzymatic property of mammalian tRNase ZL, which is that it can cleave any target RNA at any desired site by recognizing a pre-tRNA-like complex formed between the target RNA and an artificial small guide RNA (sgRNA). In this study, we designed several sgRNAs targeting human cyclin D1 mRNA to examine growth inhibition of HNSCC cells. Transfection of certain sgRNAs decreased levels of cyclin D1 mRNA and protein in HSC-2 and HSC-3 cells, and also inhibited their proliferation. The combination of these sgRNAs and cisplatin showed more than additive inhibition of cancer cell growth. These findings demonstrate that TRUE gene silencing of cyclin D1 leads to inhibition of the growth of HNSCC cells and suggest that these sgRNAs alone or combined with cisplatin may be a useful new therapy for HNSCCs.     

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.     

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.     

Suppression of HIV-1 replication by a combination of endonucleolytic ribozymes (RNase P and tRNnase ZL)

We examined the combinatorial action of RNase P and tRNase ZL-mediated specific inhibition of HIV-1 in cultured cells. We designed two short extra guide sequences (sEGS) that specifically recognize the tat and vifregions of HIV-1 mRNA and mediate the subsequent cleavage of hybridized mRNA by the RNase P and tRNase ZL components. We constructed an RNase P and tRNase ZL-associated vif and tat sEGS expression vector; which used the RNA-polymerase III dependent U6 promoter, as an expression cassette for EGS. Together, the RNase P and tRNase ZL-associated sEGS molecules allow more efficient suppression of HIV-1 mRNA production when separately applied. The possibilities offered by the vector to encode sEGS will provide a powerful tool for gene therapy.     

Intracellular mRNA cleavage by 3' tRNase under the direction of 2'-O-methyl RNA heptamers

Mammalian tRNA 3′ processing endoribonuclease (3′-tRNase) can cleave any RNA at any site under the direction of small guide RNA (sgRNA) in vitro. sgRNAs can be as short as heptamers, which are much smaller than small interfering RNAs of ~21 nt. Together with such flexibility in substrate recognition, the ubiquity and the constitutive expression of 3′-tRNase have suggested that this enzyme can be utilized for specific cleavage of cellular RNAs by introducing appropriate sgRNAs into living cells. Here we demonstrated that the expression of chloramphenicol acetyltransferase can be downregulated by an appropriate sgRNA which is introduced into Madin–Darby canine kidney epithelial cells as an expression plasmid or a synthetic 2′-O-methyl RNA. We also showed that 2′-O-methyl RNA heptamers can attack luciferase mRNAs with a high specificity and induce 3′-tRNase-mediated knock-down of the mRNAs in 293 cells. Furthermore, the MTT cell viability assay suggested that an RNA heptamer can downregulate the endogenous Bcl-2 mRNA in Sarcoma 180 cells. This novel sgRNA/3′-tRNase strategy for destroying specific cellular RNAs may be utilized for therapeutic applications.     

Suppression of HIV-1 Replication by a Combination of Endonucleolytic Ribozymes (RNase P and tRNase ZL)

We examined the combinatorial action of RNase P and tRNase ZL-mediated specific inhibition of HIV-1 in cultured cells. We designed two short extra guide sequences (sEGS) that specifically recognize the tat and vif regions of HIV-1 mRNA and mediate the subsequent cleavage of hybridized mRNA by the RNase P and tRNase ZL components. We constructed an RNase P and tRNase ZL-associated vif and tat sEGS expression vector, which used the RNA-polymerase III dependent U6 promoter, as an expression cassette for EGS. Together, the RNase P and tRNase ZL-associated sEGS molecules allow more efficient suppression of HIV-1 mRNA production when separately applied. The possibilities offered by the vector to encode sEGS will provide a powerful tool for gene therapy.     

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.     

Human cytosolic tRNase Z can downregulate gene expression through miRNA

A long form of tRNase Z (tRNase Z^L) can cleave any target RNA at any desired site under the direction of artificial small guide RNA including ∼25-nucleotide hook-shaped RNA. Here we show that human miR-103 can form a hook structure to guide target RNA cleavage by human cytosolic tRNase Z^L in vitro. In vivo analyses using luciferase mRNAs modified to contain miR-103 target sequences in their 3′ untranslated regions indicated that miR-103 downregulates gene expression through directing mRNA cleavage by tRNase Z^L. The present data suggest the possibility that human cytosolic tRNase Z^L modulates gene expression through a subset of microRNAs in the cells.     

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.     

The T loop structure is dispensable for substrate recognition by tRNase ZL

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-loop-directed 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-pre-tRNA. 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.     

RNA heptamers that direct RNA cleavage by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can recognize and cleave any target RNA that forms a precursor tRNA-like complex with another RNA. Various sets of RNA molecules were tested to identify the smallest RNA that can direct target RNA cleavage by 3' tRNase. A 3' half tRNAArgwas cleaved efficiently by 3' tRNase in the presence of small 5' half tRNAArgvariants, the D stem-loop region of which was partially deleted. Remarkably, 3' tRNase also cleaved the 3' half tRNAArgin the presence of a 7 nt 5' tRNAArg composed only of the acceptor stem region with a catalytic efficiency comparable with that of cleavage directed by an intact 5' half tRNAArg. The catalytic efficiency of cleavage directed by the heptamer decreased as the stability of the T stem-loop structures of 3' half tRNAArg variants decreased. No heptamer-directed cleavage of a 3' half tRNAArg without T stem base pairs was detected. A heptamer also directed cleavage of an HIV-1 RNA containing a stable hairpin structure. These findings suggest that in the presence of an RNA heptamer, 3' tRNase can discriminate and eliminate target RNAs that possess a stable hairpin adjacent to the heptamer binding sequence from a large complex RNA pool.     

Arabidopsis Encodes Four tRNase Z Enzymes

Functional transfer RNA (tRNA) molecules are a prerequisite for protein biosynthesis. Several processing steps are required to generate the mature functional tRNA from precursor molecules. Two of the early processing steps involve cleavage at the tRNA 5′ end and the tRNA 3′ end. While processing at the tRNA 5′ end is performed by RNase P, cleavage at the 3′ end is catalyzed by the endonuclease tRNase Z. In eukaryotes, tRNase Z enzymes are found in two versions: a short form of about 250 to 300 amino acids and a long form of about 700 to 900 amino acids. All eukaryotic genomes analyzed to date encode at least one long tRNase Z protein. Of those, Arabidopsis (Arabidopsis thaliana) is the only organism that encodes four tRNase Z proteins, two short forms and two long forms. We show here that the four proteins are distributed to different subcellular compartments in the plant cell: the nucleus, the cytoplasm, the mitochondrion, and the chloroplast. One tRNase Z is present only in the cytoplasm, one protein is found exclusively in mitochondria, while the third one has dual locations: nucleus and mitochondria. None of these three tRNase Z proteins is essential. The fourth tRNase Z protein is present in chloroplasts, and deletion of its gene results in an embryo-lethal phenotype. In vitro analysis with the recombinant proteins showed that all four tRNase Z enzymes have tRNA 3′ processing activity. In addition, the mitochondrial tRNase Z proteins cleave tRNA-like elements that serve as processing signals in mitochondrial mRNA maturation.     

RNAi-mediated inhibition of HIV-1 by targeting partially complementary viral sequences

Potent antiviral RNAi can be induced by intracellular expression of short hairpin RNAs (shRNAs) and artificial microRNAs (miRNAs). Expression of shRNA and miRNA results in target mRNA degradation (perfect base pairing) or translational repression (partial base pairing). Although efficient inhibition can be obtained, error-prone viruses such as human immunodeficiency virus type 1 (HIV-1) can escape from RNAi-mediated inhibition by mutating the target sequence. Recently, artificial miRNAs have been shown to be potent RNAi inducers due to their efficient processing by the RNAi machinery. Furthermore, miRNAs may be more proficient in suppressing imperfect targets than shRNAs. In this study, we tested the knockdown efficiency of miRNAs and shRNAs against wild-type and RNAi-escape HIV-1 variants with one or two mutations in the target sequence. ShRNAs and miRNAs can significantly inhibit the production of HIV-1 variants with mutated target sequences in the open reading frame. More pronounced mutation-tolerance was measured for targets in the 3′ untranslated region (3′ UTR). Partially complementary sequences within the 3′ UTR of the HIV-1 RNA genome efficiently act as target sites for miRNAs and shRNAs. These data suggest that targeting imperfect target sites by antiviral miRNAs or shRNAs provides an alternative RNAi approach for inhibition of pathogenic viruses.     

Kinetics of the CRISPR-Cas9 effector complex assembly and the role of 3′-terminal segment of guide RNA

CRISPR-Cas9 is widely applied for genome engineering in various organisms. The assembly of single guide RNA (sgRNA) with the Cas9 protein may limit the Cas9/sgRNA effector complex function. We developed a FRET-based assay for detection of CRISPR–Cas9 complex binding to its targets and used this assay to investigate the kinetics of Cas9 assembly with a set of structurally distinct sgRNAs. We find that Cas9 and isolated sgRNAs form the effector complex efficiently and rapidly. Yet, the assembly process is sensitive to the presence of moderate concentrations of non-specific RNA competitors, which considerably delay the Cas9/sgRNA complex formation, while not significantly affecting already formed complexes. This observation suggests that the rate of sgRNA loading into Cas9 in cells can be determined by competition between sgRNA and intracellular RNA molecules for the binding to Cas9. Non-specific RNAs exerted particularly large inhibitory effects on formation of Cas9 complexes with sgRNAs bearing shortened 3′-terminal segments. This result implies that the 3′-terminal segment confers sgRNA the ability to withstand competition from non-specific RNA and at least in part may explain the fact that use of sgRNAs truncated for the 3′-terminal stem loops leads to reduced activity during genomic editing.     

Structural insights into the targeting of mRNA GU-rich elements by the three RRMs of CELF1

The CUG-BP, Elav-like family (CELF) of RNA-binding proteins control gene expression at a number of different levels by regulating pre-mRNA splicing, deadenylation and mRNA stability. We present structural insights into the binding selectivity of CELF member 1 (CELF1) for GU-rich mRNA target sequences of the general form 5′-UGUN_xUGUN_yUGU and identify a high affinity interaction (K_d ∼ 100 nM for x = 2 and y = 4) with simultaneous binding of all three RNA recognition motifs within a single 15-nt binding element. RNA substrates spin-labelled at either the 3′ or 5′ terminus result in differential nuclear magnetic resonance paramagnetic relaxation enhancement effects, which are consistent with a non-sequential 2-1-3 arrangement of the three RNA recognition motifs on UGU sites in a 5′ to 3′ orientation along the RNA target. We further demonstrate that CELF1 binds to dispersed single-stranded UGU sites at the base of an RNA hairpin providing a structural rationale for recognition of CUG expansion repeats and splice site junctions in the regulation of alternative splicing.     

Conservation of RNA structures enables TNV and BYDV 5' and 3' elements to cooperate synergistically in cap-independent translation

The subgenomic RNA 2 of tobacco necrosis virus A (TNV sgRNA2) encodes the viral coat protein, is unpolyadenylated and presumably uncapped. Here, we show that TNV sgRNA2 is translated cap independently. This cap-independent translation requires the leader and a 140 nt element of the trailer both in wheat germ extract and in tobacco protoplasts. Similar to barley yellow dwarf virus (BYDV), the TNV 5′ and 3′ elements stimulate translation synergistically. Computer-aided phylogenetic analysis of the secondary structure of the TNV trailer revealed that the 3′ translation element is part of a major conserved stem–loop that contains similarities to structures in the BYDV 3′ translation element. These data suggest that the translation mechanisms of TNV sgRNA2 and BYDV RNA are related. To further characterize this relationship, we tested whether cooperativity exists between TNV sgRNA2 and BYDV 5′ and 3′ elements. We found that the TNV sgRNA2 5′ element stimulates translation synergistically with the BYDV 3′ element in vitro. This finding is the first evidence for conservation of structures that enable a 5′–3′ interaction stimulating cap-independent translation.