Masayuki Nashimoto

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

A long form (tRNase ZL) 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 ZL in the cells. Here we show that human cytosolic tRNase ZL modulates gene expression through 5′-half-tRNA. We found that 5′-half-tRNAGlu, which co-immunoprecipitates with tRNase ZL, 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 ZL guided by 5′-half-tRNAGlu. Furthermore, the DNA microarray data suggested that tRNase ZL is likely to be involved in the p53 signaling pathway and apoptosis.

Crystal Structure of the tRNA 3′ Processing Endoribonuclease tRNase Z from Thermotoga maritima

The maturation of the tRNA 3′ end is catalyzed by a tRNA 3′ processing endoribonuclease named tRNase Z (RNase Z or 3′-tRNase) in eukaryotes, Archaea, and some bacteria. The tRNase Z generally cuts the 3′ extra sequence from the precursor tRNA after the discriminator nucleotide. In contrast, Thermotoga maritima tRNase Z cleaves the precursor tRNA precisely after the CCA sequence. In this study, we determined the crystal structure of T. maritima tRNase Z at 2.6-Å resolution. The tRNase Z has a four-layer αβ/βα sandwich fold, which is classified as a metallo-β-lactamase fold, and forms a dimer. The active site is located at one edge of the β-sandwich and is composed of conserved motifs. Based on the structure, we constructed a docking model with the tRNAs that suggests how tRNase Z may recognize the substrate tRNAs.

Bone morphogenetic protein-2 enhances Wnt/β-catenin signaling-induced osteoprotegerin expression

Wnt/β‐catenin signaling plays an important role in the developing skeletal system. Our previous studies demonstrated that Wnt/β‐catenin signaling inhibits the ability of bone morphogenetic protein (BMP)‐2 to suppress myotube formation in the multipotent mesenchymal cell line C2C12 and that this inhibition is mediated by Id1. In this study, we examined the role of intracellular signaling by Wnt/β‐catenin and BMP‐2 in regulating the expression of osteoprotegerin (OPG) and of the receptor activator of NFκB ligand (RANKL). OPG expression was induced by Wnt/β‐catenin signaling in C2C12 cells and osteoblastic MC3T3‐E1 cells. Silencing of glycogen synthase kinase‐3β also increased OPG expression. In contrast, R expression was suppressed by Wnt/β‐catenin signaling. In a transfection assay, β‐catenin induced the activity of a reporter gene, a 1.5 kb fragment of the 5′‐flanking region of the OPG gene. Deletion and mutation analysis revealed that Wnt/β‐catenin signaling regulates transcription of OPG via a promoter region containing two Wnt/β‐catenin responsive sites. BMP‐2 enhanced Wnt/β‐catenin‐dependent transcriptional activation of the OPG promoter. In response to BMP‐2 stimulation, Smad 1 and 4 interacted with Wnt/β‐catenin responsive sites. These results show that the regulation of OPG expression is mediated through two transcription pathways that involve the OPG promoter.

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

We have been developing a unique system for the downregulation of a gene expression through cutting a specific mRNA by the long form of tRNA 3′-processing endoribonuclease (tRNase ZL) under the direction of small-guide RNA (sgRNA). However, the efficacy of this system and the involvement of tRNase ZL in the living cells were not clear. Here we show, by targeting the exogenous luciferase gene, that the efficacy of the sgRNA/tRNase ZL method can become comparable to that of the RNA interference technology and that the gene silencing is owing to tRNase ZL directed by sgRNA not owing to a simple antisense effect. We also show that tRNase ZL together with sgRNA can downregulate expression of the endogenous human genes Bcl-2 and glycogen synthase kinase-3β by degrading their mRNAs in cell culture. Furthermore, we demonstrate that a gene expression in the livers of postnatal mice can be inhibited by an only seven-nucleotide sgRNA. These data suggest that sgRNA might be utilized as therapeutic agents to treat diseases such as cancers and AIDS.

Bone morphogenetic protein-2 down-regulates miR-206 expression by blocking its maturation process

MicroRNAs (miRNAs) are small non-coding RNAs that are emerging as important post-transcriptional gene regulators. miR-206 is unique in that it is expressed only in skeletal muscle, including the myoblastic C2C12 cell line. In C2C12 cells, miR-206 expression was reduced dramatically after bone morphogenetic protein (BMP)-2 treatment. The down-regulation of miR-206 expression was also observed after co-transfection with constitutively-active Smad1 and Smad4, which are the intracellular signaling molecules of the BMP pathway. BMP-2 also reduced miR-206 expression in the presence of alpha-amanitin in a similar manner to that in the absence of alpha-amanitin. Moreover, the expression of pri-miR-206 was increased upon BMP-2 treatment for 6h compared to that in the absence of BMP-2. These results suggested that BMP-2 down-regulates miR-206 expression at the post-transcriptional level, by inhibiting the processing of pri-miR-206 into mature miR-206, and that BMP-2 could regulate miRNA biogenesis by a novel mechanism.

Structural basis for the binding of IRES RNAs to the head of the ribosomal 40S subunit

Some viruses exploit internal initiation for their propagation in the host cell. This type of initiation is facilitated by structured elements (internal ribosome entry site, IRES) upstream of the initiator AUG and requires only a reduced number of canonical initiation factors. An important example are IRES of the virus family Dicistroviridae that bind to the inter-subunit side of the small ribosomal 40S subunit and lead to the formation of elongation-competent 80S ribosomes without the help of any initiation factor. Here, we present a comprehensive functional and structural analysis of eukaryotic-specific ribosomal protein rpS25 in the context of this type of initiation and propose a structural model explaining the essential involvement of rpS25 for hijacking the ribosome.

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.

Anomalous RNA substrates for mammalian tRNA 3′ processing endoribonuclease

Mammalian tRNA 3′ processing endoribonuclease (3′ tRNase) is an enzyme responsible for the removal of a 3′ trailer from pre‐tRNA. The enzyme can also recognize and cleave any target RNA that forms a pre‐tRNA‐like complex with another RNA. To investigate the interaction between 3′ tRNase and substrates, we tested various anomalous pre‐tRNA‐like complexes for cleavage by pig 3′ tRNase. We examined how base mismatches in the acceptor stem affect 3′ tRNase cleavage of RNA complexes, and found that even one base mismatch in the acceptor stem drastically reduces the cleavage efficiency. Mammalian 3′ tRNase was able to recognize complexes between target RNAs and 5′‐half tDNAs, and cleave the target RNAs, although inefficiently, whereas the enzyme had no activity to cleave phosphodiester bonds of DNA. A relatively long RNA target, the Escherichia coli chloramphenicol acetyltransferase (CAT) mRNA, was cleaved by 3′ tRNase in the presence of appropriate 5′‐half tRNAs. We also demonstrated that an RNA complex of lin‐4 and lin‐14 from Caenorhabditis elegans can be recognized and cleaved by pig 3′ tRNase.

Human cytosolic tRNase ZL can downregulate gene expression through miRNA

A long form of tRNase Z (tRNase ZL) 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 ZL 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 ZL. The present data suggest the possibility that human cytosolic tRNase ZL modulates gene expression through a subset of microRNAs in the cells.

Inhibition of vascular endothelial growth factor expression by TRUE gene silencing

Pathogenic angiogenesis in various diseases including cancer, autoimmune diseases, and age-related macular degeneration is thought to be regressed with anti-angiogenic drugs. TRUE gene silencing is a new technology to eliminate a specific mRNA using synthetic sgRNA and cellular tRNase Z(L). To discover anti-angiogenic sgRNAs, we applied TRUE silencing to the VEGF gene. We examined eight sgRNAs for efficacy in targeting exogenous human VEGF mRNA. Many of them worked efficiently in 293 and HeLa cells. Two of them downregulated the endogenous VEGF gene expression in HeLa cells very efficiently, and the efficacy of these two sgRNAs surpassed that of siRNA extremely.