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Modulation of gene expression by human cytosolic tRNase Z(L) through 5'-half-tRNA.

Elbarbary RA, Takaku H, Uchiumi N, Tamiya H, Abe M, Takahashi M, Nishida H, Nashimoto M - PLoS ONE (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan.

ABSTRACT
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.

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Related in: MedlinePlus

Human tRNase ZL exists ubiquitously in cells.Fluorescent microscopic analyses of human 293 kidney cells, A549 epithelial lung cells, HepG2 hepatoma cells, and IMR90 lung fibroblasts. The cells were incubated with primary tRNase ZL antibodies and subsequently with an Alexa488-conjugated secondary antibody (Alexa488). Negative control pictures are shown in Figure S2. DAPI was used to stain DNA (DAPI), and MitoTracker Red was to stain the mitochondria (MitoTracker). Bar, 20 µm.
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pone-0005908-g003: Human tRNase ZL exists ubiquitously in cells.Fluorescent microscopic analyses of human 293 kidney cells, A549 epithelial lung cells, HepG2 hepatoma cells, and IMR90 lung fibroblasts. The cells were incubated with primary tRNase ZL antibodies and subsequently with an Alexa488-conjugated secondary antibody (Alexa488). Negative control pictures are shown in Figure S2. DAPI was used to stain DNA (DAPI), and MitoTracker Red was to stain the mitochondria (MitoTracker). Bar, 20 µm.

Mentions: If tRNase ZL really works with 5′-half-tRNAGlu, which is predominant in the cytoplasm, it would need to exist in the cytosol as well as in the nuclei and the mitochondria, where the tRNA 3′ processing occurs. And the above in vitro observation implies that the cytosolic form should be the Δ30 tRNase ZL rather than the full-length one. We examined the intracellular location of tRNase ZL by the indirect fluorescent method using polyclonal antibodies against a human tRNase ZL peptide (C-terminal amino acid 812–826). Fluorescent microscopic analysis showed that tRNase ZL exists everywhere in the 293 cells although the amount of the nuclear tRNase ZL appears to vary depending on the cells (Figures 3 and S2). The similar distribution patterns were observed in human A549 epithelial lung adenocarcinoma cells, HepG2 hepatoma cells, and IMR90 lung fibroblasts (Figure 3 and S2), suggesting that tRNase ZL is ubiquitous in any type of human cells.


Modulation of gene expression by human cytosolic tRNase Z(L) through 5'-half-tRNA.

Elbarbary RA, Takaku H, Uchiumi N, Tamiya H, Abe M, Takahashi M, Nishida H, Nashimoto M - PLoS ONE (2009)

Human tRNase ZL exists ubiquitously in cells.Fluorescent microscopic analyses of human 293 kidney cells, A549 epithelial lung cells, HepG2 hepatoma cells, and IMR90 lung fibroblasts. The cells were incubated with primary tRNase ZL antibodies and subsequently with an Alexa488-conjugated secondary antibody (Alexa488). Negative control pictures are shown in Figure S2. DAPI was used to stain DNA (DAPI), and MitoTracker Red was to stain the mitochondria (MitoTracker). Bar, 20 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691602&req=5

pone-0005908-g003: Human tRNase ZL exists ubiquitously in cells.Fluorescent microscopic analyses of human 293 kidney cells, A549 epithelial lung cells, HepG2 hepatoma cells, and IMR90 lung fibroblasts. The cells were incubated with primary tRNase ZL antibodies and subsequently with an Alexa488-conjugated secondary antibody (Alexa488). Negative control pictures are shown in Figure S2. DAPI was used to stain DNA (DAPI), and MitoTracker Red was to stain the mitochondria (MitoTracker). Bar, 20 µm.
Mentions: If tRNase ZL really works with 5′-half-tRNAGlu, which is predominant in the cytoplasm, it would need to exist in the cytosol as well as in the nuclei and the mitochondria, where the tRNA 3′ processing occurs. And the above in vitro observation implies that the cytosolic form should be the Δ30 tRNase ZL rather than the full-length one. We examined the intracellular location of tRNase ZL by the indirect fluorescent method using polyclonal antibodies against a human tRNase ZL peptide (C-terminal amino acid 812–826). Fluorescent microscopic analysis showed that tRNase ZL exists everywhere in the 293 cells although the amount of the nuclear tRNase ZL appears to vary depending on the cells (Figures 3 and S2). The similar distribution patterns were observed in human A549 epithelial lung adenocarcinoma cells, HepG2 hepatoma cells, and IMR90 lung fibroblasts (Figure 3 and S2), suggesting that tRNase ZL is ubiquitous in any type of human cells.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus