Elimination of Specific miRNAs by Naked 14-nt sgRNAs

tRNase ZL-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 ZL, 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 ZL 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.


Introduction
tRNase Z L is a long form of tRNA 39 processing endoribonuclease (tRNase Z, or 39 tRNase) [1,2].In human cells, nuclear full-length tRNase Z L works on precursor tRNAs to process them by removing their 39 trailer, whereas cytosolic D30 tRNase Z L appears to work on mRNAs to modulate gene expressions by cleaving them under the direction of cellular small noncoding RNAs such as 59-half-tRNA and miRNA [3,4].It has been shown that cytosolic tRNase Z L modulates the PPM1F gene expression by cleaving its mRNA under the direction of 59-half-tRNA Glu [3] and that miR-103 can downregulate gene expression through directing mRNA cleavage by cytosolic tRNase Z L [4].The cytosolic tRNase Z L together with the small noncoding RNAs appears to form a broad gene regulatory network.
We have developed a technology for suppressing the expression of a target gene by modulating this gene regulatory network under the aegis of artificial small guide RNA (sgRNA) [5][6][7][8][9].This technology 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 sgRNA and can cleave any target RNA at any desired site [10][11][12][13][14][15][16].There are four types of sgRNA, 59-half-tRNA [11], RNA heptamer [12], hook RNA [15], and ,14-nt linear RNA [16], and this technology is termed TRUE gene silencing after tRNase Z L -utilizing efficacious gene silencing.
The efficacy of TRUE gene silencing can become comparable to that of the RNA interference [7] and can surpass it in some cases [8].
miRNAs play important regulatory roles in many cellular processes, and their dysfunction appears to cause many diseases including cancer and heart disease [17].It has been shown in human cells that miR-15a and miR-16 work as tumor suppressors, while miR-17-92, miR-19a/b, miR-21, and miR-181a/b are oncogenic [18][19][20].miR-122, which is abundant in liver cells, has been shown to be a regulator of fatty-acid metabolism [21], and its expression appears to be essential for replication of hepatitis C virus [22].Thus we can expect that elimination of specific miRNAs in the cells would lead to cures of some diseases.Indeed, downregulation of the miR-19 or miR-181 level in myeloma cells by an antisense nucleic acid has been demonstrated to suppress tumor growth in nude mice [20].And oligonucleotides against miR-122 can reduce a blood cholesterol level [21] and can also suppress the replication of hepatitis C virus [22].
In this paper, we investigated if TRUE gene silencing can eliminate specific miRNAs from human cells.And we show that human tRNase Z L can cleave miRNA under the direction of 14-nt linear-type sgRNA in vitro and that naked 14-nt sgRNA can significantly downregulate miRNA expression in vivo.

In vitro RNA Cleavage Assay
In vitro RNA cleavage assays for the FITC-labeled miR-16 (2 pmol) were carried out at 37uC in the presence of the unlabeled sgRNAs (20 pmol) using histidine-tagged human D30 tRNase Z L (50 ng) in a mixture (6 ml) containing 10 mM Tris-HCl (pH 7.5), and 3.3 mM MgCl 2 .After resolution of the reaction products on a 15% polyacrylamide-8 M urea gel, the gel was analyzed with a Typhoon 9210 (GE Healthcare).
HEK293 cells were transfected with sgRNA or with sgRNA and scramble or anti-tRNase-Z L siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol, and cultured for further 42 hours.

Northern Analysis
Total RNA was extracted with ISOGEN.The RNA samples (10 mg) were separated by 15% polyacrylamide/8 M urea gel electrophoresis, and electrically transferred to a Hybond N + membrane (GE Healthcare).The membrane was ultravioletcrosslinked, probed with a 59-32 P-labeled deoxyoligonucleotide in a QuickHyb buffer (Stratagene) at 45uC, and analyzed with the Typhoon 9210.

Real-Time PCR
Total RNA was extracted from cells using ISOGEN.The cellular amounts of miR-16, miR-142-3p, and miR-206 were quantitated by a StepOne Real Time PCR System using TaqMan MicroRNA Assays and TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems).The miR-16, miR-19a/b, Bcl-2 mRNA and b-actin mRNA levels were quantitated by real-time PCR using a LightCycler 480 SYBR Green I Kit (Roche).

Western Analysis
Whole cell extracts dissolved in a buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 100 mM dithiothreitol) were separated by SDS/7.5% polyacrylamide gel electrophoresis, and transferred to a nitrocellulose membrane.The membrane was probed with polyclonal antibodies raised to a human tRNase Z L peptide (amino acid 812-826) or a monoclonal antibody against human b-actin (Sigma) using the ECL Western Blotting Detection System (GE Healthcare).

Statistical Analysis
Differences between control and experimental groups were evaluated by the Student's t-test.
These results suggest that both the length and the binding site of sgRNA are important for its efficient guiding ability.We focused on 14-nt sgRNAs that are complementary to the 1st-14th nucleotides of miRNAs for the following in vivo analyses.
In another set of the experiments, in which a scramble siRNA was used as a control, we also obtained similar results (Figure 2).Together, these results suggest that tRNase Z L is responsible at least partly for the reduction by sgR16(1-14) but not by sgR16  and that TRUE gene silencing can downregulate the miRNA expression.The mechanism by which sgR16(1-22) acts on the miR-16 level would be through binding and sequestering [20][21][22].

Naked Linear-type sgRNA can be Taken up by Living Cells
We investigated how efficiently linear-type sgRNA can be taken up by living cells without any transfection reagents and can downregulate the miRNA expression.HL60 cells were cultured in the presence of 1 mM of the naked 14-nt 39-Alexa-568-labeled sgR16(1-14) and analyzed with a fluorescent microscope.This sgRNA was indeed efficiently taken up without any transfection reagents by HL60 cells (Figure 3A).RPMI-8226 and HEK293 cells also took up the naked sgRNA efficiently (Figure 3A).We also showed that another 14-nt RNA, sgRNA14, can be taken up nakedly by HeLa, HEK293, and Jurkat cells (Figure S2A,B and  Text S1).
Furthermore, we demonstrated by polyacrylamide gel analysis of total RNA from HL60, RPMI-8226, and HEK293 cells that at least a part of the taken-up sgR16(1-14) molecules exist stably for at least 24 hours in the cells (Figure 3B,C).And at least a part of sgRNA14 molecules were also stable for at least 24 hours in HeLa, HEK293, and Jurkat cells (Figure S2C).However, the amount of sgR16(1-14) recovered from HEK293 cells that took it up with the aid of a transfection reagent was much higher than that recovered from the cells that took it up nakedly (Figure 3C), suggesting that sgR16(1-14) can be taken up more easily and/or can exist more stably in the presence of the transfection reagent.
We also carried out a similar experiment with HL60 cells using 59and 39-phosphorylated sgR16(1-14) and sgR16 .Likewise sgR16(1-14) significantly reduced the miR-16 level to 53%, but in contrast sgR16(1-22) also did work almost perfectly (Figure 4B).This was also the case in HEK293 cells (data not shown).This discrepancy would be attributed to the phosphorylation at the 39 end, which appears to make RNA more efficient in being taken up by human cells and/or more stable in the cells than RNA with no 39phosphate judging from a result of the RNA survivability test (data not shown).
Furthermore, to show that the knockdown of miR-16 is functional, we examined its endogenous target, the Bcl-2 mRNA, for the stability [30].The Bcl-2 mRNA levels in HL60 cells were upregulated to 122% and 137% by downregulating the miR-16 levels by sgR16(1-14) and sgR16 , respectively (Figure 4B,C).This result implies that sgR16(1-14) can stabilize the Bcl-2 mRNA by reducing the miR-16 level and subsequently inhibiting the mRNA transport to P-bodies.
The miR-206 expression in C2C12 cells was efficiently downregulated to 22% by sgR206 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), and the miR-19a/b level in RPMI-8226 cells was reduced to 28% by sgR19(1-14) (Figure 5B,C).These observations imply that in general we can eliminate a specific cellular miRNA at least by ,50% by using 1 mM of a naked 14-nt sgRNA on the basis of TRUE gene silencing.Our results suggest that TRUE gene silencing can be used for RNA therapy targeting disease-causing miRNA.miRNAs to be targeted would include miR-19a/b and miR-21 for cancer therapy and miR-122 for hypercholesterolemia and hepatitis C treatments.As 14-nt sgRNA can be taken up easily by the cells without any carrier reagents such as cholesterol and cationic liposome, and can guide tRNase Z L to cleave miRNA, TRUE gene silencing may be advantageous over a technology based on the inhibitory mechanism through binding and sequestering [20][21][22].The tRNase Z L level under the above transfection conditions was analyzed.b-actin was used as a loading control.(PDF)

Figure
Figure S1 The reduction in the miR-16 level by sgR16(1-14) is attributable at least partly to tRNase Z L .(A) Quantitation of the miR-16 level in HEK293 cells with a LightCycler 480 SYBR Green I Kit.The HEK293 cells were transfected with mock, 35 nM of sgR16(1-14) or sgR16(1-22) together with mock or the anti-tRNase-Z L siRNA.sgR16(1-14) and sgR16(1-22) were phosphorylated at both 59 and 39 ends.The miR-16 levels are normalized against the 5S rRNA levels.Error bars indicate s.d.(n = 3).*, P,0.001.(B) Western blot analysis.The tRNase Z L level under the above transfection conditions was analyzed.b-actin was used as a loading control.(PDF)