Construction of short tandem target mimic (STTM) to block the functions of plant and animal microRNAs
Highlights
► STTM has been demonstrated to be an effective approach to knocking down the functions of miRNAs and siRNAs in Arabidopsis. ► Construction of STTM is made simple and efficient without the need for any prior experience. ► STTM is shown effective in both plant and mammalian cell lines.
Introduction
Small RNAs including microRNA (miRNA) and small interfering RNA (siRNA), play an important role in regulating endogenous gene expression, DNA methylation and chromatin remodeling, silencing invaded viruses, and managing transposon activities [1], [2], [3], [4]. The 20–24 nt small RNAs execute their functions mainly by complementarily binding to their target sites, leading to either target mRNA degradation or translational repression [5]. The regulatory roles of small RNAs cover almost all aspects of a eukaryotic organism’s life cycle, such as growth, development, epigenetics, genome integrity, environmental stress resistance, and host defense [2], [3], [4]. With the help of the deep sequencing technique, large populations of small RNAs have been identified in plants and animals and the numbers are still expanding [6], [7]. Previous methods for the interpretation of gene function relied much on the generation and characterization of genetic mutants [8]. However, this approach is difficult to apply to the study of small RNAs due to their small sizes and their multiple members with overlapping functions as gene families. Currently, the major approaches to small RNA investigation are to generate and analyze the transgenic lines that either express miRNA-resistant targets [9], [10] or overexpress the genes that encode small RNAs [11], [12], [13]. Whereas one small RNA usually regulates several target genes [14], this method can only uncover partial functions of a particular small RNA in vivo. The increasing number of small RNAs found in various organisms requires a more effective approach to unveiling their functions and pathway networks. In this report, we introduced our newly developed approach, short tandem target mimic (STTM), to explore the functions of small RNAs by simultaneously blocking all the members in a miRNA family so as to de-repress all the target genes through a single genetic transformation event [15].
The invention of STTM was based on the discovery of target mimicry (TM), which is an endogenous mechanism that modulates miR399 activity in Arabidopsis thaliana [16]. INDUCED BY PHOSPHATE STARVATION 1 (IPS1) is a non-protein-coding gene that produces transcripts partially complementary to miR399, forming a central three-nucleotide bulge in IPS1-miR399 complexes to prevent the cleavage of IPS1 transcripts by miR399-associated/induced silencing complexes (RISCs) [16]. In this way, the miR399 activity in regulating its target gene PHOSPHATE 2 (PHO2) is subjected to the auto-modulation by IPS1 in response to phosphate starvation. Considering the fact that miR399’s abundance is uninfluenced by IPS1, it was suggested that miR399 was being sequestered by IPS1 transcripts through TM [16].
While endogenous TM was first discovered in plants [16], endogenous competing RNAs (CeRNAs) were also observed in animals recently [17], [18], [19]. Similar to plant TM, CeRNAs act as pseudo-targets to sequester specific miRNAs away from their bona fide targets and reduce the miRNA activities [17], [20]. Currently, the study of CeRNAs in animals is at its beginning [17], [19], and there is no established approach to constructing an artificial CeRNA that can be applied in animals. As was demonstrated by the example of the mouse miR-30d in this study, STTM is also able to function as an artificial CeRNA to block or to down-regulate any miRNAs of interest in animals. The following protocol is described in detail for the efficient construction and expression of STTMs in plants and animals, respectively.
Section snippets
Effective construction of STTMs to be expressed in plants and animals
STTM is an artificial short (∼100 nt) non-coding RNA that can be expressed through genetic engineering (Fig. 1). The central part of STTM was composed of two small RNA binding sites and a spacer/linker (Fig. 1C) [15]. The two binding sites are complementary to target small RNAs except for the central three-nucleotide bulge (Fig. 1C) [15]. This bulge stuck out between the 10th and 11th nucleotide of the targeted small RNA, so that the binding sites could trap small RNAs without being cleaved by
Materials and reagents
- 1.
Vectors to be used: pOT2, pFGC5941-PacI (Fig. 3A), pEGP-miR (Fig. 4A, Cell Biolabs), pEGP-miR Null Control (Cell Biolabs), and the modified pCambia1300-PacI and pCambia2300-PacI (not shown);
- 2.
Primers synthesized in Integrated DNA Technology (IDT): STTM construction primers (Table 1), the origin deletion primers (Table 2) and the STTM30ad-88nt-Linker.
- 3.
PCR amplification and DNA cloning: long amplification Tag DNA polymerase (2.5 U/μl), dNTPs and restriction enzymes including SwaI, PacI, BamHI and NheI
STTM165/166-48nt triggered the degradation of miR165/166 and transformed the normally developed plant into anomalous in transgenic Arabidopsis
The STTM165/166-48nt, which was designed and constructed according to the protocol, was transformed into Arabidopsis plants through a flower dipping approach. Compared to the control plants, the transgenic plants showed extreme anomalies (Fig. 5). Furthermore, the miR165/166 level is likely degraded and the expression of most miR165/166 target genes such as PHB, PHV, REV are largely up-regulated [15]. The deep sequencing also confirmed that the copy number of miR165/166 are almost close to zero
Conclusions
STTM is a simple but effective tool to block the function of small RNAs of interests in plant and mammalian cells. In both systems, the target small RNAs showed significant reductions upon introduction of STTM either through transgenics or transfection, suggesting a destruction of small RNA identities triggered by STTM. In plants, STTM can be expressed as a short non-coding RNA for functional blockage of miRNAs, while in animals, such an STTM structure can be expressed inside an intron and
Acknowledgments
This work was supported, in whole or in part, by USDA-NRI grants 2006-35301-17115 and 2006-35100-17433, NSF grants (MCB-0718029: Subaward No. S-00000260 and IOS-1048216/IOS-1219316), and Michigan Technological University to G.T. The work was also supported in part by NIH K01 DK078648 and R03 DK084166 to X.T. We thank Dr. Peter Nelson for providing luciferase reporter plasmids and Lyssa Scheunemann for a critical reading of this article.
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