Construction of short tandem target mimic (STTM) to block the functions of plant and animal microRNAs

Abstract

Small RNAs are widespread in plants and animals. They largely include microRNAs (miRNAs) and short interfering RNAs (siRNAs), and they play key roles in gene and chromatin regulations. Here we describe in detail the method for an effective construction of the recently developed short tandem target mimic (STTM) technology to block small RNA functions in plants and animals. STTM is a powerful technology complementing the previous target mimic (TM) in plants and the miRNA sponge, as well as the recently defined endogenous competing RNA (CeRNA) in animals. We expect STTM will not only be effective in blocking small RNA functions in plants but will also become a popular approach in animals.

SINAT E3 Ligases Control the Light-Mediated Stability of the Brassinosteroid-Activated Transcription Factor BES1 in Arabidopsis

“…To create the knockdown lines of SINATs , a “two-hit” artificial miRNA (amiRNAs) strategy was used as described (Ji et al, 2011; Tang et al, 2012; Teotia et al, 2016). Briefly, two artificial microRNAs (amiRNAs: amiR1 and amiR2) were constructed using a 23 35S promoter and a 35S terminator in pOT2-Poly-Cis vector, resulting in pOT2- SINATs- amiR1-amiR2.…”

Short tandem target mimic rice lines uncover functions of miRNAs in regulating important agronomic traits

“…STTM vectors were constructed as described by Tang et al (19). For the OX-miR398 vector, a PCR fragment amplified from genomic DNA using primers OE-miR398a-F and OX-miR398a-R was cloned into the BamHI/SpeI sites of the binary vector pTCK303 (53) between the maize Ubi 1 promoter and the Nos terminator.…”

“…STTMs were developed as an improvement on target mimicry and contain two imperfect miRNA target sites with a CTA trinucleotide bulge in the center linked by a 31-to 96-nt short spacer (17). STTMs can trigger efficient degradation of the targeted miRNAs in Arabidopsis and mice (17,19). Here, we report an extensive functional study of rice miRNAs using STTM technology.…”

MicroRNAs as pharmacological targets in diabetes

“…In turn, overexpression of HNF1b improves insulin sensitivity in db/db mice [118]. Thus, deregulated miR-802 may contribute the development of obesity-associated impairment of glucose metabolism, which can be corrected by modulating miR-802 levels through down regulation of this miRNA by various in vitro and in vivo technologies such as injecting antagomiR or expressing recently developed small tandem target mimic (STTM) [116, 119]. …”