A Resource for Inactivation of MicroRNAs Using Short Tandem Target Mimic Technology in Model and Crop Plants

Abstract

microRNAs (miRNAs) are endogenous small non-coding RNAs that bind to mRNAs and target them for cleavage and/or translational repression, leading to gene silencing. We previously developed short tandem target mimic (STTM) technology to deactivate endogenous miRNAs in Arabidopsis. Here, we created hundreds of STTMs that target both conserved and species-specific miRNAs in Arabidopsis, tomato, rice, and maize, providing a resource for the functional interrogation of miRNAs. We not only revealed the functions of several miRNAs in plant development, but also demonstrated that tissue-specific inactivation of a few miRNAs in rice leads to an increase in grain size without adversely affecting overall plant growth and development. RNA-seq and small RNA-seq analyses of STTM156/157 and STTM165/166 transgenic plants revealed the roles of these miRNAs in plant hormone biosynthesis and activation, secondary metabolism, and ion-channel activity-associated electrophysiology, demonstrating that STTM technology is an effective approach for studying miRNA functions. To facilitate the study and application of STTM transgenic plants and to provide a useful platform for storing and sharing of information about miRNA-regulated gene networks, we have established an online Genome Browser (https://blossom.ffr.mtu.edu/designindex2.php) to display the transcriptomic and miRNAomic changes in STTM-induced miRNA knockdown plants.

MicroRNAs and new biotechnological tools for its modulation and improving stress tolerance in plants

“…Then, these 21-nt amiRNAs are methylated by HEN1, which subsequently undergoes nucleus-cytoplasm transport by HST to P-bodies and becomes integrated into the RISC, where it will act in a manner similar to canonical miRNAs on PTGS. (b) Target mimicry strategy to deplete specific miRNAs (Peng et al, 2018;Zhang et al, 2017). The constitutive or transient expression of the target mimic gene driven by a specific promoter is transcribed by RNA polymerase II in the nucleus.…”

“…Thus, STTM sequester miRNAs from the endogenous target mRNA resulting in its up-regulation (Franco-Zorrilla et al, 2007). Several STTMs targeting the MIR genes in model and crop plants have been recently engineered and constitutively expressed as transgenes for the comprehensive functional analysis of miRNAs (Peng et al, 2018;Zhang et al, 2017). In addition, STTMs have been optimized to enhance lossof-function phenotypes caused by artificial single target mimics.…”

MicroRNAs meet with quantitative trait loci: Small powerful players in regulating quantitative yield traits in rice

“…Several genetic engineering technologies have been deployed to study the functions of miRNAs, such as target mimic (MIM) (Franco‐Zorrilla et al, ) and short tandem target mimic (STTM) (Yan et al, ). In addition, large scale studies involving MIMs and STTMs for functional interrogation of miRNAs have been done in Arabidopsis, rice and other species (Peng et al, ; Todesco et al, ; Zhang, Zhang, et al, ), and offers a useful platform and resource for such studies. In recent years, targeted genome modification technologies, such as ZFNs (Zhang et al, ), TALENs (Li, Liu, Spalding, Weeks, & Yang, ), CRISPR/Cas9 (Li, Teng, Li, & Zhou, ), and CRISPR/Cas13 (Ali, Mahas, & Mahfouz, ; Aman et al, ), were successfully used for plant genome editing.…”

“…Therefore, traditional over‐expression of miRNAs or downregulating them through methods such as MIM, STTM, CRISPR/Cas9 and/or CRISPR/Cas13 technology can be further used to study the roles of these miRNAs in regulating rice grain development. Using STTM technology, we already identified the roles of miR159, miR167, and miR1432 in controlling rice seed development (Peng et al, ; Zhao et al, ). Furthermore, majority of the miRNAs mentioned in this review, negatively regulate rice yield‐associated agronomic traits, therefore lowering their expression in rice may enhance the yield.…”

Protocol: low cost fast and efficient generation of molecular tools for small RNA analysis

“…sRNA levels can be increased either by directly overexpressing the endogenous precursor [3] or by the use of molecular tools such as artificial miRNAs (amiRs), which substitutes the 21 nt sequence of an endogenous pri-miRNA for our miRNA of interest [4,5]. Conversely, molecular tools such as Target Mimics (MIMs or TMs) [6,7], Short Tandem Target Mimic (STTMs) [8,9], or molecular SPONGES (SPs) [10] are used to reduce plant miRNA levels with varying degrees of efficacy, which depend mainly on the miRNA family targeted [10]. Such techniques are extensively employed in both model (Arabidopsis) and crop plants (e.g.…”