Artificial microRNA (amiRNA) technology allows researchers to use as few as 21 nucleotides (NT) to guide effective silencing of specific transcripts. However, not all artificially designed amRNA constructs will choose the expected ~21nt guide chain amIGAs. The selection of miRNA guide strands from mature miRNA duplexes has been studied in detail in human and insect systems, but not much has been done on plants. Here, the authors compared DNA-viral vectors (tomato-mottled virus, ToMoV-based), RNA-viral vectors (tobacco mosaic virus, TMV-based), and non-viral binary vectors expressing amiRNAs in plants. We then used deep sequencing and mutation analysis to show that when excluding structural factors caused by base mismatch in mature amRNA doubles, the nucleotide composition of the mature amRNA region determines the choice of the guide strand. We found that strands with excess purines were preferentially selected as guide strands, and artificial miRNAs that were not mismatched in the amien RNA double strands were mainly loaded into AGO2, rather than into AGO1 as most plant endogenous miRNAs do. By analyzing the target effect, we also showed that amiRNA can provide the expected RNAi effect only when the target strand is selected as the guide strand and displays an AGO load. Thus, by removing mismatches in mature amiRNA double strands and designing the desired guide strand to contain excess purines, the guide strand selection of amienas can be better controlled to achieve functional RNAi effects.
Artificial microRNAs (amiRNAs) are used to modulate the resistance of plants and animals to viruses as potential biomarkers for disease. MiRNAs are processed from endogenous noncoding RNAs and are important regulators of gene expression. In plants, primary miRNAs (pri-miRNAs) exhibit specific secondary structures that are first processed into precursor miRNAs (pre-miRNAs) and then enter the nucleus via DCL1 to form miRNA double strands, repositioning the miRNA double strands into the cytoplasm. Subsequently, the miRNA bistrends are loaded into the AGO protein in the RISC complex and one of the strands is selected as the guide strand. When a miRNA binds to a RISC encounters an RNA of interest, the latter is degraded or translated to inhibit. In plants and animals miR/miR* (guide chain/follower chain) bichains are similar, with a length of about 20-24 nt, the two chains are not completely complementary, and have a 3' end overhang of 2 nt.
AmiRNA technology for plant applications provides an alternative and improved approach to gene silencing and uses endogenous pri-miRNA backbones, but replaces miRNAs and miRNAs* with artificially designed amiRNA/amiRNA* sequences of the target transcript.
Figure 1: Predictive folding structure of pri-miR319a and partial pri-amiRNAs.
(a) 5p and 3p arm miRNA double stranded bodies. The mature miR319a double-chained region contains 3 mismatches between 5p and 3p chains. (b) The boot chains amIRA2 and amIRA2c, removing the mismatch sequence structure between the 5p and 3p chains from amira2.
Figure 2: MiR319a backbone backbone is sheared to generate amiRNA and cloned into three different vectors. The two viral vectors TRBO and TAV are in the binary vector pCB301 vector, and the non-viral vector is the binary vector pGWB2. Both TRBO and pGWB2 are driven by the 35S promoter of the cauliflower mosaic virus.
Figure 3: Constructing sequence tissues of TAV-amiRNA-EGFP_target vectors and TAV-EGFP_target (empty vectors). The binary vector pCB301 can be used for all TAV vector cloning. TAV-amiRNA or EGFP with OCS terminator sequence and simultaneous 35S promoter sequence driver is cloned to the T-DNA right boundary (RB) and left boundary (LB) sequence CR of the binary vector. The Common Area of Viruses (CR) is represented by a blue box. Viral genes: AC1 (Rep; replication-related proteins), AC2 (TrAP; transcriptional activating proteins), AC3 (REn; replication enhancers), and AC4 are indicated in light blue. The upstream and downstream sequences of the pri-miR319a skeleton are represented in gray. The amiRNA-target sequence is cloned in the 3'-terminal UTR region of the EGFP sequence.
Transform the constructed vector into Agrobacterium tumefaciens and inject into the leaves of the Tobacco Ben's plant, perform Northern blot hybridization and PCR analysis respectively to confirm replication of recombinant TRBO and TAV viral vectors.
Original link:
https://onlinelibrary.wiley.com/doi/10.1111/pbi.13786
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