Bioscience Methods 2025, Vol.16, No.6, 280-288 http://bioscipublisher.com/index.php/bm 282 mRNA is degraded. Sometimes, it does not cut but blocks the initiation or extension of translation, depending on the type of miRNA. It is worth noting that some mirnas with a length of 22 nucleotides can also trigger a chain reaction of Phasirnas, as if opening up another regulatory channel. This mechanism is particularly important in regulating the developmental process or responding to stress, making the regulation of gene expression more flexible (Dong et al., 2022; Ding and Zhang, 2023; Xu and Chen, 2023). Figure 1 Regulation of miRNA biogenesis, RISC loading, and action in plants. MIRNAgenes are transcribed by RNA Polymerase II (Pol II) and fold into stem-loop structures called pri-miRNAs. pri-miRNAs are mainly processed by DCL1 from either “base-to-loop” or “loop-to-base” direction. Nascent miRNA/miRNA duplexes are methylated by the small RNA methyltransferase HEN1. RISC loading occurs in the nucleus, but may also occur in the cytoplasm. miRNAs mediate gene silencing through either target cleavage or translation inhibition. Some miRNAs can trigger the production of secondary siRNAs through sequential actions of RDR6 and DCL2/4, generating 21-22 nt secondary siRNAs called phasiRNAs and easiRNAs, which in turn repress gene expression via PTGS (for phasiRNAs) or TGS (for easiRNAs) (D). It is important to note that although the steps in the model are separate, they could be closely coupled. Factors involved in the regulation of MIR transcription, pri-miRNA processing, and RISC assembly are shown in parts (A-C), respectively (Adopted from Wang et al., 2019) 3.3 Specificity of microRNA-target gene recognition in plants When it comes to miRNA and target recognition, what usually comes to mind is a "perfect match". The recognition of plant mirnas is indeed very picky. In most cases, only when the bases correspond one-to-one
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