microRNA production in Arabidopsis

Abstract

In plants, microRNAs (miRNAs) associate with ARGONAUTE (AGO) proteins and act as sequence-specific repressors of target gene expression, at the post-transcriptional level through target transcript cleavage and/or translational inhibition. MiRNAs are mainly transcribed by DNA-dependent RNA polymerase II (POL II) and processed by DICER LIKE1 (DCL1) complex into 21∼22 nucleotide (nt) long. Although the main molecular framework of miRNA biogenesis and modes of action have been established, there are still new requirements continually emerging in the recent years. The studies on the involvement factors in miRNA biogenesis indicate that miRNA biogenesis is not accomplished separately step by step, but is closely linked and dynamically regulated with each other. In this article, we will summarize the current knowledge on miRNA biogenesis, including MIR gene transcription, primary miRNA (pri-miRNA) processing, miRNA AGO1 loading and nuclear export; and miRNA metabolism including methylation, uridylation and turnover. We will describe how miRNAs are produced and how the different steps are regulated. We hope to raise awareness that the linkage between different steps and the subcellular regulation are becoming important for the understanding of plant miRNA biogenesis and modes of action.

Keywords: AGO; Arabidopsis; DCL; miRNA biogenesis; microprocessors.

Figure 1

Overview of miRNA biogenesis, miRISC assembling and export, and modes of action in plants. MIR genes are transcribed by POL II and regulated by multiple cotranscriptional regulators (such as mediator, DCL1 complex, TREX-2, ect.). The C-terminus of POL II can be phosphorylated by kinases during the transcription process. Pri-miRNA is processed at the dicing bodies (DCL1, HYL1 and SE). Pre-miRNA is subsequently processed by DCL1 into an imperfect miRNA/miRNA* duplexes. HEN1 methylates the 3’ end of the miRNA/miRNA*. Mature miRNAs are loaded into AGO1 protein in the nucleus. MiRISCs are exported into the cytoplasm through a CRM1 (EXPO1)/NES-dependent manner via the TREX-2/NUP1 facilitation pathway. EMA1 and TRN1 can negatively and positively regulate miRNA loading, respectively. Translocation of miRNAs from the nucleus to the cytoplasm directs target transcript cleavage and translation repression. AGO1 mediates cleavage of miRNA targets sequences, and followed by degradation of cleaved fragments. Translation repression occurs on membrane-bound polysomes (MBPs), and requires endoplasmic reticulum (ER)-localized AMP1. Degradation of miRNA begins with the removal of the methyl group at the 3’ end by the SDN1 family of 3’-5’ exonucleases, followed at 3’ end uridylation by the nucleotidyl transferases HESO1 and/or URT1. In addition, SDN1 and HESO1/URT1 can act on both AGO-bound miRNAs and free miRNAs in the cytoplasm. Free miRNAs are also degraded by SDN1 directly. For the target mRNAs cleaved by miRISC, RICE1 is responsible for degrading the uridylated target mRNA 5’ fragment. The 5’-3’ exonuclease XRN4 can degrade the 3’ fragment of target RNA.

Figure 2

Diagram of the principal steps in microRNA (miRNA) biogenesis. Positive and negative modulators are marked with light pink and light purple ellipses, respectively. (A) Regulation of MIR transcription. Multiple transcription regulators are required to regulate POL II-mediated MIR transcription. The activity of POL II is also regulated by phosphorylation of its C-terminal structural domain (CTD). (B) Structures of pri-miRNAs affect their processing patterns. DCL1 recognizes a small bulge near the base of the stem-loop secondary structure of pri-miRNA. Base-to-loop or sequential base-to-loop processing of pri-miRNAs. Some pri-miRNAs are cut first at ∼15 nt distal to the loop to release miRNA/miRNA* duplex, but some pri-miRNAs with long stems are initially cut at 15 nt distal to the loop, and each subsequent cut is ∼21 nt to release miRNA/miRNA*. Loop-to-base or sequential loop-to-base processing pattern of pri-miRNAs. DCL1 recognizes a small loop at the end of the stem, and cut at 15 nt toward the base of the stem. Some pri-miRNAs with long stem loops are processed through multiple cleavages from the proximal to the loop at 21 nt intervals. Bidirectional processing of pri-miRNAs with a multibranched terminal loop. (C) Regulation of pri-miRNA processing. Processing pri-miRNAs require the use of core dicing bodies (DCL1, SE, and HYL1) as well as several other accessory components. The abundance and activity of DCL1, SE, and HYL1 are regulated by several protein factors and miRNAs. Core microprocessor of phosphorylation status affects the efficiency of pri-miRNA processing. Phosphorylation of SE mediated by PRP4KAs and followed be degraded by the 20S proteasome. CBP80 and CBP20 are subunits of the nucleocapsid binding complex (CBC), which interact with SE. They bind to the m⁷G cap structure of the 5’ end pri-mRNA to prevent degradation. The phosphorylation of DCL1 is mediated by DAWDLE (DDL) to affect pri-miRNA processing. The dephosphorylated and phosphorylated forms of HYL1 are mediated by a series of phosphatases (CPL1, PP4/SMEK) and kinases (MPK3 and SnRK2). In addition, the microprocessor can be synergized by many proteins to promote pri-miRNA processing (marked with light pink ellipse), while CHR2 inhibits its processing.