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Fangyue Guo:
Requirement of a deeply conserved ARGONAUTE phospho-cluster for target-mediated microRNA degradation in plants

Date: 30-09-2021    Supervisor: Peter Brodersen

miRNAs are a type of small RNAs with a length of 21-22 nts that negatively regulate gene expression post-transcriptionally. By loading into Argonaute proteins, miRNAs are assembled into the RNA-induced silencing complex (RISC) that can target mRNAs with base-pairing complementarity to the miRNA for their regulation. In Arabidopsis, the PIWI domain of Argonaute1 (AGO1) possesses endonucleolytic activity towards target mRNA in an extensive target-pairing manner. In addition to regulate the target by direct endonucleolysis, the cleavage fragments can become substrates for RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), which processes the single-stranded mRNA fragments to generate double-stranded RNAs (dsRNAs). The dsRNA is, in turn, cut into small and phased secondary short interfering RNAs (siRNAs) by DICER-like 4 (DCL4) and DCL2. These secondary siRNAs may target new mRNA molecules of the same gene, creating silencing amplification loops, or other mRNAs in trans, adding complexity to the regulation by a single miRNA.

Phosphorylation is one of the most common post-translational modifications of protein. In my main project, I investigated a conserved phosphorylation cluster in a flexible loop of the C-terminal of Arabidopsis AGO1, S1001/S1003/S1005/S1008, which is in the proximity of the miRNA and mRNA interaction site. Because the negative charge of phosphate groups could induce repulsive force towards target mRNA, phosphorylation of the cluster may influence the target mRNA association with AGO1. By performing RNA immunoprecipitation, we observed a significant increase in target binding by AGO1 phospho-dead mutants, in which the four serines have been replaced by alanines (4xA). In addition, a noticeably decrease in target-binding was displayed by the AGO1 phospho-mimic mutant, with four aspartic acid residues (4xD) that mimic the negative charge of phosphorylated serines. Similar results were obtained from in vitro slicer assays, as they showed that the AGO1 phospho-mimic mutant (4xD) exhibits less slicer activity, probably caused by a distinctly diminished target mRNA binding, and reduced association with the 5’-cleavage fragments.

The C-terminal phospho-cluster is highly conserved between the miRNA-Argonautes of human, C. elegans and Arabidopsis. Consistent with the phosphorylation cycles from other Argonautes in human and C. elegans (Golden et al., 2017, Quévillon-Huberdeau, 2017), our results also suggest that target binding triggers AGO1 phosphorylation by casein kinase, which then phosphorylate AGO1 to promote target mRNA release, finally AGO1 become dephosphorylated by phosphatase and is ready to be loaded in new target mRNA.

Target mRNA with extensive base-pairing but a central bulge in the 9-11th nt of miRNA that can trigger miRNA degradation, is called target-mediated miRNA degradation (TDMD). Those mRNA that cannot be sliced because of the central bulge is termed as target mimic mRNA. (Lang et al. 2018) proposed that TDMD requires the F-box protein HWS and (Mei et al., 2019) observed that AGO1 is stably mimicry target-bound in hws mutants, which suggests target-bound and phosphorylated AGO1 is a substrate for HWS. Inspired by the above, we hypothesized that the Cterminal phospho-cluster is required for TDMD and involves the F-box protein HWS. To investigate this, we first introduced three target mimic lines, MIM156, MIM160, and MIM164 (Todesco et al., 2010) in the AGO14xA and AGO14xD. Similar to hws being introduced to MIM160, significant suppression of the MIM160 phenotype and distinct increase of the miR160 level were observed from AGO14xA. Secondly, we analysed an endogenous TDMD pair, miR399 and its target mimic INDUCED BY PHOSPHATE STARVATION1 (IPS1), a non-coding RNA that can be induced under phosphate starvation (Franco-Zorrilla et al., 2007). Again, we observed that miR399 levels were dramatically elevated in AGO14xA, resembling the hws feature, suggesting that miR399 escaped from IPS1-induced degradation. Based on these results, we proposed that TDMD requires the target-bound and the phosphorylated AGO1.

In the second chapter, we have investigated the links between the RNA helicase DDX3 and AGO1 phosphorylation. Arabidopsis has three DDX3 homologs called RH11, RH37, and RH52. The fact that the rh11/rh37/rh52 triple mutant is not viable, suggests that DDX3 activity is essential for plant development.