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Investigation of the plant miRNA pathway

Periodic Report Summary 1 - PLANMIP (Investigation of the plant miRNA pathway)

The project objectives were to provide answers to the following questions:

1. What is the membrane compartment(s) to which AGO1 and miRNAs localize?

2. What is the function of membrane-anchored RISC, and how is RISC recruited to membranes?

3. What specific genes influence inhibition of the miRNA pathway following inhibition of isoprenoid
biosynthesis?

In the four months that have elapsed from project start to project termination (due to the award of an ERC Starting Grant to the researcher) the following work and results have been obtained:

AGO1 localization
We have shown that the N-terminal domain alone is sufficient to drive GFP reporter localization to distinct cytoplasmic bodies whose identity remains unknown to us. We have used westerns to identify tissues where AGO1 is particularly highly expressed, and are continuing our efforts on immunofluorescence and elctron microscopy on those tissues.
AGO1 membrane recruitment
a) Construction of HPB-AGO1
The construct had still not been completed by 31/12/2012 despite employment of different strategies. Work is continuing to get this important construct made. ago1-3/mcca mutant lines have been selected and are ready for transformation.
b) AGO1 yeast two-hybrid screening.
The constructs have been made and shown to be expressed as Gal4 fusions in yeast by western analysis. Plating on selective media has shown that they do not cause reporter autoactivation. A screen has been succesfully conducted with the N-terminal domain of AGO1, and a single colony positive for activation of all three reporters has been recovered. Work is ongoing to characterize this by retransformation, sequencing etc.
c) ago1 suppressor screens
We have introduced a miR398 sensor into ago1-38 by crossing, but have not yet carried out the mutagenesis.
Isoprenoid/miRNA crosstalk
This part of the project was only initiated by ordering and growing Arabidopsis ecotypes during the four months covered here.

The potential impact of the expected results of the project reaches far. The links between membrane and isoprenoid biology on the one side and RNA silencing on the other remain nearly fully unexplored, yet our recently published results clearly demonstrate the existence of such links (Brodersen et al. (2012), PNAS 109, 1778-83). For example, the recent demonstration that inhibition of miR-33 in primates influences serum levels of HDL and VLDL triglycerides in conjunction with our demonstration of miRNA pathway inhibition by statins could shed some light on atheroprotective action on this very widely used group of drugs.