Functional analysis of microRNA 840 in Arabidopsis

Abstract

Three mechanisms have been implicated for plant microRNAs (miRNAs) to regulate gene expression, i.e. directing target RNA cleavage, transcriptional silencing and translational repression. A great number of target genes of plant miRNAs were predicted using different algorithms and verified through experimental methods.

In the present work, one miRNA from Arabidopsis, microRNA 840 (miR840) was initially analyzed through the high-through put sequencing and bioinformatic method (Rajagopalan et al., 2006). MiR840 expresses from the complementary strand of its predicted target gene, AtWhirly3. AtWhirly3 encodes a homolog of the potato transcriptional regulator p24. MiR840 is located between 2 genes, At2g02740 (Whirly 3, Why 3) and At2g02750 (Pentatricopeptide Repeat Protein, PPR). Whirly 3 protein belongs to the Whirly protein family which is one of the main interesting topics in our group. Understanding the potential regulative function of miR840 related to Whirly 3 gene is important to deeply analyze the Whirly 3 protein.

Searching the TAIR (http://www.arabidopsis.org) and TIGR (http://plantta.jcvi.org), the two databases give different annotation versions for this AtWhirly3 gene, differing at the length of the 3’ untranslated region. According to the annotation from TAIR, the target of miR840 is only at the 3’ untranslated region (3’ UTR) of Why 3 gene, so this means miR840 only down-regulate the Why 3 gene. But the information from TIGR gives another annotation, Why 3 gene (At2g02740) and its neighbor gene, a PPR gene (At2g02750) overlap with their 3’ UTR where the target site of the miR840 is. This means the miR840 may also be able to down-regulate both the Why 3 and PPR gene, depending on what annotation you take. Therefore experimental analysis is necessary to determine the miR840 target. My master project here is to perform the biological experiments for analyzing the function of miR840.

To verify the target genes and study the biological function of miR840. 4 vectors have been firstly constructed and then introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation (1: overexpressed precursors of miR840; 2: overexpressed mutated miR840 target for directing the miR840 lost its normal function; 3: overexpressed Why 3; 4: overexpressed PPR). The mutated miR840 was constructed by the mechanism of target mimicry which through artificial insertion of a mismatch-loop into the cleavage site at the miRNAs target, for making mutated miRNAs targets. The mutated target sites will direct the miRNAs cannot cleave their targets (miRNAs lost their normal function).

The expression level of the transgenic plants will be analyzed, the changes of the expression levels observed in the transgenic plant lines could imply the possible role of miR840.

The subcellular location of the PPR (At2g02750) protein which is still unknown yet, so the bioinformatic method was used to predicted its subcellular location.