demonstrated that translational repression is frequently used by
plant miRISCs even on mRNAs, which under different conditions
could be a subject of slicing. These findings imply that it is not the
degree of miRNA/target complementarity, but rather the biochemical properties of miRISCs (posttranslational modifications
of AGOs, interacting protein partners) and perhaps other still
poorly understood factors (e.g., dynamic properties of the target
RNA) that determine the mechanism of repression employed [5].
Recently, we have reported the cloning of Nicotiana benthamiana Argonaute proteins [6]. To characterize these molecules, we
have developed an in vivo transient reporter system. Our system is
based on the Agrobacterium-mediated transient expression assay
developed earlier to analyze miRNA-dependent gene silencing [7–
9]. The three components of the system are the following: (1) sensor transcript, (2) miRNA expression cassette, and (3) AGO expression cassette (Fig. 1a). These elements are incorporated into binary
plasmid vectors to allow their efficient co-delivery into tobacco
leaves by agroinfiltration. The sensor plasmid encodes a GFP-Renilla luciferase fusion protein under the control of a constitutively
active CaMV 35S promoter. miRNA target sites are inserted into
various positions of the sensor mRNA (3
0 UTR or ORF). The
second component of the reporter system encodes a miRNA. This
plasmid also constitutively produces GFP protein, which is used as
an internal control to normalize for the varying efficiencies of
agroinfiltration. The AGO protein under study is expressed from
the third binary plasmid. The effector complexes—assembled from
the co-expressed AGO proteins and miRNAs—interact with the
sensor mRNAs via the miRNA target sites. Monitoring the expression of the sensor mRNA allows the assessment of gene silencing.
Using the above system, detailed characterization of
N. benthamiana AGO2 has been performed [6]. We have shown
that AGO2 can directly repress translation via various miRNA
target site constellations (ORF, 3
0 UTR). In addition, we have
observed that AGO2 is able to inhibit its target RNAs via both
slicing-dependent and -independent fashion. Several functionally
important amino acid residues of AGO2 have also been identified
that affect its small RNA loading, cleavage activity, translational
repression potential, and antiviral activity.
In this chapter we provide a detailed description of our in vivo
reporter system. The use of this system allows the rapid initial
assessment of the activities of both wild-type and mutant forms of
AGO proteins. Based on the results, further biochemical studies
(cleavage assays, binding assays, etc.) can be initiated to better
understand the mechanisms these molecules employ to regulate
gene expression.
432
Ka ´ roly Fa ´ tyol
plant miRISCs even on mRNAs, which under different conditions
could be a subject of slicing. These findings imply that it is not the
degree of miRNA/target complementarity, but rather the biochemical properties of miRISCs (posttranslational modifications
of AGOs, interacting protein partners) and perhaps other still
poorly understood factors (e.g., dynamic properties of the target
RNA) that determine the mechanism of repression employed [5].
Recently, we have reported the cloning of Nicotiana benthamiana Argonaute proteins [6]. To characterize these molecules, we
have developed an in vivo transient reporter system. Our system is
based on the Agrobacterium-mediated transient expression assay
developed earlier to analyze miRNA-dependent gene silencing [7–
9]. The three components of the system are the following: (1) sensor transcript, (2) miRNA expression cassette, and (3) AGO expression cassette (Fig. 1a). These elements are incorporated into binary
plasmid vectors to allow their efficient co-delivery into tobacco
leaves by agroinfiltration. The sensor plasmid encodes a GFP-Renilla luciferase fusion protein under the control of a constitutively
active CaMV 35S promoter. miRNA target sites are inserted into
various positions of the sensor mRNA (3
0 UTR or ORF). The
second component of the reporter system encodes a miRNA. This
plasmid also constitutively produces GFP protein, which is used as
an internal control to normalize for the varying efficiencies of
agroinfiltration. The AGO protein under study is expressed from
the third binary plasmid. The effector complexes—assembled from
the co-expressed AGO proteins and miRNAs—interact with the
sensor mRNAs via the miRNA target sites. Monitoring the expression of the sensor mRNA allows the assessment of gene silencing.
Using the above system, detailed characterization of
N. benthamiana AGO2 has been performed [6]. We have shown
that AGO2 can directly repress translation via various miRNA
target site constellations (ORF, 3
0 UTR). In addition, we have
observed that AGO2 is able to inhibit its target RNAs via both
slicing-dependent and -independent fashion. Several functionally
important amino acid residues of AGO2 have also been identified
that affect its small RNA loading, cleavage activity, translational
repression potential, and antiviral activity.
In this chapter we provide a detailed description of our in vivo
reporter system. The use of this system allows the rapid initial
assessment of the activities of both wild-type and mutant forms of
AGO proteins. Based on the results, further biochemical studies
(cleavage assays, binding assays, etc.) can be initiated to better
understand the mechanisms these molecules employ to regulate
gene expression.
432
Ka ´ roly Fa ´ tyol