Chapter 25
In Vivo Reporter System for miRNA-Mediated RNA Silencing
Ka ´ roly Fa ´ tyol
Abstract
Argonaute proteins play a central role in the evolutionarily conserved mechanisms of RNA silencing.
Programmed by a variety of small RNAs, including miRNAs, they recognize their target nucleic acids
and modulate gene expression by various means. Argonaute proteins are large complex molecules. Therefore, to better understand the mechanisms they use to regulate gene expression, it is necessary to identify
regions of them bearing functional importance (protein-protein interaction surfaces, acceptor sites of
posttranslational modifications, etc.). Identification of these regions can be performed using a variety of
mutant screens. Here we describe a transient reporter assay system, which is suitable to carry out rapid
functional assessment of mutant Argonaute molecules before proceeding to their more detailed biochemical characterization.
Key words Argonaute, miRNA, In vivo reporter system, RNA silencing, Translational repression,
Slicing
1 Introduction
The basic mechanisms of miRNA-mediated posttranscriptional
gene regulation—including the production, processing, and execution stages—are highly conserved from animals to plants [1–4]. Primary miRNAs are usually produced as polII transcripts. RNase
III-like enzymes (called DCLs in plants) process these transcripts,
resulting in the release of mature miRNA/miRNA∗ duplexes.
These duplexes are subsequently loaded into Argonaute proteins
(AGOs), producing miRISCs (RNA-induced silencing complexes).
Using the simple rules of Watson-Crick base pairing, the miRNAprogrammed miRISCs recognize their substrate RNAs and regulate their expression by two major mechanisms: transcript cleavage
(slicing) and translational repression. Earlier it was assumed that the
degree of complementarity between the miRNA and its target
determines which of the above mechanisms is used. Since in plants
the miRNA/target complementarity is nearly perfect, plant miRISCs were presumed to employ slicing as their main mode of action.
Newer data however have contested this assumption. It was
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_25, © Springer Science+Business Media, LLC, part of Springer Nature 2020
431
In Vivo Reporter System for miRNA-Mediated RNA Silencing
Ka ´ roly Fa ´ tyol
Abstract
Argonaute proteins play a central role in the evolutionarily conserved mechanisms of RNA silencing.
Programmed by a variety of small RNAs, including miRNAs, they recognize their target nucleic acids
and modulate gene expression by various means. Argonaute proteins are large complex molecules. Therefore, to better understand the mechanisms they use to regulate gene expression, it is necessary to identify
regions of them bearing functional importance (protein-protein interaction surfaces, acceptor sites of
posttranslational modifications, etc.). Identification of these regions can be performed using a variety of
mutant screens. Here we describe a transient reporter assay system, which is suitable to carry out rapid
functional assessment of mutant Argonaute molecules before proceeding to their more detailed biochemical characterization.
Key words Argonaute, miRNA, In vivo reporter system, RNA silencing, Translational repression,
Slicing
1 Introduction
The basic mechanisms of miRNA-mediated posttranscriptional
gene regulation—including the production, processing, and execution stages—are highly conserved from animals to plants [1–4]. Primary miRNAs are usually produced as polII transcripts. RNase
III-like enzymes (called DCLs in plants) process these transcripts,
resulting in the release of mature miRNA/miRNA∗ duplexes.
These duplexes are subsequently loaded into Argonaute proteins
(AGOs), producing miRISCs (RNA-induced silencing complexes).
Using the simple rules of Watson-Crick base pairing, the miRNAprogrammed miRISCs recognize their substrate RNAs and regulate their expression by two major mechanisms: transcript cleavage
(slicing) and translational repression. Earlier it was assumed that the
degree of complementarity between the miRNA and its target
determines which of the above mechanisms is used. Since in plants
the miRNA/target complementarity is nearly perfect, plant miRISCs were presumed to employ slicing as their main mode of action.
Newer data however have contested this assumption. It was
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_25, © Springer Science+Business Media, LLC, part of Springer Nature 2020
431