[12–16]. This is particularly useful when the cis-acting elements,
necessary to recruit the RBP to a transcript, are not well known or if
the protein functions within a complex whose components have
not been fully characterized. Tethering of a protein of interest
affects the properties of the reporter RNA that can then be
measured using an appropriate readout. Since proteins could have
more than one cellular function, it is generally preferred to tether a
single functional domain of a protein to the reporter. Importantly,
tethering of an individual domain helps to study its role in isolation
and minimizes any secondary effects arising due to binding of the
protein of interest to its endogenous targets.
In this tethering assay (Fig. 1), the reporter is composed of a
Renilla luciferase coding sequence followed by 24x-MS2 stemloops in its 3
0 UTR. The reporter is stably integrated into HeLa
cells and is placed under a doxycycline-inducible promoter
[17, 18]. The cells are transfected with a plasmid expressing the
fusion protein of MS2 coat protein (MCP) fused to green fluorescent protein (GFP) and a protein or protein domain of interest
(POI). The reporter can be visualized using the GFP signal in living
cells and its movement can be measured by single-particle tracking
(SPT). Since the reporter transcripts lack any known cis-acting
elements that promote RNA localization, the mRNAs predominantly undergo diffusional movement (Fig. 1). When the protein
domain that is fused to MCP-GFP recruits a molecular motor, its
mobility gets altered and can be measured by calculating the mean
squared displacements, diffusion coefficients, and velocity of the
reporter RNA molecule (Fig. 1). In order to establish the assay, it is
necessary to quantify the effect of tethering known motorinteracting domains to a transcript. For monitoring actin-based
transport, the RILPL2 RH1 domain was chosen because of its
well-characterized interaction with myosin Va [19]. For
microtubule-based transport, the N-terminal domain of SKIP
that has been shown to bind to kinesin light chain 2 (KLC2) was
selected [20].
This chapter contains instructions on how to perform the
tethering assay for RNA mobility. It describes how to acquire
single-mRNA imaging data and analyze the images in order to
quantify RNA mobility. We conclude with a description of the
analysis of the positive controls (RILPL2-RH1 and SKIP).
2 Materials
2.1 Sample
Preparation
1. HeLa cells stably expressing a reporter mRNA carrying
24x-MS2 stem-loops in the 3
0 untranslated region, under a
doxycycline-inducible promoter [17, 18].
Tethering Assay for RNA Mobility
271
necessary to recruit the RBP to a transcript, are not well known or if
the protein functions within a complex whose components have
not been fully characterized. Tethering of a protein of interest
affects the properties of the reporter RNA that can then be
measured using an appropriate readout. Since proteins could have
more than one cellular function, it is generally preferred to tether a
single functional domain of a protein to the reporter. Importantly,
tethering of an individual domain helps to study its role in isolation
and minimizes any secondary effects arising due to binding of the
protein of interest to its endogenous targets.
In this tethering assay (Fig. 1), the reporter is composed of a
Renilla luciferase coding sequence followed by 24x-MS2 stemloops in its 3
0 UTR. The reporter is stably integrated into HeLa
cells and is placed under a doxycycline-inducible promoter
[17, 18]. The cells are transfected with a plasmid expressing the
fusion protein of MS2 coat protein (MCP) fused to green fluorescent protein (GFP) and a protein or protein domain of interest
(POI). The reporter can be visualized using the GFP signal in living
cells and its movement can be measured by single-particle tracking
(SPT). Since the reporter transcripts lack any known cis-acting
elements that promote RNA localization, the mRNAs predominantly undergo diffusional movement (Fig. 1). When the protein
domain that is fused to MCP-GFP recruits a molecular motor, its
mobility gets altered and can be measured by calculating the mean
squared displacements, diffusion coefficients, and velocity of the
reporter RNA molecule (Fig. 1). In order to establish the assay, it is
necessary to quantify the effect of tethering known motorinteracting domains to a transcript. For monitoring actin-based
transport, the RILPL2 RH1 domain was chosen because of its
well-characterized interaction with myosin Va [19]. For
microtubule-based transport, the N-terminal domain of SKIP
that has been shown to bind to kinesin light chain 2 (KLC2) was
selected [20].
This chapter contains instructions on how to perform the
tethering assay for RNA mobility. It describes how to acquire
single-mRNA imaging data and analyze the images in order to
quantify RNA mobility. We conclude with a description of the
analysis of the positive controls (RILPL2-RH1 and SKIP).
2 Materials
2.1 Sample
Preparation
1. HeLa cells stably expressing a reporter mRNA carrying
24x-MS2 stem-loops in the 3
0 untranslated region, under a
doxycycline-inducible promoter [17, 18].
Tethering Assay for RNA Mobility
271