Chapter 16
A Single-Molecule RNA Mobility Assay to Identify Proteins
that Link RNAs to Molecular Motors
Varun Bhaskar, Min Jia, and Jeffrey A. Chao
Abstract
mRNA transport and localization is a key aspect of posttranscriptional gene regulation. While the transport
of many mRNAs is thought to occur through the recruitment of molecular motors, it has been a challenge
to identify RNA-binding proteins (RBPs) that directly interact with motors by conventional assays. In order
to identify RBPs and their specific domains that are responsible for recruiting a motor to transport granules,
we have developed a single-molecule RNA mobility assay that enables quantifying the effect of a tethered
RBP on the movement of an RNA. We demonstrate that tethering of RNAs to myosin or kinesin through
their well-characterized interacting proteins results in quantitative differences in RNA mobility. This
methodology provides a framework for identifying RBPs that mediate associations with motors.
Key words Tethering assay, Myosin, Kinesin, RNA transport, Single-molecule RNA imaging, Live
cell
1 Introduction
mRNA transport and cytoplasmic localization is a key aspect of
posttranscriptional gene regulation. While multiple mechanisms
have been described that result in asymmetric RNA localization,
active transport is thought to be the predominant mode by which
mRNAs achieve specific subcellular localization [1–6]. Active transport of an mRNA involves the assembly of a ribonucleoprotein
particle and its subsequent transport by a molecular motor including the kinesin, dynein, and myosin family of motors [7–11]. Therefore, the active transport of mRNA necessitates physical linking of
the target mRNA to a molecular motor for its transport and
subsequent anchoring at its destination [7–11]. Due to the complex architecture of the transport granule and the presence of
multiple RNA-binding proteins (RBPs) within the granule, it has
been a challenge to identify RBPs that directly bind to motors by
conventional assays [9]. In order to identify RBPs and their specific
domains that are responsible for recruiting a motor to a transport
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_16, © Springer Science+Business Media, LLC, part of Springer Nature 2020
269
A Single-Molecule RNA Mobility Assay to Identify Proteins
that Link RNAs to Molecular Motors
Varun Bhaskar, Min Jia, and Jeffrey A. Chao
Abstract
mRNA transport and localization is a key aspect of posttranscriptional gene regulation. While the transport
of many mRNAs is thought to occur through the recruitment of molecular motors, it has been a challenge
to identify RNA-binding proteins (RBPs) that directly interact with motors by conventional assays. In order
to identify RBPs and their specific domains that are responsible for recruiting a motor to transport granules,
we have developed a single-molecule RNA mobility assay that enables quantifying the effect of a tethered
RBP on the movement of an RNA. We demonstrate that tethering of RNAs to myosin or kinesin through
their well-characterized interacting proteins results in quantitative differences in RNA mobility. This
methodology provides a framework for identifying RBPs that mediate associations with motors.
Key words Tethering assay, Myosin, Kinesin, RNA transport, Single-molecule RNA imaging, Live
cell
1 Introduction
mRNA transport and cytoplasmic localization is a key aspect of
posttranscriptional gene regulation. While multiple mechanisms
have been described that result in asymmetric RNA localization,
active transport is thought to be the predominant mode by which
mRNAs achieve specific subcellular localization [1–6]. Active transport of an mRNA involves the assembly of a ribonucleoprotein
particle and its subsequent transport by a molecular motor including the kinesin, dynein, and myosin family of motors [7–11]. Therefore, the active transport of mRNA necessitates physical linking of
the target mRNA to a molecular motor for its transport and
subsequent anchoring at its destination [7–11]. Due to the complex architecture of the transport granule and the presence of
multiple RNA-binding proteins (RBPs) within the granule, it has
been a challenge to identify RBPs that directly bind to motors by
conventional assays [9]. In order to identify RBPs and their specific
domains that are responsible for recruiting a motor to a transport
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_16, © Springer Science+Business Media, LLC, part of Springer Nature 2020
269