Chapter 11
A Protocol for Non-biased Identification of RNAs Transferred
Between Heterologous Mammalian Cell Types Using RNA
Tagging, Cell Sorting, and Sequencing
Sandipan Dasgupta and Jeffrey E. Gerst
Abstract
Intercellular communication is a major hallmark of multicellular organisms and is responsible for coordinating cell and tissue differentiation, immune responses, synaptic transmission, and both paracrine and
endocrine signaling, for example. Small molecules, peptides, and proteins have all been studied extensively
as mediators of intercellular communication; however, RNAs have also been shown recently to transfer
between cells. In mammalian cells, microRNAs, tRNAs, short noncoding RNAs, mRNA fragments, as well
as full-length mRNAs have all been shown to transfer between cells either by exosomes or by membrane
nanotubes. We have previously described nanotube-mediated cell-cell transfer of specific mRNAs between
heterologous mammalian cell types cultured in vitro. Here, we describe a simple method for the unbiased
and quantitative identification of the complete range of transferred mRNAs (i.e., the mRNA transferome)
in one population of mammalian cells following co-culture with another population. After co-culture, the
individual cell populations are sorted by magnetic bead-mediated cell sorting and the transferred RNAs are
then identified by downstream analysis methods, such as RNA sequencing. Application of this technique
not only allows for determination of the mRNA transferome, but can also reveal changes in the native
transcriptome of a cell population after co-culture. This can indicate the effect that co-culture and
intercellular transfer of mRNA have upon cell physiology.
Key words mRNA, MS2, MS2-binding sequence, β-Actin, Magnetic sorting, MACS, RNA sequencing, Co-culture, RNA transfer, Membrane nanotubes, miRNA, lncRNA
1 Introduction
Multicellular organisms are complex entities composed of multiple
cell types and to ensure functional operation of the organism the
various cell types need to communicate with one another. The
languages in which cells talk to each other include those of secreted
peptides and proteins (e.g., hormones, cytokines, chemokines, cell
adhesion molecules), small molecules (e.g., nitric oxide, carbon
monoxide, neurotransmitters, steroids), and nucleic acids (e.g.,
DNA). In addition, it has been recently shown that RNAs can be
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_11, © Springer Science+Business Media, LLC, part of Springer Nature 2020
195
A Protocol for Non-biased Identification of RNAs Transferred
Between Heterologous Mammalian Cell Types Using RNA
Tagging, Cell Sorting, and Sequencing
Sandipan Dasgupta and Jeffrey E. Gerst
Abstract
Intercellular communication is a major hallmark of multicellular organisms and is responsible for coordinating cell and tissue differentiation, immune responses, synaptic transmission, and both paracrine and
endocrine signaling, for example. Small molecules, peptides, and proteins have all been studied extensively
as mediators of intercellular communication; however, RNAs have also been shown recently to transfer
between cells. In mammalian cells, microRNAs, tRNAs, short noncoding RNAs, mRNA fragments, as well
as full-length mRNAs have all been shown to transfer between cells either by exosomes or by membrane
nanotubes. We have previously described nanotube-mediated cell-cell transfer of specific mRNAs between
heterologous mammalian cell types cultured in vitro. Here, we describe a simple method for the unbiased
and quantitative identification of the complete range of transferred mRNAs (i.e., the mRNA transferome)
in one population of mammalian cells following co-culture with another population. After co-culture, the
individual cell populations are sorted by magnetic bead-mediated cell sorting and the transferred RNAs are
then identified by downstream analysis methods, such as RNA sequencing. Application of this technique
not only allows for determination of the mRNA transferome, but can also reveal changes in the native
transcriptome of a cell population after co-culture. This can indicate the effect that co-culture and
intercellular transfer of mRNA have upon cell physiology.
Key words mRNA, MS2, MS2-binding sequence, β-Actin, Magnetic sorting, MACS, RNA sequencing, Co-culture, RNA transfer, Membrane nanotubes, miRNA, lncRNA
1 Introduction
Multicellular organisms are complex entities composed of multiple
cell types and to ensure functional operation of the organism the
various cell types need to communicate with one another. The
languages in which cells talk to each other include those of secreted
peptides and proteins (e.g., hormones, cytokines, chemokines, cell
adhesion molecules), small molecules (e.g., nitric oxide, carbon
monoxide, neurotransmitters, steroids), and nucleic acids (e.g.,
DNA). In addition, it has been recently shown that RNAs can be
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_11, © Springer Science+Business Media, LLC, part of Springer Nature 2020
195