Chapter 3
Visualization of Endoplasmic Reticulum-Associated mRNA
in Mammalian Cells
Jingze J. Wu and Alexander F. Palazzo
Abstract
In eukaryotes, most mRNAs that encode secretory or membrane-bound proteins are translated by ribosomes associated with the surface of the endoplasmic reticulum (ER). Other such mRNAs are tethered to
the ER by mRNA receptors. However, there has been much debate as to whether all mRNAs, regardless of
their encoded polypeptide, are anchored to the ER at some low level. Here we describe a protocol to
visualize ER-associated mRNAs in tissue culture cells by single-molecule fluorescence in situ hybridization
(smFISH). Using this protocol, we have established that a subset of all mRNAs, regardless of whether they
encode secretory or cytosolic proteins, are ER associated in a ribosome-dependent manner.
Key words mRNA, Single-molecule localization, Cell extraction, Endoplasmic reticulum, Ribosomes
1 Introduction
In eukaryotes translation can occur in one of the two general
compartments: free floating in the cytosol and attached to the
surface of the endoplasmic reticulum (ER) [1]. Generally, it was
believed that mRNAs encoding either cytosolic or nuclear proteins
were translated exclusively in the former while mRNAs that
encoded secretory, organellar, or membrane-bound proteins were
mostly translated in the latter. The major difference between the
two is that on the ER, ribosomes bind to the Sec61 translocon,
which enables the newly synthesized polypeptide chain to traverse
the membrane as it is translated, allowing for transmembrane
domains to partition into the membrane, or luminal proteins to
partition into the ER [2]. As a result, the newly synthesized protein
adopts the right topology.
Generally, it is believed that the partitioning of an mRNA to
one of these two compartments is dictated by the properties of the
nascent polypeptide chain. In particular, any mRNA-ribosomenascent polypeptide chain complex that contains a hydrophobic
stretch of amino acids will recruit the signal recognition particle
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_3, © Springer Science+Business Media, LLC, part of Springer Nature 2020
35
Visualization of Endoplasmic Reticulum-Associated mRNA
in Mammalian Cells
Jingze J. Wu and Alexander F. Palazzo
Abstract
In eukaryotes, most mRNAs that encode secretory or membrane-bound proteins are translated by ribosomes associated with the surface of the endoplasmic reticulum (ER). Other such mRNAs are tethered to
the ER by mRNA receptors. However, there has been much debate as to whether all mRNAs, regardless of
their encoded polypeptide, are anchored to the ER at some low level. Here we describe a protocol to
visualize ER-associated mRNAs in tissue culture cells by single-molecule fluorescence in situ hybridization
(smFISH). Using this protocol, we have established that a subset of all mRNAs, regardless of whether they
encode secretory or cytosolic proteins, are ER associated in a ribosome-dependent manner.
Key words mRNA, Single-molecule localization, Cell extraction, Endoplasmic reticulum, Ribosomes
1 Introduction
In eukaryotes translation can occur in one of the two general
compartments: free floating in the cytosol and attached to the
surface of the endoplasmic reticulum (ER) [1]. Generally, it was
believed that mRNAs encoding either cytosolic or nuclear proteins
were translated exclusively in the former while mRNAs that
encoded secretory, organellar, or membrane-bound proteins were
mostly translated in the latter. The major difference between the
two is that on the ER, ribosomes bind to the Sec61 translocon,
which enables the newly synthesized polypeptide chain to traverse
the membrane as it is translated, allowing for transmembrane
domains to partition into the membrane, or luminal proteins to
partition into the ER [2]. As a result, the newly synthesized protein
adopts the right topology.
Generally, it is believed that the partitioning of an mRNA to
one of these two compartments is dictated by the properties of the
nascent polypeptide chain. In particular, any mRNA-ribosomenascent polypeptide chain complex that contains a hydrophobic
stretch of amino acids will recruit the signal recognition particle
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_3, © Springer Science+Business Media, LLC, part of Springer Nature 2020
35