(SRP) [3]. The SRP in turn mediates the recruitment of the
mRNA-ribosome-nascent polypeptide chain complex to the SRP
receptor which is present on the surface of the ER [4]. Once it has
reached the ER, the ribosome directly binds to the surface of the
translocon [5, 6]. The nascent chain is then transferred from the
SRP to the translocon which allows it either to translocate across
the membrane into the lumen of the ER or to diffuse through a
lateral gate and incorporate into the membrane [2]. The idea that
the SRP drives much of the membrane association of mRNAs has
been validated through the examination of ER-derived ribosomeprotected mRNA footprints [7].
Despite all this, the strict division between the cytosol and ER is
not absolute. Many mRNAs that encode secretory proteins are
translated by free ribosomes in the cytosol and their translational
products are post-translationally targeted to the secretory pathway
[8]. It is likely that these mRNAs encode nascent polypeptides that
inefficiently recruit the SRP because they have short and/or mildly
hydrophobic polypeptides. In addition, we and others have documented that a small fraction of all mRNAs that encode cytosolic or
nuclear proteins are anchored to the ER by ribosomes [9–11]. This
ER association is due to the initiation of translation by transloconbound ribosomes. It turns out that non-translating ribosomes can
associate with translocons and are capable of initiating translation
[12–14]. In this case anchoring of mRNAs to translocon-bound
ribosomes would occur before the emergence of any nascent polypeptide. These ribosomes would then translate the mRNAs on the
surface of the ER. Due to the absence of any hydrophobic stretch,
the nascent polypeptide would not be able to access the translocon.
Instead the polypeptide would stay in the cytosol, despite the fact
that it is synthesized from a translocon-bound ribosome. Indeed,
since ribosomes that synthesize cytosolic loops of polytopic
membrane-bound proteins remain associated to the translocon
[15], it is clear that translocon-bound ribosomes can synthesize
long polypeptide stretches that remain in the cytosol.
In addition to this, we and others have documented that
mRNAs can associate with RNA-binding proteins that are present
on the surface of the ER [16–19]. This allows for alternative ways of
associating with the ER that are independent of the encoded
polypeptide.
Here, we present a protocol to interrogate whether given
mRNAs are anchored to the ER by single-molecule fluorescent in
situ hybridization (smFISH), a powerful technique that allows the
detection of mRNAs using a pool of probes (Fig. 1). Using appropriate controls (Fig. 2), we have shown that smFISH can be used to
readily detect target mRNAs with high confidence. The resulting
smFISH foci can be easily analyzed by most imaging software (for
example, see Fig. 3) allowing the researcher to assess the number of
a particular mRNA per cell. We have coupled this imaging
36
Jingze J. Wu and Alexander F. Palazzo
mRNA-ribosome-nascent polypeptide chain complex to the SRP
receptor which is present on the surface of the ER [4]. Once it has
reached the ER, the ribosome directly binds to the surface of the
translocon [5, 6]. The nascent chain is then transferred from the
SRP to the translocon which allows it either to translocate across
the membrane into the lumen of the ER or to diffuse through a
lateral gate and incorporate into the membrane [2]. The idea that
the SRP drives much of the membrane association of mRNAs has
been validated through the examination of ER-derived ribosomeprotected mRNA footprints [7].
Despite all this, the strict division between the cytosol and ER is
not absolute. Many mRNAs that encode secretory proteins are
translated by free ribosomes in the cytosol and their translational
products are post-translationally targeted to the secretory pathway
[8]. It is likely that these mRNAs encode nascent polypeptides that
inefficiently recruit the SRP because they have short and/or mildly
hydrophobic polypeptides. In addition, we and others have documented that a small fraction of all mRNAs that encode cytosolic or
nuclear proteins are anchored to the ER by ribosomes [9–11]. This
ER association is due to the initiation of translation by transloconbound ribosomes. It turns out that non-translating ribosomes can
associate with translocons and are capable of initiating translation
[12–14]. In this case anchoring of mRNAs to translocon-bound
ribosomes would occur before the emergence of any nascent polypeptide. These ribosomes would then translate the mRNAs on the
surface of the ER. Due to the absence of any hydrophobic stretch,
the nascent polypeptide would not be able to access the translocon.
Instead the polypeptide would stay in the cytosol, despite the fact
that it is synthesized from a translocon-bound ribosome. Indeed,
since ribosomes that synthesize cytosolic loops of polytopic
membrane-bound proteins remain associated to the translocon
[15], it is clear that translocon-bound ribosomes can synthesize
long polypeptide stretches that remain in the cytosol.
In addition to this, we and others have documented that
mRNAs can associate with RNA-binding proteins that are present
on the surface of the ER [16–19]. This allows for alternative ways of
associating with the ER that are independent of the encoded
polypeptide.
Here, we present a protocol to interrogate whether given
mRNAs are anchored to the ER by single-molecule fluorescent in
situ hybridization (smFISH), a powerful technique that allows the
detection of mRNAs using a pool of probes (Fig. 1). Using appropriate controls (Fig. 2), we have shown that smFISH can be used to
readily detect target mRNAs with high confidence. The resulting
smFISH foci can be easily analyzed by most imaging software (for
example, see Fig. 3) allowing the researcher to assess the number of
a particular mRNA per cell. We have coupled this imaging
36
Jingze J. Wu and Alexander F. Palazzo