ribosome–mRNA complexes from the superior colliculus (SCs) in
the midbrain, where RGC axons terminate, and enables transla-
tional profiling of distal RGC axons at different developmental
stages in vivo by deep sequencing [5] (Fig.1a).
Major technical problems of TRAP-based approaches stem
from nonspecific binding of mRNAs expressed from cells that do
not express the tagged ribosome during the affinity-purification
step, which inevitably generates false-positive signals, particularly
when combined with sensitive detection methods such as deep
sequencing. This becomes particularly problematic when the
mRNAs bound to tagged ribosomes represent only a small fraction
of the total mRNA in the dissected tissue. For example, the mRNAs
translated in distal RGC axons represent a very small fraction of the
total mRNAs present in the superior colliculus, where many neu-
rons and glia contain their own mRNAs. We addressed this problem
by “differential expression analysis” on biological replicates of Cre-
positive and -negative groups, which controls for all potential
causes of nonspecific mRNA binding [5] (Fig.1a).
Another key problem of TRAP-based approaches is that they
purify all ribosome-bound mRNAs, not specifically translated
mRNAs. As ribosome-bound mRNAs include not only translated
mRNAs but also translation-stalled mRNAs, such as those bound
to the translational repressor FMRP [10], TRAPed mRNAs do not
necessarily represent the translatome (i.e., the entire set of trans-
lated mRNAs). To address this point, we carried out in vitro ribo-
some run-off assay to distinguish translated mRNAs from
translation-stalled mRNAs. When allowed to continue translational
elongation in vitro in the presence of rabbit reticulocyte lysate and
translation initiation inhibitors, only the mRNAs that were being
translated at the time of tissue preparation resume translation and
finally “run-off” the tagged ribosome (Fig.1b). Quantitative anal-
ysis of TRAPed mRNAs with and without run-off unbiasedly iden-
tifies translated mRNAs [5].
Here, we describe a detailed protocol for the axon-TRAP-
RiboTag of RGC axons in vivo and a method for in vitro ribosome
run-off to validate whether axon-TRAPed mRNAs were being
translated. Although the protocol is designed for mouse RGC
axons, this approach might be applied to other axons, subcellular
compartments and cells.2 Materials
2.1 Tissue
Preparation
- Mouse embryos or adult mice generated through crosses
between a homozygotic RiboTag mouse and a Cre-expressing
mouse (seeNote 1). If a hemizygotic Cre-expressing mouse is
used for the crossing, Cre-negative littermates can be used as
the negative control for TRAP.
Isolation of In Vivo Axonal Translatome 87